The Otorhinolaryngologist and the Early Diagnosis of Mucopolysaccharidosis
D. de Araujo Torres1, M. Pires de Mello Valente1, P. Pires de Mello1, A. Lopes Barth1, and D. Dain Gandelman Horovitz1
1Fernandes Figueira Institute, Rio de Janeiro-RJ
Purpose: Although the clinical presentation of Mucopolysaccharidoses (MPS) may be very heterogeneous, showing variable severity of phenotypes, similar events should motivate clinical suspicion for clinical diagnosis. Overall, the head and neck structures are impacted very early in the disease, usually with recurrent airway infections, otitis media with effusion, hearing loss, delayed speech-language development, severe adenotonsillar hypertrophy, sleep disturbances and other respiratory problems. This study aims to estimate the role of the otorhinolaryngologist in the early diagnosis of MPS through clinical history data recovery from patients followed at Fernandes Figueira Institute / Fiocruz Rio de Janeiro—Brazil, reference center for the treatment of rare diseases. Methodology: A retrospective review was performed from the medical records in addition to interviews with the patients’ guardians, taking into account the history of ENT complaints, number of prior consultations, and also surgery and anesthetic procedures. Results: 24 patients were included, 5 with MPS I, 8 with MPS II, 4 with MPS IV-A and 7 with MPS VI. 43.5% reported that patients presented otitis/ear pain prior to MPS diagnosis; 65.2% have shown symptoms of snoring/apnea; 39.1% have informed about their suspicions of hearing loss, 54.2% reported that there was delay in language acquisition and in 58.3% airway symptoms were among the first manifestations of the disease. These figures show the high prevalence of ear-nose and throat (ENT) involvement since the beginning of the natural history of MPS. In this sample, 70.8% were suspected as MPS by medical geneticists, and in none of the cases the otorhinolaryngologist contributed to early diagnosis, although the number of appointments with this specialist was 1.7 per patient, with a maximum of 10 consultations for the same individual before the MPS diagnosis. One patient underwent adenotonsillectomy and bilateral myringotomy before the diagnosis of MPS, which exposed him to greater risks since surgical procedures in this group of patients require specific care. Conclusion: Otorhinolaryngologists are among the first medical specialists who evaluate MPS patients when the underlying diagnosis is not yet known. Nevertheless, these experts have not been contributing to its earlier confirmation, which could lead to early introduction of specific treatment, preventing irreversible damage. Thus, it is crucial to train these specialists in order to provide early diagnosis of MPS, with a significant impact on the prognosis of MPS patients.
Trachea and Bronchi Evaluation in Mucopolysaccharidoses
D. de Araujo Torres1, M. Pires de Mello Valente1, P. Pires de Mello1, A. Lopes Barth1, D. Dain Gandelman Horovitz1
1Fernandes Figueira Institute, Rio de Janeiro-RJ
Purpose: It is well established that airways complications are among the most common early and lethal manifestations of mucopolysaccharidosis (MPS). Around 20% of surgical MPS patient mortality are due to respiratory tract complications. However, not much is known on the anatomical characteristics of the airway in this patient group, the exact sites of involvement and variability between the different subtypes of MPS. Methodology: We evaluated the airways with a flexible 3,5 mm bronchoscope, trespassing the glottis and viewing the anatomy of the lower airways in 22 patients (4 MPS I, 7 MPS II, 1 MPS III-A, 3 MPS IV-A and 7 MPS VI). 19 patients had been on enzyme replacement therapy (ERT) for 1-10 years by the time of assessment; the remaining were a young MPS I patient who underwent transplantation, one MPS IV-A not yet on ERT and the MPS III-A patient. Results: Tracheal deformities were present in 81.8% (18/22) of the exams and bronchial deformities in 63.6% (14/22). We found different types of structural involvement, ranging from reduced latero-lateral tracheal diameter, loss of the structure of cartilage rings, tortuosity of the trachea to severe tracheomalacia. Among patients with MPS I, 75% (3/4) had tracheal and bronchial deformities. The only MPS I with normal architecture is still very young and underwent transplantation at an early age. In the MPS II, these rates were 85.7% (6/7) for trachea and 42.8% (3/7) regarding bronchi. Only the youngest MPS II patient (4.4 years) did not show lower airway deformity. Also in the MPS II subgroup, a 9 year-old boy has shown progressive structural involvement of the lower airways, with significant malacia of both trachea and bronchial tree; he progressed to collapse and airway obstruction distal to the tracheostomy tube, requiring several cannula exchanges, with increasingly long lengths; he is currently with a longer cannula close to the carina, although still presenting collapse of the distal respiratory tree and significant respiratory compromise. Airway disease still showed clear progression despite ERT introduced 4 years ago. Of the MPS IV-A group, 100% (3/3) had involvement of the trachea and bronchi, while in MPS VI patients 85.7% (6/7) for trachea and 71.4% (5/7) for bronchi. The only MPS VI patient with lower airways within the normal range has a milder disease phenotype. Only one MPS III-A patient was evaluated, and results were quite distinct from other MPS subtypes: anatomical changes in airways were more discrete, but, on the other hand, significant salivary stasis in valleculae, piriform sinus and ventricular bands. These findings are consistent with the neurodegeneration process in MPS III, leading to lack of swallowing coordination. Conclusion: Significant and impressive trachea and bronchi deformities were found, which varied according to the subtype of disease, severity of the phenotype and age of the patient. There appears to be correlation between severity with age and disease duration. The ERT contribution to reduction of disease progression in the lower airways is still unknown, as we have been following some patients with severe progression and compromise of the trachea and bronchi architecture, despite adequate enzyme therapy.
Natural History and Clinical Characteristics of Taiwanese Patients with Mucopolysaccharidosis II: Data from the Hunter Outcome Survey (HOS)
H. Lin1, C. Chuang1, M. Chen1, S. Lin2, P. Chiu3, D. Niu4, F. Tsai5, W. Hwu6, Y. Chien6, J. Lin7, S. Lin1
1Mackay Memorial Hospital, Taipei
2Chi Mei Medical Center, Tainan
3Kaohsiung Veterans General Hospital, Kaohsiung
4Taipei Veterans General Hospital, Taipei
5China Medical University Hospital, Taichung
6National Taiwan University Hospital, Taipei
7Chang Gung Memorial Hospital, Taoyuan
Purpose: Hunter syndrome (mucopolysaccharidosis II) is a rare, X-linked disorder caused by a deficiency in the lysosomal enzyme iduronate-2-sulfatase. This leads to accumulation of glycosaminoglycans in the lysosomes of many organs and tissues, causing progressive cellular dysfunction and clinical signs and symptoms. In Asian countries there is a higher incidence of Hunter syndrome compared with other types of mucopolysaccharidoses. The Hunter Outcome Survey (HOS) is an international, multicentre, long-term, observational study collecting data on participating patients with a confirmed diagnosis of Hunter syndrome. This is the first description of clinical characteristics and surgical history in a large population of patients with Hunter syndrome in Taiwan using HOS data. Methods: From July 2007 to March 2015, 61 patients were enrolled in HOS sites in Taiwan. Patients’ records were collected and analysed for medical history, demographic information, signs and symptoms of organ involvement, type of surgery (at any time) and age at first surgery, as well as age at and cause of death (if a patient had died). Results: Of the 61 patients, 44 were alive at HOS entry and were followed prospectively; 17 had died before HOS entry and available data were entered retrospectively. Median age (10th, to 90th percentiles) at onset of symptoms was 2.5 (0.2, 5.5) years, and at diagnosis was 3.4 (1.2, 11.9) years (n = 55). A total of 44% of patients (21/48) had received enzyme replacement therapy (ERT) with idursulfase (Elaprase®, Shire); median age at ERT initiation was 14.9 (5.0, 24.9) years. Median age at most recent visit in HOS was 15.2 (6.6, 25.0) years. The most prevalent clinical manifestations were facial dysmorphism (98%), claw hands (96%), joint stiffness (95%), abdominal hernia (91%), hepatomegaly (86%), and valvular heart disease (86%) (n = 55). The earliest presenting manifestations included hernia, facial dysmorphism and claw hands, with median ages of onset of 3.0, 4.3 and 4.7 years, respectively. Forty-seven patients (77%) experienced at least one surgical procedure with the most common being hernia repair (54%), ear tube insertion (25%), adenoidectomy (20%) and tonsillectomy (16%). Hernia repair was the earliest type of surgery (median age 4.1 [0.5, 7.9] years). Nineteen patients underwent 20 surgical procedures before receiving a confirmed diagnosis of Hunter syndrome; this included 16 hernia repairs (median age, 0.9 [0.4, 4.7] years) and two ear tube insertions (median age, 2.2 [2.1, 2.2] years). At the time of this analysis, 41% of patients (25/61) had died, and median survival based on Kaplan–Meier estimates (95% confidence interval) was 18.0 (14.2, 25.9) years. Respiratory failure (44%) and cardiac failure or arrest (24%) were the leading causes of death. Conclusions: We found that abdominal hernia was the earliest presenting manifestation experienced by patients with Hunter syndrome in Taiwan, and hernia repair was the most common surgery performed before diagnosis. HOS is a valuable source of information on the natural history and management of Hunter syndrome, which should help to improve early diagnosis and, ultimately, clinical outcomes and quality of life. Funding: Shire sponsors the Hunter Outcome Survey and funds medical writing support.
A MPS II Reference Center Experience: 2002-2016
G. Motta1, D. Miguel1, P. Correa1, A. Acosta1
1Prof. Edgard Santos University Hospital, SALVADOR/Bahia
Purpose: Hunter syndrome is a genetic disorder where iduronate-2-sulfatase (I2 S), an enzyme that degrades glycosaminoglycans, is absent or deficient. Clinical manifestations vary widely in severity and involve multiple organs and tissues. An attenuated and a severe phenotype are recognized depending on the degree of cognitive impairment. Early diagnosis is vital for disease management. Clinical signs common to children with Hunter syndrome include inguinal hernia, frequent ear and respiratory infections, facial dysmorphisms, macrocephaly, bone dysplasia, short stature, sleep apnea, and behavior problems. The aim of this study is present the experience of a Reference Center on treatment of Hunter Syndrome since 2002. Methodology: All patients are followed by this Reference Center since diagnosis of Hunter Syndrome. The Diagnosis was made by screening urinary glycosaminoglycans and confirmed by measuring I2 S activity and/or analyzing I2 S gene mutations. The presented data was collected by meetings records and, prospectively, during appointments when some information was missing. All data was collected after consent form signature. Results: Seventeen MPS type II patients were followed for the last 14 years, of which 11 are alive and six have already died. Among the living patients, the average current age is 17.1 years; and of those who have died, the average age of death was 14.2 years. The average age at diagnosis of all patients was 6.8 years and the average age of onset of symptoms was at 2.2 years. The average delay on MPS II diagnosis was 4.6 years from the onset of symptoms. Of all 17 patients followed, Ten (58.8%) presented any degree of cognitive impaiment and fifteen have received Enzyme Replacement Therapy (ERT) for an average of 5.1 years (0.6-13.0 years). Conclusion: Our patients had age of onset of symptoms similar to those observed in the literature (1.5 years). Compared to the data of Hunter Outcome Survey—HOS (47.6%), our sample had a higher frequency of patients with cognitive impairment. Our patients had a higher age at diagnosis (more than twice the age reported in the literature). The delay in the diagnosis of our patients was approximately four times higher than that described in HOS. The main causes of death and average age on death of the presented patients follow a similar profile to that previously reported.
Long Term Follow-Up of Cognitive Development and Brain MRI Characteristics of Dutch MPS II Patients
A. Vollebregt1, B. Ebbink1, J. van den Hout1, M. Lequin2, I. Plug1, F. Aarsen3, A. van der Ploeg1
1Erasmus MC, Rotterdam
2UMC Utrecht-WKZ, Utrecht
3Sophia Children’s Hospital, Rotterdam
Purpose: Mucopolysaccharidosis type II (Hunters disease, OMIM, MPS II) is a lysosomal storage disorder in which the deficiency of iduronate-2-sulphatase results in the storage of the glycosaminoglycans heparan and dermatan sulphate in multiple organs. Typical clinical characteristics of these patients are coarse facial features, a short stature, hepatosplenomegaly, cardiac valve abnormalities and ivory skin lesions. Around 75% of the MPS II patients develop the neuropathic form of the disease. These patients will develop cognitive impairment and die young. Mildly affected MPS II patients, do not develop evident intellectual disability and are able to survive into adulthood. Our aims were to gain insight in the progression of psychomotor and behavioural problems and to correlate the brain MRI abnormalities to IQ scores in MPS II patients. By using this approach we hope to find predictors for neurological outcome in order to distinguish the mild and severe patients at an early stage. Methodology: All MPS II patients referred to our centre for ERT treatment were included. Cognitive development was tested by using the Griffth mental development scales (mental age of 0-8 yrs), Wechsler intelligence scale for children (6-16 years) and Wechsler adult intelligence scales (> 16 years). For patients with uncompensated hearing loss the Snijders-Oomen nonverbal intelligence test was used. If clinically feasible, a brain magnetic resonance imaging (MRI) was performed on a yearly basis. All available brain MRIs were qualitatively analysed by two observers using a MPS specific protocol. In this protocol the observers scored the severity of 25 different MRI characteristics and localisations e.g. Virchow-Robin spaces, white matter lesions, arachnoidal cysts, ventricle enlargement and bone structure abnormalities. Results: Nineteen MPS II patients were included into this study. The age of the patient at last visit ranged from 4 to 50 years. Brain MRIs were available in sixteen patients. A total of 51 MRIs were analysed, with a maximum of 8 MRIs per patient. Neuropsychological data were available for fifteen patients. In these patients, a total of 85 different neuropsychological measurements had been performed in a follow- up period from 1-9.7 years (mean = 5 years). Nine out of 14 patients (64%), with a current age ranging from 7-20 years, never reached a Griffith mental age above 4 years and showed severe decline in cognitive development. The other 5 patients all have an IQ above 70. However, due to the fact that follow-up is limited due to their current age (4-7 years), presently we cannot predict if these patients ultimately will develop the non-neuropathic or neuropathic form of the disease. We related the results of neurocognitive results to the brain MRI pathology. These results will be presented. Conclusion: Our approach enabled us to gain insight in the relationship between neurocognitive development and brain MRI pathology in this relatively large group of patients and identified multiple brain MRI characteristics related to disease progression in MPS II.
Diagnosis and Treatment of Individuals With MPS II Hunter Disease in the United Kingdom
S. Thomas1, A. Morrison1
1The Society for Mucopolysaccharide Disease (MPS Society), Amersham
Objectives: To understand how and when MPS II is diagnosed and treated in the UK and to determine the prevalence of central nervous system (CNS) involvement and concomitant diagnoses. Methods: Individuals with MPS II resident in the UK were identified by the MPS Society and invited to take part in the survey via telephone interview. A specifically designed questionnaire was used to assess the individual’s diagnoses and treatment as well as their educational attainment and need for support from primary through to further education. Results for the diagnosis and treatment section of the survey only are presented here. Interviews took place in December 2015 and January 2016. Results: Diagnosis Forty one individuals were recruited to the study, ranging in age from 1 to 36 years (mean 12 years). Age at diagnosis of MPS II ranged from 6 days to 7 years (mean 2.5 years). Diagnosis was earlier in the younger individuals, mean 1.8 years (range 6 days to 3 years) in the under 8 year olds, compared to a mean of 2.9 years (range 6 months to 7 years) in those aged 8 years and over. Sixty five percent (27/41) had seen other physicians or specialists before their MPS II diagnosis; paediatrician (n = 24), ear, nose and throat (n = 10), audiologist (n = 4), speech and language (n = 3), others (n = 9). Thirty one percent (13/41) had seen more than one specialist before MPS II was diagnosed. Diagnosis was confirmed by enzyme activity test in urine (n = 6), blood (n = 9) or both (n = 25). There was only one report of diagnosis by genetic testing. Additional diagnoses were reported in 7 individuals after their MPS II diagnosis. Attention deficit hyperactivity disorder (n = 5) and autism spectrum disorder (n = 3) were the most common. Of the 41 individuals surveyed, 54% (n = 22) reported CNS involvement and 37% (n = 15) reported no CNS involvement. A review of these individual’s data indicated all but 3 had CNS involvement with 48% (n = 20) having severe progressive CNS involvement. Treatment Ninety percent (n = 37) were being or had been treated with enzyme replacement therapy (ERT) including 7 individuals receiving or having received intravenous ERT and a clinical trial for intrathecal ERT. The mean age at start of treatment was 5.9 years (range 8 weeks to 27 years). Children born since the reimbursement of ERT (2007 in England, Scotland and Northern Ireland) started treatment at a mean age of 2.4 years (range 8 weeks to 4 years). Four individuals had received hematopoietic stem cell transplants. One individual had received no treatment. Conclusion: MPS II is being diagnosed at an earlier age, but it can still take up to 3 years to obtain a definitive diagnosis. Individuals are often referred to one or more specialists before an MPS II diagnosis is reached. Most individuals are being treated with ERT and normally start treatment before the age of three.
Sanfilippo Syndrome Registry Project and Natural History Studies: An Example of Patients, Parents and Researchers Collaborating for a Cure
J. Wood1, P. Levy2, K. Brown3
1Jonah’s Just Begun-Foundation to Cure Sanfilippo, Brooklyn, New York
2The Children’s Hospital at Montefiore, Bronx, New York
3Patient Crossroads, San Mateo
In the past 5 years, two clinical trials, for Sanfilippo type A, have been conducted, namely Shire’s enzyme replacement therapy and Lysogene’s gene therapy. Abeona recently posted their intentions https://clinicaltrials.gov/ct2/show/NCT02716246?term=abeona&rank=1 Meanwhile we watch the progress of MPSIII A and B programs sponsored by: Alexion, Armagen, Biomarin and Estevee. Furthermore a handful of other academic researchers, supported by patient organizations are throwing their hats in the ring. For the other Sanfilippo types C and D the research is at preclinical stages including gene-therapy (Type C) and Enzyme Replacement Therapy (Type D). These recent scientific advancements towards treatments for Sanfilippo Syndrome indicate that it is time for us to collect and analyze information on Sanfilippo patients in a single centralized registry as part of the PatientCrossroadsCONNECT website (https://connect.patientcrossroads.org/?org=SanfilippoRegistry). In addition it is important we understand how the disease progresses and what differences there may be between the different sub-types. This requires natural history studies (NHS) which can help us in determining the clinical outcome measures, identify potential surrogate endpoints via defined assessments including standardized clinical, biochemical, neurocognitive, behavioral, developmental, and imaging measures. From our experiences such data collected from NHS studies are not shared between researchers except when published as papers at a much later date. Sanfilippo Syndrome has a very small patient population and the participation in multiple NHS (which may be occurring simultaneously) places an unrealistic burden on patients and families. Sanfilippo Syndrome is ultra-rare and patients are geographically diverse. Providing patients and families an outlet to find pertinent information pertaining to Sanfilippo such as where Natural History Studies and clinical trials are taking place or making themselves known by participating in a centralized registry is essential. With the use of RareConnect platform we hope to bring families from around the world closer together and give them access to information that they may not have access to otherwise (https://www.rareconnect.org/en/community/sanfilippo-syndrome). We will describe how the data collected from the NHS studies for Type C and D at The Children’s Hospital at Montefiore will be available to other qualified institutions to prevent repetition. Such NHS studies and registries can also help in identifying participants for clinical trials. We will illustrate how close collaborations between parent/patient led disease organizations and clinical researchers is essential to ensure our limited funding and time is well spent as we try to identify treatments.
Natural History of Siblings with Mucopolysaccharidosis type IIIA (Sanfilippo A Syndrome)
A. Köhn1, A. Meyer2, K. Kossow1, C. Mühlhausen1, K. Ullrich1, N. Muschol1
1University Medical Center Hamburg-Eppendorf, Hamburg
2Asklepios Medical Center North, Hamburg
Background: Deficiency of the lysosomal enzyme sulfamidase (SGSH) leads to the neurodegenerative disorder mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo syndrome), which is characterized by progressive loss of motor, speech and cognitive abilities. Occurrence of symptoms is greatly variable. Even within relatives or siblings there is evidence for a large heterogeneity in the course of disease. Therefore it is difficult to provide a reliable prediction of the natural course of disease in an individual patient. Methods and patients: The natural history of six pairs of siblings with MPS IIIA was assessed using a four-point scoring system (FPSS), quantifying the patient's motor, speech, and cognitive skills over time. Molecular genetic analyses were performed in ten out of twelve patients. Results: Affected siblings showed a similar progression of the disease. First symptoms as well as first regression steps or even preservation of specific abilities were alike in affected siblings. The severity of the course of disease was in accordance with certain gene mutations in the SGSH gene. Conclusions: Similar courses of disease in affected siblings suggest strong genotype-phenotype correlations in MPS IIIA. This has an impact on the predictability of the course of disease, on advices for parents of children with MPS IIIA and might influence therapeutic decisions.
Morquio A Registry Study (MARS): Design and Baseline Characteristics of Enrolled Patients
N. Guffon1, G. Baujat2, M. Bober3, B. Burton4, L. Clarke5, P. Garcia6, R. Giugliani7, C. Hendriksz8, C. Lavery9, J. Raiman10, S. Graham11, E. Jurecki11, Z. Sisic11, A. Waite11
1Hôpital Femme Mère Enfant, CERLYMM, HCL, Lyon
2Hôpital Necker Enfants Malades (AP-HP), Paris
3AI duPont Hospital for Children, Wilmington
4Lurie Children’s Hospital & NWU Feinberg, Chicago
5Children’s and Women’s Centre of British Columbia, Vancouver
6Hospital Pediatrico de Coimbra
7Med Genet Serv HCPA, Dep Genet UFRGS & INAGEMP, Porto Alegre
8Salford Royal NHS Foundation Trust, Salford
9MPS Society, Amersham
10Birmingham Children’s Hospital, Birmingham
11BioMarin Pharmaceutical Inc., Novato
Purpose: The Morquio A Registry Study (MARS; ClinicalTrials.gov #NCT02294877) is a voluntary, multicenter, multinational, observational registry for patients with a confirmed diagnosis of Morquio A syndrome (mucopolysaccharidosis IVA; MPS IVA). The purpose of MARS is to characterize and describe the Morquio A population as a whole, including the heterogeneity, progression, and natural history of Morquio A, study safety and long-term effectiveness of elosulfase alfa, and help the Morquio A medical community with the development of recommendations for monitoring subjects and reporting on subject outcomes to optimize care. Methodology: Demographic, clinical, laboratory, and safety data are collected upon entry into the registry and at 6 and 12 month intervals thereafter from all or some of the assessments dependent upon the individual’s standard care for up to 10 years. Additional data are collected in an ERT pregnancy and lactation sub-study and a separate clinical trial follow-up sub-study. The goal of the clinical trial follow-up sub-study is to assess the long-term safety and efficacy of elosulfase alfa ERT in patients who previously participated in either the extension of the Phase 3 pivotal trial of elosulfase alfa (ClinicalTrials.gov #NCT01415427) or the pediatric clinical trial of elosulfase alfa for patients less than 5 years of age (ClinicalTrials.gov #NCT01515956). Results: As of October 2015, 38 patients have enrolled in MARS, 21 of whom are reported to have received at least one dose of elosulfase alfa. Median age at enrollment is 12.0 years. Median (min, max) age at diagnosis is 3.3 (1, 49) years in ERT-treated patients and 4.2 (1, 12) years in treatment-naïve patients. Conclusion: Through standardized collection of data, MARS will assist in systematically filling the current knowledge gaps around Morquio A disease progression and characterizing clinical outcomes in both the presence and absence of ERT, ultimately facilitating optimal monitoring and care for individuals with Morquio A syndrome.
Obstructive Airway in Morquio A Syndrome, the Past, the Present and the Future
S. Tomatsu1, L. Averill1, M. Theroux1
1Alfred I. duPont Hospital for Children, Wilmington
Purpose: Patients with severe tracheal obstruction in Morquio A syndrome are at risk of dying of sleep apnea and related complications. Tracheal obstruction also leads to life-threatening complications during anesthesia as a result of the difficulty in managing the upper airway due to factors inherent to the Morquio A syndrome, compounded by the difficulty in intubating the trachea. A detailed description of the obstructive pathology of the trachea is not available in the literature probably due to lack of a homogenous group of Morquio A patients to study at any one particular center. We present a series of cases with significant tracheal obstruction who were unrecognized due to the difficulty in interpreting tracheal narrowing airway symptoms. Our goal is to provide the guidelines in the management of these patients that allow earlier recognition and intervention of tracheal obstruction. Methods: Sagittal MRI images of the cervical spine of 28 Morquio A patients (12 ± 8.14 years) showed that19/28 (67.9%) patients had at least 25% tracheal narrowing and that narrowing worsened with age (all 8 patients over 15 years had greater than 50% narrowing). Results: Eight out of 28 patients were categorized as severe (>75%) tracheal narrowing when images were evaluated in neutral head and neck position. Of the 19 patients with tracheal narrowing, compression by the tortuous brachiocephalic artery was the most common cause (n = 15). Evidence of such tracheal narrowing was evident as early as at 2 years of age. The etiology of tracheal impingement by the brachiocephalic artery in Morquio A appears to be due to a combination of the narrow thoracic inlet crowding structures and the disproportionate growth of trachea and brachiocephalic artery in relationship to the chest cavity leading to tracheal tortuosity. Conclusion: Tracheal narrowing, often due to impression from the crossing tortuous brachiocephalic artery, increases with age in Morquio A patients. Greater attention to the trachea is needed when evaluating cervical spine MRIs as well as other imaging and clinical investigations, with the goal of establishing a timely treatment protocol to reduce the mortality rate in this patient population.
Morquio Syndrome Type A Airway and Anesthetic Considerations
S. Tomatsu1, M. Theroux1, W. Mackenzie1, C. Goff1
1Alfred I. duPont Hospital for Children, Wilmington
Purpose: Patients with Morquio A syndrome have short stature, kyphoscoliosis, and restrictive lung due to thoracic bone deformity. Patients with Morquio A syndrome have a higher incidence of airway abnormalities which results in an increased risk of complications from anesthesia, even when undergoing short procedures. However, to date there was no systemic description on the anesthesia issue in Morquio A. Method: We retrospectively reviewed our experience of anesthesia from 1993 to 2014 and identified 37 patients with Morquio A syndrome who have undergone general anesthesia procedures. We collected data including demographics, the otolaryngology procedures, difficulty of the airways, intubation techniques used, extubation success, need for tracheotomy, and postoperative admissions including ICU care. Thirty-seven patients underwent 195 surgical procedures. Complete data analyses were available for 149 procedures. Results: Twenty-two of the thirty-seven patients (59%) were identified as having a difficult airway. Nine of 22 patients (41%) with difficult airways were consistently difficult to intubate requiring multiple attempts for intubation for every anesthetic they received. Seven of 9 patients (78%), who were difficult to intubate previously, had their cervical spinal fusion. Physical characteristics indicative of difficulty with mask ventilation and intubation included restricted mouth opening and a short neck with a limited range of motion. Twenty-eight of 37 patients (76%) had pectus excavatum or carinatum. Conclusion: Preoperative planning and cooperation among the anesthesiology, pulmonology, and otolaryngology should provide safe airway management during surgical procedures. Airway management techniques such as video laryngoscopes help the physcian achieve this goal. Necessity for multiple surgical procedures in most patients with Morquio A makes it a compassionate and safer approach to combine airway examination with another surgical procedure.
Clinical Characterization of Morquio Syndrome Patients at Southwestern Región on Colombia
L. Moreno Giraldo1, A. Escudero Rodriguez1, J. Satizábal Soto1, A. Sánchez Gómez1
1Genomics, Cali
Introduction: Morquio Syndrome (Mucopolysaccharidosis-MPS IVA) is a lysosomal storage disorder due to a mutation in the GALNS gene on chromosome 16q24.3. It is inherited in an autosomal recessive mode, which causes a deficiency in the enzyme N- acetylgalactosamine-6-sulfatase (GALNS). Colombia does not have reliable data on the prevalence and incidence of this disease, only isolated cases have been reported. Besides, archaeological findings suggest the presence of this disorder in our population since pre-Hispanic times and the existence of a founder effect The natural history of disease shows great heterogeneity in the clinical manifestations among patients showing a diversity of bone and joint abnormalities including short stature with neck and short trunk, abnormal gait, genu valgus, hip dysplasia, chest keel, hypermobility ligamentous, and abnormalities in the spinal cord. Among the skeletal manifestations are not committed to respiratory obstruction and / or restriction of the airway, cardiac abnormalities (valvular disease, conduction disturbances and heart rhythm), nervous system disorders with cervical myelopathy, spinal compression; eye disorders, otic, buccal; dental abnormalities and organ enlargement, abdominal hernias. Methodology: Descriptive observational cross-sectional study was perfomed in a cohort of 12 patients with clinical characterization by meon of enzyme assays and molecular diagnosis of Morquio syndrome at Colombian Southwest. anthropometric measurements were evaluated: weight, height (according to tables Growth charts for Patients affected With Morquio type A disease) and BMI (Indicede Body Mass) and taking into account the recommendations of the International Guide for monitoring and treatment of Morquio syndrome (1). The results of diagnostic aids for the characterization of patients were reviewed in each of the medical records. Results: 58% (7 patients) were female and 42% (5 patients) were male. Age ranged between 2 and 28 years. In the weight/age indicator, 1 patient was found with risk of obesity, and 2 in malnutrition state. At the height/age indicator 100% of the patients were normal. Finally, 3 patients were associated with overweight and 2 with severely obese by BMI. 100% had complete medical history with assessment by specialties: genetics, physical medicine, ophthalmology, audiology, dentistry and physical therapy. The signs-symptoms manifested in all patients were associated with osteoarticular deformity, short stature and gait disturbance. The findings in vertebral hypoplasia by MRI, platyspondyly, kyphosis, and ligamentous laxity thickening was found in 42% (5 patients), and two cases with marked decrease of the spinal canal and compression level C2-C3. 25% (3 patients) have reported radiography long bones and pelvis where the genus valgus and bilateral hip dysplasia was a common finding. At cardiovascular level, it was found 1 patient with mild mitral insufficiency and 1 patient with mitral and aortic sclerosis. In the test of 6-minute walk, the most common manifestation was the maximum fatigue of members (10/10 Borg Scale). In spirometry all patients had restrictive changes moderate to severe with an average oxygen saturation of 94%. At neurological examination, all patients had normal intelligence and just one patient with signs of myelopathy. In the ophthalmologic evaluation, all patients had different degrees of refractive errors and 2 patients with opacity of the anterior chamber. In the otolaryngology evaluation, only two patients had altered audiometry with mild bilateral hearing loss and other manifestations of sensory severe type. No clinical or sonographic findings of abdominal hernias were found. Conclusions: Our population with Morquio syndrome show a fair distribution on gender. Some nutritional disorders were registered in our sample with some bias on high BMI. The protocol of clinical therapeutics in our population shows a multidisciplinary approach. As other studies, patients showed a high frequency of osteoarticular disorders. Being an inherited-metabolic multisystem heterogeneous and progressive disease requires early diagnosis, early treatment, ongoing monitoring and rehabilitation to improve the quality and life expectancy, through adequate, complete, and transdisciplinary approach are desirable in order to get the best results on patient life.
Antropometric and Metabolic Characterization on MPS Patients (Morquio Syndrome) at the Colombian Southwestern
L. Moreno Giraldo1, A. Escudero Rodriguez1, J. Satizábal Soto1, A. Sánchez Gómez1
1Genomics, Cali
Introduction: Morquio Syndrome-A (mucopolysaccharidosis IV-A) is a lysosomal storage disease inherited in an autosomal recessive manner. It is caused by genetic deficiency in the enzyme N-acetylgalactosamine-6-sulfatase (GALNS) due to mutation in the GALNS gene that is located on chromosome 16q24.3. The enzyme deficiency results in the tissue accumulation of glycosaminoglycans (GAGs) chondroitin-6-sulfate and keratan sulfate (KS). At Colombia the incidence and prevalence of the disease is unknown. Affected patients with Morquio-A syndrome appear normal at birth, but they will be at an advanced stage of the disease within a few years, where gradually development of deep skeletal-articular abnormalities and a series of respiratory, spinal, cardiac, visual, and dental manifestations, organomegaly, nutritional disorders and growth-development is characteristic. Attenuated and severe for patients with Morquio A phenotypes are classified according to genotype and size. Patients with Morquio-A with severe short stature should avoid obesity and the risks associated with it (diabetes mellitus type 2, dyslipidemia and hypertension, metabolic syndrome components described by Reaven in 1980, associated with cardiovascular risk. No previous studies of nutritional assessment and insulin resistance associated with Mucopolysaccharidosis IV-A are reported in literature. This epidemiological situation promotes the search for metabolic and anthropometric markers that identify early risk to develop metabolic syndrome in this population. Methodology: A Descriptive, cross sectional study on 11 patients with enzymatic and molecular diagnosis of Mucopolysaccharidosis IVA was performed. For each patient plasma fasting levels of total cholesterol, triglycerides, LDL-low density lipoprotein, HDL high-density lipoprotein, glucose, basal insulin; index of insulin resistance (HOMA-IR), and anthropometric measurements (height, weight, and calculate body mass index) were calculated. Growth charts based on a population of patients with Morquio-A enrolled in the International Register of Morquio were used. Results: Of the 11 patients, 54.5% (6) were female and 45.4% (5) were male. Age ranged between 13 and 28 years, mean weight was and 26 kg ranged 17.6-43 kg. The average size was 97 cm (92- 104 cm); the mean BMI was 27.6 kg / m2 (19.92 - 47.65 kg / m2). No changes in blood glucose levels, plasma insulin, and HOMA-IR were registered. Regarding lipid levels, we found 1 patient with hypercholesterolemia. With respect of weight / age indicator, a patient found at risk for obesity and two at risk of malnutrition. At the height / age indicator 100% of the population was normal, for body mass index (BMI) three patients were found in overweight and two severely obese. Conclusions: Patients afflicted with Morquio Syndrome-A have a growth deficit compared to normal children, which is associated with increased incidence of obesity and overweight that lead to an increasing on cardiovascular risk. Childhood obesity is a growing public health problem worldwide. No previous studies of nutritional assessment and insulin resistance in patients with Mucopolysaccharidosis type IVA have been reported in colombia. Using adapted growth charts and biochemical markers’ assessment could helpto monitor the disease and evaluate the clinical efficacy of treatment. Nutritional, anthropometric and biochemical assessments are essential for active surveillance of the disease and prevention of possible complications.
Activity of Daily Living for Morquio A Syndrome
E. Yasuda1, Y. Suzuki2, S. Tomatsu1, T. Shimada1
1Alfred I. duPont Hospital for Children, Wilmington
2Gifu University, Gifu
Purpose: The aim of this study was to evaluate the activity of daily living (ADL) and surgical interventions in patients with mucopolysaccharidosis IVA (MPS IVA). The factor(s) that affect ADL are age, clinical phenotypes, surgical interventions, therapeutic effect, and body mass index. Method: The ADL questionnaire comprises three domains: “Movement,” “Movement with cognition,” and “Cognition.” Each domain has four subcategories rated on a 5-point scale based on the level of assistance. The questionnaire was collected from 145 healthy controls and 82 patients with MPS IVA. The patient cohort consisted of 63 severe and 17 attenuated phenotypes (2 were undefined); 4 patients treated with hematopoietic stem cell transplantation (HSCT), 33 patients treated with enzyme replacement therapy (ERT) for more than a year, and 45 untreated patients. Results: MPS IVA patients show a decline in ADL scores after 10 years of age. Patients with a severe phenotype have a lower ADL score than healthy control subjects, and lower scores than patients with an attenuated phenotype in domains of “Movement” and “Movement with cognitive function.” Patients, who underwent HSCT and were followed up for over 10 years, had higher ADL scores and fewer surgical interventions than untreated patients. ADL scores for ERT patients (2.5 years follow-up on average) were similar with the-age-matched controls below 10 years of age, but declined in older patients. Surgical frequency was higher for severe phenotypic patients than attenuated ones. Surgical frequency for patients treated with ERT was not decreased compared to untreated patients. Conclusion: In conclusion, we have shown the utility of the proposed ADL questionnaire and frequency of surgical interventions in patients with MPS IVA to evaluate the clinical severity and therapeutic efficacy compared with age-matched controls.
Aspects of Functionality in MPS IV-A and MPS VI Children
N. Cavalcanti1, D. Horovitz1, A. Barth1
1Instituto Nacional Fernandes Figueira Fiocruz, Rio de Janeiro
Introduction: Mucopolysaccharidoses (MPS) are lysosomal storage diseases, in which symptoms include progressive involvement of the musculoskeletal system, along with the involvement of other body organs and systems. MPS types IV-A and VI phenotypes have important differences, as ligamentous laxity present in MPS IV-A, as opposed to restricted joints and soft tissues in MPS VI. Purpose: To study functionality in MPS IV-A and VI, their differences and similarities, the functional difficulties in each type, and to anticipate how appropriate therapeutics could enable the resolution of functional problems that usually arise from the expected disease progression. Methods: A longitudinal study was conducted in nine children 8 to 15 years old with MPS (IV-A: n = 5; VI: n = 4), evaluated between January 2014 and February 2016 by the Physical Therapy Department of the National Institute of Women’s Child and Adolescent Health Fernandes Figueira (Fiocruz / Rio de Janeiro / Brazil). Four evaluations were carried out, except in two children, who were evaluated only twice or three times each. The Physical Performance Measure Function of MPS I 1 was applied, consisting of 10 functional tasks and two three minutes walks, in normal and fast pace. Records of 34 evaluations were inserted in EpiInfo 7, with the average of each functional activity and distance walked. Results: Important differences in the two walking paces were observed, with a 38% higher average distance in fast walking in MPS VI (VI = 226,3 meters (m); IV-A = 163,8 m) and 40% higher distance in the normal walk in MPS VI (VI = 205,6 m; IV-A = 146,5 m). Differences were above 20% in the average time to perform the functional tasks of putting on a pullover shirt and stand to squat and return (IV-A = 25 “; VI = 33”), and above 10% in the floor to stand (IV-A = 23 “; VI = 20”) and fine grasp tasks (IV-A = 35 “; VI = 40”). There were no significant differences found in the following activities: putting on a backpack on, stand and reach and shuttle run. Conclusion: Children with MPS VI obtained superior performance in walking activities while in functional tasks there was a slight predominance in the execution speed in MPS IV-A. We believe that the walking difficulty in MPS IV-A may be related to deviations in the alignment of the lower limbs (LL) and dysostoses often present, both associated with nociceptive episodes in lower limbs. Early prevention and intervention measures should be taken, and orthopedic reviews and physical therapy focused to the use of orthotics, surgical patches, muscular work and proper positioning. No significant statistical difference was found in functional tasks, which was surprising, in view of a supposed greater mobility noticed in MPS IV-A. Further studies and more detailed analysis are needed with a larger patient sample to confirm this study's findings and to increase knowledge regarding this important aspect of quality of life in MPS.1 Haley SM, Fragala Pinkham MA, Dumas HM, Ni P, Skrinar AM, Cox GF. A physical performance measure for individuals with mucopolysaccharidosis type I. Dev Med Child Neurol. 2006 Jul;48(7):576-81.
Keywords
Mucopolysaccharidosis, Functionality.
Bone Mineral Density in Morquio Syndrome Type B
F. Kubaski1, H. Kecskemethy1, T. Harcke1, S. Tomatsu1
1Alfred I. duPont Hospital for Children, Wilmington
Purpose: Mucopolysaccharidosis IVB is caused by the deficiency of β-galactosidase (GLB1) which is responsible for the catabolism of keratan sulfate and GM1 ganglioside. Patients with MPS IVB have characteristic skeletal dysplasia with growth retardation although skeletal abnormalities seen in MPS IVB are not as severe as seen in MPS IVA, with less height stunting and overall more typical functional abilities. Several reports of bone mineral density (BMD) are present in patients with MPS IVA; however, there is no report for MPS IVB. Methods: In this prospective study of BMD in three patients (2 females) with MPS IVB, BMD was acquired by dual-energy X-ray absorptiometry (DXA) at whole body (WB), lumbar spine (LS), and lateral distal femur (LDF). Demographics, activity of daily living, height Z-score, Tanner score, and laboratory results were evaluated. Results: Three patients with MPS IVB (mean age 26.9 years; 17.7 to 31.7 years), were evaluated. All were ambulatory: two patients were full-time ambulators without assistance and one patient ambulated with a walker or a manual wheelchair. One patient sustained 2 fractures caused by trauma (fall and MVA). Valid DXA scans were not available at WB for any patient, LS for only one patient, and LDF for all 3 patients. WB and hip were invalid due to the presence of prosthetic hip hardware in every subject. For the one patient with a valid LS, the Z-score was normal (−0.8). BMD in three regions of LDF investigated from three patients was low, and LDF Z-scores in three regions from distal to proximal were −3.1 (range: −2.9 to −3.6) for R1, −2.3 (range −2.0 to −2.5) for R2, and −2.1 (range −2.0 to −2.3) for R3. Conclusion: Patients with MPS IVB have low BMD in LDF even with full-time ambulation. Routine body sites to assess BMD are problematic since anatomical abnormalities precluded a valid measurement of the LS and prosthetic hips. Neither the hip nor WB provides valid BMD. The LDF is the only body site consistently available in all patients. Patients do not have low-energy fractures despite low BMD.
Clinical and Radiological Characteristics of Hip Disease in Mucopolysaccharidosis Type VI
E. Oussoren1, J. Bessems1, V. Pollet1, I. Plug1, A. Devos1, G. Ruijter2, A. van der Ploeg1, M. Langeveld1
1Erasmus University Medical Center, Rotterdam
2Erasmus MC, University Medical Center, Rotterdam
Purpose: Mucopolysaccharidosis type VI (MPS VI) leads to extensive skeletal abnormalities among which the hip problems are the earliest to develop and the most debilitating [1-5]. Methodology: To gain insight in the development of hip pathology in MPS VI, a retrospective study into the features of MPS VI hip disease was carried out. Clinical burden of hip disease was assessed by a questionnaire. In all available hip X ray images, the acetabular index or sharp angle, the acetabular head index and continuity of the Shenton line were measured. Abnormalities in hip morphology were described. Results: A total of 143 X ray images of 14 ERT treated MPS VI patients (age range at first image 2.0 to 21.1 years) were assessed. Median follow up duration was 6.1 years. Only two patients suffered from hip pain, but 10 patients complained of early exertional exhaustion due to their abnormal gait. In all patients, at all ages, the acetabulum and os ileum were dysplastic. Coverage of the femoral head improved over time, but remained insufficient, without loss of congruency of the hip joints. The femoral head appeared normal in the X rays of the youngest patients, but over time the ossification pattern was abnormal in all patients. Conclusion: Hip abnormalities in MPS VI develop very early in life, most likely during fetal development. As such, response to treatment is poor. Surgical interventions should be left as late as possible, since the final shape and angle of the femoral head differs significantly between patients.
Acknowledgments
We thank Allianz Global assistance for having provided part of the data shown in this abstract, the Fondazione Pierfranco and Luisa Mariani always supporting the clinical work in Monza, Vera Marchetti wonderful secretary of the center in Monza, the nurses and doctors in Genova, Bari and Monza for their daily work which made the treatment of these patients feasible.
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
The author(s) received no financial support for the research, authorship, and/or publication of this article.
References
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Minimal Estimated Incidence of MPS I, II, IV-A and VI in Brazil and Comparison to the Rest of the World
A. Federhen1, M. Burin2, S. Leistner-Segal2, U. Matte2, K. Tirelli2, A. Facchin2, G. Pasqualim2, F. Bender2, C. Rafaelli2, R. Giugliani2
1Post-Graduation Program on Child and Adolescent Health, Porto Alegre
2MPS Brazil Network/Medical Genetics Service, Porto Alegre
Purpose: The MPS BRAZIL-NETWORK (MBN) was created in 2004 to provide easy access to diagnosis and to help in management of MPS diseases in Brazil. To achieve these goals, the MBN provides information about the MPS, making it broadly available; provides the laboratory tests needed for the investigation of MPS in suspected subjects; supports educational initiatives and increases awareness about the MPS; provides specialized training for technicians and professionals; facilitates the access of patients to diagnostic and treatment centers; foster research initiatives in the area and builds a comprehensive registry of Brazilian MPS cases. For the past eleven years, the MBN has been trying to draw a picture of disease epidemiology in the country. Between From 2004 and 2013, 1069 MPS patients were identified, including 197 with MPS I (18%), 298 with MPS II (28%), 127 with MPS IV-A (12%) and 250 with MPS VI (23%). Methodology: In order to estimate the minimal incidence of MPS to whom enzyme replacement therapy is available (MPS I, II, IV-A and VI), the number of MPS patients identified by MBN was divided by the total number of live births from 1994 to 2012. Only patients who were born in this period were considered. Similar studies of incidence and prevalence of MPS diseases were performed in countries like Canada, Israel, The Netherlands, Australia, Portugal, Sweden, Norway, Denmark, Tunisia, Taiwan, Saudi Arabia, France, Greece, United Kingdom, Estonia and Czech Republic. The overall estimated incidence (by 100,000 live births) in these countries ranged from 0.11 to 4.0 for MPS I, from 0.13 to 1.48 for MPS II, from 0.07 to 4.0 for MPS IV-A and from 0.05 to 12.5 for MPS VI. Results: According the Brazilian Health System database, from 1994 to 2012 (19 years) 56,587,867 live births occurred in Brazil. Among all the patients diagnosed by the MBN, 138 MPS I, 220 MPS II, 65 MPS IV-A and 177 MPS VI patients were born during this period. So, the minimal incidence (by 100,000 live births) estimated for MPS I was 0.24, similar to Taiwan but much smaller than the incidence reported in other countries. Regarding MPS II, the minimal incidence was estimated as 0.38, similar to Sweden, Norway, Denmark, Tunisia and Czech Republic. Considering only male live births (28,948,394) in the same period in Brazil, the minimal incidence of MPS II was 0.75, much similar to the incidence estimated in British Columbia (0.9) and in West Australia (0.6) for male live births. The minimal incidence for MPS IV-A in Brazil was 0.11, similar only to the incidence founded in West Australia. Regarding MPS VI, the minimal incidence was estimated as 0.31, similar to West Australia, Tunisia and Estonia and higher the incidence reported in The Netherlands, Sweden, Norway, Denmark and Czech Republic. Conclusion: Further studies, in progress, will contribute to estimate more accurately the incidence of MPS in Brazil. Since Brazil is a large country, to create and keep a database which truly reflects the epidemiology of disease in the country is a challenge in which the MBN is working on, as this information is important to support public policies aiming the better management of these treatable rare diseases.
Epidemiology and Clinic Profile of Mucopolysaccharidosis type IIIC in Paraiba, Brazil
P. Medeiros1, S. Santos2, H. Melo1, G. Oliveira1, T. Oliveira1
1Hospital Universitário Alcides Carneiro, Campina Grande
2Universidade Estadual da Paraíba, Campina Grande
Purpose: Mucopolyssacharidosis type IIIC (MPS IIIC, Sanfilippo Syndrome) is caused by cellular acetyl-CoA α-glucosamide acetyltransferase deficiency, being one of the most rare MPS subtypes. MPS IIIC cases represent 11% of all MPS III disorders in Europe and 18% in Spain (total number of patients: 55). MSP IIIC prevalence varies according to geographic region, being estimated in 0.21 cases (per 100,000 newborns) in Netherlands, 0.07 cases in Australia and 0.12 in Portugal. However, no prevalence data for MPC IIIC cases is known for Brazilian population, although the estimated frequency lies around 0.77% (one case amongst 130 studied patients). We detected 8 MPS IIIC cases in Paraiba state, located in Brazil southwest region, which corresponds to 90% of all identified cases. Brazilian southwest region is well known to its high frequency in consanguinity marriages, which is estimated between 6-41%. In addition, a founder effect for Morquio Syndrome (MPS IV-A) was also detected in the same region. Methodology: In order to identify MPS IIIC clinic and epidemiologic profile of in Paraiba state, seventy-eight medical records from patients were revised, altogether with application of a questionnaire during individual interview sessions performed between years 2013 and 2015. Results: We found three female and four male MSP IIIC patients in the cohort analyzed, with mean age of 17.5 years old (min 11, max 22 years). Mean enzymatic diagnostic age was 11 years old (min 6, max 21 years). Consanguinity was observed in only one couple having two affected children. It was found that psychomotor development in the first year of life was normal for all MSP IIIC patients analyzed. First signs and symptoms arouse between 2.5 and 7.5 years old, and among a total of 7 patients it was detected behavioral alterations in 6 of them, one patient presented delay to acquire psychomotor abilities, and other 3 patients showed convulsions starting at 6, 16 and 21 years old. Siblings of consanguine couples showed heterogenic evolution. The younger son, which had a delay in his neuropsychomotor development, also presented fast disease involution and passed away when he was 14 years old, although his 23 years old brother is still alive. Conclusion: MPS IIIC disorder is the most frequent mucopolysaccharidosis subtype in Paraiba state in Brazil Northwest. Its high prevalence is considered a strong evidence to support a founder effect hypothesis considering the great majority of patients from this region. Additional investigation is necessary to strengthen this hypothesis and to better explain the increased number of patients seen in this territory.
A Register for Lysosomal Storage Diseases in Switzerland by the Example of Mucopolysaccharidosis
A. Wiesbauer1
1University of Bern
Background: The different forms of mucopolysaccharidosis are genetic lysosomal storage diseases and are among the rare diseases. There are no epidemiological data for Switzerland so far. Research question: In the thesis I pilot the Swiss Register for lysosomal storage diseases by the example of mucopolysaccharidosis. This involved the following steps: I identified patients in Switzerland with a diagnosis of MPS and extracted their data from the medical records and transferred them to the MPS-register. I determined the frequency of subtypes of MPS in Switzerland and compared it with data from other countries. The age of patients at the onset of symptoms and at diagnosis allowed the calculation of the prediagnostic interval. I described the investigations which led to a diagnosis and examined the data on quality of life in the medical history. Methodology: Through contact with the three centers for lysosomal storage diseases in Bern, Lausanne and Zurich, I was able to identify 51 patients with MPS, collect 39 histories electronically and import them into a database, which should be used later for other lysosomal diseases. To test this option, I imported data of seven patients with other LSDs. Results: Currently (June 2014), 39 patients are living in Switzerland with a diagnosed mucopolysaccharidosis. The total-prevalence is 1: 200,000. 5 patients have MPS I, 12 MPS II, 9 MPS III, 10 MPS IV, 2 MPS VI and MPS VII. The average age of patients at the onset of symptoms was 8 (MPS I), 40 (MPS II), 21 (MPS III), 18 (MPS IV) and 31 (MPS VI) months. The average age at diagnosis was 15 (MPS I), 56 (MPS II), 54 (MPS III), 38 (MPS IV) and 69 (MPS VI) months. The average delay between the first symptoms and the diagnosis was, depending on type 7-38 months. The medical examinations leading to diagnosis have been described. Depending on the type a diag nosis was made only after time-consuming detours. The quality of life is not accurately detected, however indirect indicators are described. Strengths and weaknesses of the Swiss register and the current database could be demonstrated and proposals for further improvements of the register made. Discussion: The results are similar to those found in comparable studies abroad. However, there are significant differences. Strengths and weaknesses of the register have been exemplified. Any future extension requires a prior evaluation of the needs of scientists who will use it for their research.
Newborn Screening for Mucopolysaccharidoses
F. Kubaski1, S. Tomatsu1, R. Mason1, Y. Suzuki2, T. Orii2, S. Yamaguchi3
1Alfred I. duPont Hospital for Children, Wilmington
2Gifu University, Gifu
3Shimane University, Izumo
Purpose: Mucopolysaccharidoses (MPS) are a group of inborn errors of metabolism that are progressive and usually result in irreversible in skeletal, visceral and/or brain damage, highlighting a need for early diagnosis. Methods: This pilot study analyzed 2,638 dried blood spot (DBS) from newborns and 13 DBS from newborn patients with MPS (MPS I, n = 6; MPS II, n = 2; MPS III, n = 5). Disaccharides were produced from polymer GAGs by digestion with chondroitinase B, heparitinase, and keratanase II. Heparan sulfate (0 S, NS) and mono- and di-sulfated KS were measured by liquid chromatography tandem mass spectrometry (LC/MS/MS). A robust measure of variation, median absolute deviation (MAD), was used to determine cutoffs to distinguish patients from controls. Results: The median and MAD for each GAG in controls were as follows: ΔDiHS-0 S, 28 and 9 ng/mL; ΔDiHS-NS, 6 and 3 ng/mL; mono-sulfated KS, 143 and 45 ng/mL; di-sulfated KS, 27 and 17 ng/mL; ratio di-sulfated KS in total KS, 15 and 2%. All patients with MPS I, II, and III showed results above the cutoffs (median + 7X MAD) for ΔDiHS-0 S and ΔDiHS-NS with 3 and 5% false positives respectively. By combining ΔDiHS-0 S and ΔDiHS-NS the rate of false positives was reduced to 2.7%, still distinguished all patients from controls. Conclusions: The study indicated that ΔDiHS-0 S and ΔDiHS-NS are good biomarkers for newborn screening for MPS I, II, and III, as the combination of ΔDiHS-0 S and ΔDiHS-NS allowed the discrimination of controls from MPS patients, with 2.7% false positive samples.
Newborn Screening for Mucopolysaccharidoses—Outcomes of a Comparative Effectiveness Study and Prospective Screening for MPS I
D. Matern1, K. Sanders1, D. Gavrilov1, J. Lacey1, R. Majumdar1, J. Hopwood2, K. Raymond1, P. Rinaldo1, S. Tortorelli1, D. Oglesbee1
1Mayo Clinic, Rochester
2South Australian Health and Medical Research Institute, Adelaide
Backround: Newborn screening (NBS) for MPS I was recently added to the US Recommended Uniform (Newborn) Screening Panel (RUSP). Three states (IL, KY, MO) have already begun screening for MPS I by tandem mass spectrometry (MS/MS) or fluorometry using a digital microfluidics platform (DMF). To prevent a recurrence of the significant variability in performance that characterized the application of MS/MS in NBS, we implemented three high-throughput screening assays for the simultaneous measurement of multiple biomarkers in dried blood spot (DBS) specimens to determine the best approach to NBS for up to 13 different LSDs. Methodology: Our laboratory conducted a comparative effectiveness study employing MS/MS, DMF and an immunoassay to screen 100,000 de-identified NBS samples for MPS I and additional lysosomal and other conditions. To confirm presumptive positive screening results for the de-identified samples, molecular genetic analysis of the relevant genes was performed. Results: All assays proved to be sensitive but revealed a surprising prevalence for some of these conditions hitherto considered rare: MPS I 1:3,600; MPS IIIA 1:33,000; MPS IIIB 1:100,000; MPS VI: 1:25,000 newborns; and MPS II 1:5,300 male newborns. Of note, most of the mutations found among these cases have not been described before and are therefore considered variants of uncertain clinical significance. Conclusions: Following completion of the study, a web-based application based on the Region 4 Collaborative (R4 S) project for MS/MS data sharing and comparison has been created (https://clir.mayo.edu/) to facilitate newborn screening result review and improve screening performance. Since February 2016, the MS/MS based assay, R4 S tools and relevant 2nd tier assays (incl. for glycosaminoglycans) have been used in our laboratory to prospectively screen KY newborns for MPS I, Pompe disease and Krabbe disease. It is expected that ca. 25,000 newborns will have been screened by July 2016.
Targeted-Population Screening for Mucopolysaccharidoses—A Highly Efficient Tool for the Diagnosis of Patients
Z. Lukacs1, P. Nieves Cobos1, S. Murko1, R. Santer1, A. Gal1
1Hamburg University Medical Center, Hamburg
Background: Lysosomal storage diseases present with highly variable symptoms and usually with different severity, depending on the individual age of onset. Diagnosis is sometimes further delayed because of the relative rarity of the diseases which does not make them prime candidates in diagnostic algorithms. Recently, dried blood specimens have been introduced for enzyme activity measurement of a number of lysosomal enzymes and thereby, help to facilitate testing. Methods: Dried blood cards have been shipped from distant health care providers (Middle East, Russia, Southern Europe, South Africa, among others) to the metabolic laboratory at the Hamburg University Medical Center. Patients showed symptoms compatible with mucopolysaccharidoses (MPS), esp. coarse face, hepatosplenomegaly, and rheuma-like symptoms without inflammation. The samples were tested for MPS I, MPS II (male) and MPS VI using fluorometry or tandem mass spectrometry. Results: Among 4705 samples tested to date, 7.8% have tested positive for MPS I, 3.5% tested positive for MPS II, 4.8% for MPS VI and 2.7% showed enzyme activities compatible with mucolipidoses II/III. In addition, ten samples were suspicious for multiple sulfatase deficiency. Among the first 200 samples that were received in the laboratory all positive samples have also been assessed molecular genetically. Enzymes results for MPS I were confirmed in 85% of patients, while all MPS II and MPS VI cases were confirmed. Conclusion: Targeted-population screening of patients with symptoms remotely associated with the distinct lysosomal storage diseases using DBS proved a highly efficient approach in expediting the diagnosis of patients. In addition, it is cost effective, especially in comparison to whole-population screening and circumvents ethical problems that arise from testing a (potentially) healthy population.
Arylsulfatase B Activity Determination for Screening and Diagnosis of Mucopolysaccharidosis VI—Six Years of Experience in São Paulo, Brazil
V. D’Almeida1, M. Braga1, J. Yamamoto1, A. Esteves1, V. Pereira1, A. Martins2
1Laboratory of Inborn Errors of Metabolism, Sao Paulo
2Center of Reference in Inborn Errors of Metabolism, Sao Paulo
Purpose: Mucopolysaccharidosis VI is one of the lysosomal storage diseases that could be treated by enzyme replacement therapy, which reinforces the need of accurate diagnostic methods for this disorder, allowing a better outcome with earlier initiation of therapy. Methodology: Since October 2009, we performed analysis of arylsulfatase B from dried blood spot (DBS) and leukocytes samples of patients suspected of Mucopolysaccharidosis type VI at the Laboratory of Inborn Errors of Metabolism, in São Paulo, Brazil. The analysis was performed using a synthetic fluorogenic substrate according to the method described by Civallero et al. 2006. Results: We received samples from 1058 patients from the different regions of the country. The mean age was 9 years (SD 10; range from 0 to 68 years old) and 57.7% of the patients were males. The DBS results were considered normal when enzyme activity was higher than 3.17 µmol/L blood/hour. Among all DBS samples we identified 6.5% of positive results. Concerning leukocytes samples, we analyzed 116 samples and 41.4% had positive results (enzyme activity lower than 7.5 nmol/mg prot/hour). During this period, 83 DBS samples were also tested in leukocytes. Among them, we could confirm the results in 87% of the samples (39.8% of positive cases of MPS VI). We had some samples rejected due to low quality (4.7%). Conclusion: Considering the high rate of agreement between DBS and leukocytes, our experience showed the relevance of using DBS in MPS VI diagnosis.
Screening for Mucopolysaccharidoses in Patients With Short Stature of Unknown Etiology
J. Franco1, G. Spinosa1
1Hospital Candido Fontoura, Sao Paulo
Introduction: Mucopolysaccharidoses (MPS) comprise a group of genetic lysosomal storage disorders (LSD) that result from the deficiency of one or more of the enzymes required for glycosaminoglycan (GAG) catabolism. The accumulation of these molecules leads to cellular destruction, causing progressive tissue and organ dysfunction. The multisystem manifestations and the somatic disease cause significant burden, especially osteoarticular and neurological disabilities. Diagnosing this group of diseases is challenging in many cases. The misdiagnosis is due to lack of awareness of health care professionals, to poor access to screening and to their variable clinical spectrum (Siqueira et al, 2015). Purpose: The goal of the present study was to verify the frequency of MPS in a sample of Endocrinology with short stature of unknown etiology. Methodology: It was conducted retrospectively from November 2014 to December 2015, 836 outpatients medical reports of short stature was reviewed in a Pediatric Endocrinology Service of São Paulo, Brazil. Among those 898, almost 100 cases were selected for a second clinical assessment with a medical geneticist. The main inclusion criterion was short stature that had unknown etiology associated to osteoarticular signs (trigger fingers, joint stiffness, or other symptom as history of hernia). After a clinical reassessment, 67 dried blood spots (DBS) on filter paper were done, 21 cases were positive and samples were recollected for enzymatic assay in leucocytes. Three patients (0,33% positivity rate) were confirmed which was previously undetected (2 MPS II and 1 MPS III). Conclusion: This study shows that these diseases are underdiagnosed, even among growth experts and that systematic screening can help identify patients who may benefit from early treatments already available for several MPS types.
A Cross-specialty Collaboration Platform between Cardiologists and Medical Geneticists Based on High-risk Criteria for Mucopolysaccharidosis Confirmative Diagnosis
H. Lin1, S. Lin1, C. Hung1, C. Chuang1, S. Huang1, R. Tu1, D. Niu2, C. Hou1
1Mackay Memorial Hospital, Taipei, Taiwan
2Taipei Veterans General Hospital, Taipei, Taiwan
Purpose: The mucopolysaccharidoses (MPSs) are inherited lysosomal storage disorders caused by the absence of functional enzymes that contribute to the degradation of glycosaminoglycans (GAGs) which cause damages in various organs including the heart. Cardiac abnormalities have been observed in patients with MPS of any types, with the most documented abnormalities being cardiac valve thickening, valvular regurgitation and stenosis, and cardiac hypertrophy. The incidence of all types of MPS varied in different populations ranging from 1.95 to 4.50 in 100,000 live births. In Taiwan, the collective birth incidence of all MPS patients was 2.04 per 100,000 live births. The aim of the study was to establish a cross-specialty collaboration platform between Cardiologists and Medical Geneticists based on high-risk criteria for further MPS confirmative diagnosis. Methodology: In cardiovascular clinics, adult patients (more than 18 years of age) with valvular heart disease detected by echocardiographic examinations, and of unknown cause determined by Cardiologists were prospectively enrolled in this study between June 2014 to August 2015. Urine samples were collected, and quantification as well as two-dimensional electrophoresis of urinary GAGs were checked, followed by a determination of disaccharides, eg. dermatan sulfate (DS), heparan sulfate (HS), and chondroitin sulfate (CS), in the GAGs using liquid chromatography-mass spectrometry (LC-MS/MS) for the MPS patient screening. Written informed consent was obtained from each of the subjects. The study was approved by the ethics committee of Mackay Memorial Hospital, Taipei, Taiwan. Results: One hundred and three patients (M/F: 47/56) with valvular heart disease of unknown cause were enrolled. The mean age was 47.2 ± 15.6 years. The median age was 46.2 years. The age ranged from 18.8 to 72.2 years. Urine DMB ratio of all these 103 patients were within normal reference range (5.2±2.5 mg/mmol creatinine). Urinary DS, CS and HS concentrations determined by LC-MS/MS detection were also within normal reference ranges. Conclusion: Although no suspected MPS patient was screened out by urinary GAGs quantification in this study, the situation reflected the rare nature of this disease. We smoothly established a cross-specialty collaboration platform between Cardiologists and Medical Geneticists based on high-risk criteria for MPS patient screening. Further larger cohort for MPS high-risk group screening will be warranted to alert more awareness of different medical specialties.
The Clinical Benefit of Gag Electrophoresis as a First Line Simple Screening Test for the Diagnosis of Mucopolysaccharidoses: A Real Life Study of an MPS IIIA Case
H. Derin1, H. Caksen1, T. Tanyalcin2, K. Harvey3, D. Burke3
1Pediatric Neurology, Meram Medical Faculty, Konya
2Tanyalcin Medical Laboratory, Izmir
3Enzyme Unit, Chemical Pathology, Great Ormond Street Hospital, London
The purpose of this study is to share the clinical importance of urine GAG (glycosaminoglycan) as a simple screening test for the diagnosis of mucopolysaccharidoses. A10-year-old male patient (51750- S Cetin) was admitted to paediatric neurology outpatient clinic with aphasia, inability to walk, seizures and swallowing difficulty. There was no a history of perinatal hypoxia. The parents do not describe irritability, temper tantrums or sleep disturbance. Consanguinity is present in parents with first-degree cousin marriage. Developmental delay was recognized in the first 1.5 years of his life. The patient's 12-year-old brother cannot walk either and can not sit without support and he receives antiepileptic therapy for his epilepsy. The patient was found to have coarse facial features with mental retardation, hypertrichosis, joint stiffness-contractures, kyphosis and scoliosis. Brainstem auditory evoked response test and ocular examination was normal. Chromosomal analysis revealed 46 XY karyotype. Cranial magnetic resonance imaging revealed a diffuse cerebral atrophy and ventricular dilatation in both hemispheres. Electroencephalography showed epileptiform abnormalities with focal and secondary generalization. Based on the clinical and electroencephalography findings, the patient’s antiepileptic medications were reviewed. As a diagnostic screening test, urine GAG levels were measured and GAG electrophoresis was performed http://iem.sagepub.com/content/3/2326409815613805.full.pdf+html.
Cellulose acetate GAG electrophoresis showed a clear heparan sulfate band that is thicker and heavier when compared to chondroitin sulfate band. Refer to the picture; the 4th row from top is the MPS III A patient. QC TR control sample at row 6 from top is the control sample with including keratan, chondroitin, heparan and dermatan sulfate standards. Total GAG concentration in urine is 287 mg/L; 27 mg GAG/mmol creatinine, the reference range for this age is 3-11 mg/mmol creatinine. White blood cell heparin sulphamidase activity was undetectable; reference range for MPSIIIA cases 0-0.4 nmol/17h/mg ptn, plasma a-N-Acetylglucosaminidase activity was 42 nmol/hr/ml (12-73) and plasma total beta-hexosaminidase activity was found to be 1.37 umol/hr/ml (0.41 -1.7), the latter 2 enzymes are in the unaffected range. Beta -galactosidase activity in homogenate was within the unaffected range at 109 nmol/hr/mg ptn. These results are consistent with a diagnosis of mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo disease). In this study, it is clear that abnormal amounts of heparin sulfate in urine can be identified by the simple screening method GAGE (GAG electrophoresis). A diagnosis of Sanfilippo syndrome was confirmed by determining the absence of a specific lysosomal enzyme required for the degradation of heparin sulfate in lysosomes (heparin sulphamidase). Further confirmation and characterization by mutation analysis is recommended.
Mass Spectrometry Analysis of Disaccharides for Morquio Syndrome by Enzymatic Digestion of Keratan Sulfate
P. Lavoie1, C. Auray-Blais1, M. Boutin1
1CIUSS-CHUS de l’Estrie, Hospital Fleurimont, Sherbrooke
Purpose: Morquio A syndrome (Mucopolysaccharidosis (MPS) IVA) is an autosomal recessive lysosomal storage disorder caused by a defect of the enzyme N-acetylgalactosamine-6-sulfatase, which plays a key role in the catabolism of keratan sulfate (KS), a glycosaminoglycan. The objectives of this research project were to: 1) implement and validate a mass spectrometry methodology for the analysis of two main disaccharides related to KS; 2) to establish normal reference values; 3) to analyze urine samples from a group of untreated patients with Morquio A syndrome and other types of mucopolysaccharidoses; 4) to evaluate the sensitivity and specificity of the assay compared to the total GAG measurement DMB-based spectrophotometric test. Methodology: One hundred microliters of urine was added to ten microliters of a keratanase II solution, followed by the addition of one hundred microliters of an ammonium acetate buffer and the internal standard working solution. Incubation was performed for three hours at 40°C. The detection of two KS disaccharides (Galβ1-4GlcNAc(6 S), or LacNAc S1 and Gal(6 S)β1-4GlcNAc(6 S), or LacNAc S2) was achieved using a Xevo TQ-S (Waters Corp.) tandem mass spectrometer coupled to an Acquity I-Class ultra-performance liquid chromatography system (see Figure 1). Results: Abnormal values of LacNAc S1 and LacNAc S2 normalized to creatinine were obtained for all patients with Morquio A syndrome, confirming the high sensitivity of the test. The unreliability of the DMB-based spectrophotometric methodology was confirmed with normal results obtained from urine samples of some Morquio A patients. Conclusion: Considering that elevations of both disaccharides were also found in some patients with MPS types I, II, and VI, we recommend that this KS test be used for high-risk screening of Morquio IVA patients. Abnormal cases would afterwards be confirmed by enzyme activity. This test will be useful for monitoring and long term follow up of patients with Morquio A syndrome under enzyme replacement therapy treatment.
Enzymatic digestion of keratan sulfate by keratanase II.
Multiplex Methodology for the Analysis of Dermatan Sulfate, Heparan Sulfate, Keratan Sulfate and Chondroitin Sulfate by UPLC-MS/MS
C. Auray-Blais1, P. Lavoie1
1CIUSSS-CHUS de l’Estrie, Sherbrooke, QC
Purpose: Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders resulting from primary defects in enzymes involved in the degradation of glycosaminoglycans (GAGs). As shown in Table 1, different urinary GAG profiles are observed depending on MPS types. A mass spectrometry methodology based on the methanolysis of GAGs and analysis of related disaccharides was devised and validated. Dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and chondroitin sulfate (CS) were specifically targeted. The primary objective of this research project was to improve the detection, diagnosis and monitoring of patients affected with different types of MPSs, such as Hurler/Scheie (MPS I/H, I/S, IH/S), Hunter (MPS II), Sanfilippo (MPS III), Morquio (MPS IV) and Maroteaux-Lamy (MPS VI) syndromes. Methodology: Twenty-five microliters of urine were evaporated under a nitrogen flow. Thereafter, five hundred microliters of methanolic hydrochloric acid (3 N) was added and samples were incubated for 1 hour at 65°C. Samples were dried under a nitrogen flow, then resuspension was performed using two hundred microliters of a solution containing all deuterated internal standards. Analyses were done using an Acquity I-Class system equipped with a BEH Amide column for ultra-performance liquid chromatography coupled to a Xevo TQ-S (Waters Corp.) tandem mass spectrometer. Results: Intraday precision and accuracy results were good with RSDs at ≤ 7.6% and biases ranging from −12.0% to 18.4%. Interday precision and accuracy results showed RSDs at ≤ 8.7% and biases ranging from −12.8% to 14.2%. Different types of mucopolysaccharidoses were easily differentiated according to the urinary GAG profile. A longitudinal study prior to and after enzyme replacement therapy (ERT) treatment showed reduced levels of DS and HS over time in an 8 month-old MPS I patient and a 12 year-old MPS II patient. Conclusion: The quantitation of urinary disaccharides related to DS, HS, KS, and CS using mass spectrometry in patients with mucopolysaccharidoses is a useful tool for high-risk screening studies, and for the diagnosis and monitoring of patients under ERT.
Enzyme Deficiencies and Related GAG Profiles in Mucopolysaccharidoses.
Expected GAG elevation
MPS Type
Disease Name
Deficient enzyme
DS
HS
CS
KS
Hyaluronan
MPS IH
Hurler
α-Iduronidase
•
•
MPS IHS
Hurler-Scheie
α-Iduronidase
•
•
MPS IS
Scheie
α-Iduronidase
•
•
MPS II
Hunter
Iduronate sulfatase
•
•
MPS IIIA
Sanfilippo A
Heparan-N-sulfatase
•
MPS IIIB
Sanfilippo B
α-N-acetylglucosaminidase
•
MPS IIIC
Sanfilippo C
AcetylCoA α-glucosamine acetyltransferase
•
MPS IIID
Sanfilippo D
N-acetylglucosamine 6-sulfatase
•
MPS IVA
Morquio A
Galactosamine-6-sulfate sulfatase
•
•
MPS IVB
Morquio B
β-Galactosidase
•
MPS VI
Maroteaux-Lamy
N-acetylgalactosamine 4-sulfatase
•
MPS VII
Sly
β-Glucuronidase
•
•
MPS IX
Natowicz
Hyaluronidase 1
•
Mucopolysaccharidoses and Other Lysosomal Diseases: Relative Frequency Reported From Data of a Brazilian Reference Center
R. Giugliani1, A. Federhen1, K. Tirelli1, J. de Mari1, F. Bender1, F. Medeiros1, A. Scholz1, F. Bittencourt1, R. Guidobono1, M. Burin1
1Medical Genetics Service, Porto Alegre
The Medical Genetics Service of Hospital de Clínicas de Porto Alegre, located in the Southern part of Brazil, has the most comprehensive laboratory for the diagnosis of lysosomal storage diseases (LSDs). From 1982 to 2015, this laboratory was able to identify 3,286 cases of LSDs (average of almost 100 cases/year), diagnosed on samples of 60,000 high risk patients from all Brazilian regions referred for investigation. Screening methods included quantitation and electrophoresis of urinary GAGs, thin layer chromatography of urinary oligosaccharides and syalyloligosaccharides, assay of chitotriosidase activity in plasma, and other selected procedures performed according to the clinical suspicion. Diagnoses were confirmed by specific fluorimetric, colorimetric or radioisotopic enzyme assays, and/or by the identification of the pathogenic mutations, usually in blood samples. The Mucopolysaccharidoses (MPS) were the most common LSD diagnosed (considering all types). The most MPS types diagnosed were MPS II (419 cases), MPS VI (285 cases), MPS I (276 cases), MPS IVA (175 cases) and MPS III B (102 cases). Other LSDs were identified as Gaucher Disease (726 cases), Acid Sphingomyelinase Deficiency/ASMD (213 cases), GM1 Gangliosidosis (176 cases), Metachromatic Leucodystrophy (150 cases), Fabry disease (118 cases) and Krabbe disease (101 cases). These results indicate that LSDs, although individually rare, may be frequent when investigation is concentrated in reference laboratories. Large numbers of cases enable centers to obtain experience in managing these conditions, and also to perform natural history studies, and to participle in clinical trials. It is important to mention that the majority of patients identified could benefit from the therapeutic alternatives already available or in clinical development for these conditions.
Mutational Cluster Analysis on a Mucopolysaccharidosis Cohort at Southwestern Region in Colombia
A. Sánchez Gómez1, J. Satizábal Soto1, L. Moreno Giraldo1, A. Escudero Rodriguez1
1Genomics, Cali
Introduction: Even though mucopolysaccharidosis diseases are considered with a low incidence in general populations the Southwestern region of Colombia has showed an increasing on incidence of mucopolysaccharidosis diseases at recent years. Several authors have proposed different mechanisms to explain this increasing. Health local agencies are looking for evidences to confirm this phenomena due to high cost on therapeutic treatment s for these kind of diseases. Our research group has screened gene variants on a set of patients suffering any type of Mucopolysaccharidosis. With the results of this screening we found a pattern of transitional mutations C>T or T>C along gene sequences that leads to proposed a molecular mechanism involved in de novo development of pathological sequence variants. Mehodology: Gene sequencing was performed on a group of 11 patients diagnosed with any of the Mucopolysaccharidosis described in literature, to characterized variants in sequences. For new SNPs, SIFT and PolyPhen2 analysis were ran to determine pathological effect. Results: A total of 69 gene variants were reported, from those 28 were characterized as transitions C>T and T>C. For those 28, 5 were not registered previously. 3 were associated as pathogenic and explained the symptoms in the patients. Just 6 gene variants were located at intron regions and none of them were associated with alterations on gene splicing or truncated proteins. Finally, 7 variants were synonymous and 17 did not show protein shift. Several authors have reported a preference on mutations along CpG islands with a prevalence of nearly 80% in transitional mutations. Conclusions: A possible explanation of this behavior would be related with epigenetic mechanisms like DNA methylation to bias gene expression on altered sequences to alleviate any pathogenic effect. We consider this finding very valuable to propose a broad screening of methylation patterns along mucopolysaccharidosis genes to elucidate molecular etiopathogenesis.
Mutational Profile of Brazilian MPS I Patients
G. Pasqualim1, E. Poletto1, U. Matte2, R. Giugliani3
1Postgraduate program in Genetics and Molecular Biology, Porto Alegre
2Gene Therapy Center, Porto Alegre
3Medical Genetics Service, Porto Alegre
Purpose: Mucopolysaccharidosis type I (MPS I) is a recessive lysosomal storage disorder caused by mutations in the α-L-iduronidase (IDUA) gene. Molecular diagnosis can confirm clinical suspicion and allows genetic counselling. Moreover, to some extent, permits a correlation with phenotype and can guide the choice of treatment. Hence, knowledge about mutation profile of a determined population can accelerate and reduce the costs of diagnosis as well as facilitate choice of treatment. Although some mutations are very common worldwide, local frequencies can vary significantly. Therefore, in this study we analyse the IDUA mutation profile in Brazilian patients. Methodology: Whole blood genomic DNA was extracted from EDTA tubes either by standard salting-out method or commercial kits according to manufacturer’s instructions (Wizard® Genomic DNA Purification from Promega, Easy-DNA or iPrep PureLink gDNA Blood Kit, from Thermo Fisher Scientific). Exons and flanking intronic regions of IDUA were then amplified by PCR. Following purification with PEG 8000 20% NaCl 2.5 M and quantification with Low Mass Reader (Thermo Fisher Scientific, USA), sequencing was done in ABI3500 genetic analyzer using BigDye Terminator v3.1 (Applied Biosystem Thermo Fisher Scientific, USA). Sequences were analyzed by comparison to genomic reference sequence (GenBank accession number NG_008103) and all alterations were confirmed by reverse strand sequencing. Results: From 2006 to 2016, 115 patients had confirmed MPS I diagnosis. Amongst them, we were unable to determine the second causative mutation by Sanger sequencing in 2 samples with confirmed clinical and biochemical diagnosis. One patient from a neonatal screening program was a compound heterozygous for the a pseudodeciency allele (c.246C>G [p.(H82Q)]). Nonsense mutations were the most common class of alterations, corresponding to 45% of total. By far the most frequent mutation identified was p.W402X, corresponding to 38% of alleles (32 homozygous and 24 heterozygous patients). Next, p.Q70X, p.R621X e R619X showed a combined frequency of 5%. Missense mutations consisted of 40% of total alleles. In particular, p.P533 R was the second most frequent of all mutations with frequency of 16%. It was followed by p.G208D with 4% and p.R383 H, p.R89Q, L18P e p.Y625C, all with 3%. Finally, small deletions such as c.46_57del12, c.144_146delGAG, c.1333_1335delGAC and 1044_1049delCGACAA had a combine frequency of 9%. No gross deletions, gross insertions or complex rearrangements were found. Conclusion: The mutations p.W402X and p.P533 R are the most frequent mutations in Brazilian patients with a combined frequency of 54%. Target analysis of the exons affected by these mutations (9 and 11) as well as exons 2, 14 and 8 can cover more than 70% of genetic variations found in this population. Thus, such approach could reduce time and costs of diagnosis.
Array-CGH Analysis as an Alternative Method for the Detection of Female Carrier of Genomic Rearrangements Within the IDS Gene Region
M. Riegel1, N. Ortigara1, A. Brusius-Facchin1, S. Leistner-Segal1, P. Rozenfeld2, R. Giugliani1
1Serviço de Genética Médica, HCPA, Porto Alegre
2Facultad de Ciencias Exactas, Universidad Nacional de La Pla, Buenos Aires
Purpose: To evaluate the use of high-resolution array-based Comparative Genomic Hybridization (Array-CGH) for the detection of genomic rearrangements in the IDS gene region in female relatives of selected MPS II patients. Methodology: Chromosome microarray analysis was performed on DNA samples from 7 female relatives of MPS II patients with known deletions or duplications within the IDS gene region. The customized oligonucleotide platform using the one million microarray (Agilent Technologies Inc., Santa Clara, CA) was applied to each of the samples (from subjects to be tested, and controls) in order to identify chromosomal rearrangements in the Xq28 region and to determine the genomic breakpoints. The arrays were analyzed through the Agilent Feature Extraction for CytoGenomics. Graphical overview was obtained using the Agilent CytoGenomis analysis software. Results: The array-CGH data demonstrated partial or complete deletions in 3 of the 7 samples analysed. Additionally. The microarray analysis revealed a contiguous duplicated region on Xq28 in one of the samples. Conclusion: This report outlines an alternative approach for the detection of female relatives of MPS II patients with IDS gene deletions or duplications. For a specific group of mutations such as complete gene deletions including contiguous genes and gene inversions/rearrangements, traditional sequencing or even newer technologies as massive parallel sequencing, would fail to identify female carriers of intragenic or whole-gene deletions/duplications. Furthermore, genomic positions of the breakpoints within the Xq28 region cannot be determined by traditional methods. The array-CGH detection of female carriers not only provides valuable information for genetic counseling and clinical management, but also enables the detection of genomic rearrangements in and around the IDS region. Furthermore, array-CGH may be particularly useful as complementary diagnostic test to the gene sequencing analyses in families where intragenic deletions, duplications, and rearrangements are causative. As gene CGH array is a technology just recently being used in the metabolic and lysosomal disorder investigation, the intragenic deletions/duplications detected need to be confirmed. Methods such as breakpoints PCR, Southern blot, real time PCR, or confirmation with dye swap array CGH can be considered to determine the exact breakpoints involved in the gene rearrangements.
Molecular Findings of Malaysians With Mucopolysaccharidosis Type II
H. Leong1, A. Abdul Rashid1, Y. Leong1, M. Haniffa1, W. Ong1, G. Ch’ng1, W. Keng1, Y. Wong2, H. Chew1, L. Ngu1
1Genetics Department, Kuala Lumpur
2DNA Laboratories Sdn Bhd, Selangor
Mucopolysaccharidosis type II (MPS II) or Hunter syndrome is an X-linked inherited lysosomal storage disorder with a wide spectrum of clinical severity caused by deficiency of iduronate-2-sulfatase (IDS). Mutational analysis of the IDS gene was performed on 14 Malaysians with MPS II from 13 families and mutations were detected in all the patients studied. The results showed allelic heterogeneity. There were five patients with missense mutations: c.262C>T (Arg88Cys), c.263G>A (Arg88His), c.329G>A (Arg110Lys), c.776T>C (Leu259Pro), c.1079T>G (Ile360Arg). Two patients had small deletions causing frameshift leading to premature termination: c.601_602delAG (Ser201HisfsX2) and c.1608_1609delTA (Tyr536X). One patient had a splicing mutation (c.240+1G>T). Three had nonsense mutations: c.73G>T (Gly25X), c.801G>A (Trp267X) and c.1569T>G (Tyr523X). Three patients from two families showed recombinant mutations between the IDS gene and pseudogene (IDS2). Four of the mutations were novel (c.73G>T, c.329G>A, c.1079T>G and c.1569T>G). In silico analysis showed these mutations to be likely pathogenic. Conclusion: The identification of mutations in Malaysian patients with MPS II is foremost important for genetic counseling, carrier status determination and prenatal diagnosis.
Effect of IDS Variants on CNS Involvement in Mucopolysaccharidosis Type II
A. Vollebregt1, M. Hoogeveen-Westerveld1, G. Ruijter1, M. Kroos1, I. Plug1, A. van der Ploeg1, P. Pijnappel1
1Erasmus MC, Rotterdam
Purpose: Hunter syndrome is a lysosomal storage disorder caused by deficiency of iduronidate-2-sulphatase (IDS), resulting in accumulation of glycosaminoglycans (GAGs). Two patient populations can be distinguished, based on the presence or absence of CNS involvement. The aim of this study is to use an integrated approach to study the relation between the IDS genotype and the central nervous system (CNS) phenotype in the Dutch MPS II population. Methodology: The patients’ phenotype was determined by two physicians who evaluated the level of education, behaviour and available IQ data. Urine glycosaminoglycan (uGAG) levels were measured by a novel mass spectrometry method specific for heparin and dermatan sulfate. Effects of IDS variants on enzyme activity and intracellular processing were determined. The IDS cDNA was cloned into an expression vector, IDS variants were introduced by site directed mutagenesis, and constructs were transfected into HEK293 cells. Analysis included enzyme activity and immunoblot assays. Results: Intellectual disability was severe in 9 patients, intermediate in 1, mild in 1 and absent in 6. Regardless of the level of intellectual disability, the uGAG levels were elevated to similar extents and showed a strong age-dependency. We identified 15 unique IDS variants in 17 patients. All variant cDNA constructs showed strong impairment of IDS protein expression or processing and they all show similarly reduced IDS enzymatic activities. Conclusion: The pathogenic effect of IDS variants in vitro and the uGAG levels do not correlate with the severity of CNS involvement in MPS II patients. We speculate that the CNS phenotype of MPS II may be explained by the genotype in combination with modifying factors.
Molecular Results for the Whole Set of Patients.
No
Locus
Nucleotide shift
A.A shift
Literature report
Enzymatic activity
Codón
POLYPHEN 2
MUTATION TASTER
1
Ex11
c.1156C>T (homo)
p.R386C
Ogawa, 1995
–
CGT, TGT
0.999
0,999
2
Ex09
c.974G>A (het)
p.W325*
Wang, 2010
0,7 µmol/l/h
Stop Codon
0,999
0,999
Ex03
c.280C>T (het)
p.R94C
Ogawa, 1995
TCT TCC
1,000
0,999
3
Ex11
c.1156C>T (het)
p.R386C
Ogawa, 1995
0,4 µmol/l/h
CGT, TGT
0.999
0,999
Ex09
c.901G>T (het)
p.G301C
Bunge, 1997
GGC,TGC
0,999
0.999
4
Ex09
c.901G>T (homo)
p.G301C
Bunge, 1997
0,3 µmol/l/h
GGC,TGC
0,999
0.999
5
Ex11
c.1156C>T (het)
p.R386C
Ogawa, 1995
0,3 µmol/l/h
CGT, TGT
0,999
0,999
Ex09
c.901G>T (het)
p.G301C
Bunge, 1997
GGC,TGC
0,999
0.999
6
Ex09
c.901G>T (het)
p.G301C
Bunge, 1997
0,3 µmol/l/h
GGC,TGC
0,999
0.999
Ex11
c.1156C>T (het)
p.R386C
Ogawa, 1995
CGT, TGT
0,999
0,999
7
Ex09
c.901G>T (het)
p.G301C
Bunge, 1997
0,3 µmol/l/h
GGC,TGC
0,999
0.999
Ex11
c.1156C>T (het)
p.R386C
Ogawa, 1995
CGT, TGT
0.999
0,999
8
Ex11
c.1156C>T (homo)
p.R386C
Ogawa, 1995
0,5 µmol/l/h
CGT, TGT
0.999
0,999
9
Ex11
c.1156C>T (homo)
p.R386C
Ogawa, 1995
0,9 µmol/l/h
CGT, TGT
0.999
0,999
10
Ex11
c.1156C>T (homo)
p.R386C
Ogawa, 1995
0,3 µmol/l/h
CGT, TGT
0.999
0,999
11
Ex11
c.1156C>T (homo)
p.R386C
Ogawa, 1995
0,3 µmol/l/h
CGT, TGT
0.999
0,999
12
Ex05
c.425A>T (het)
p.H142L
N/A
0,3 µmol/l/h
GCA, GCT
0,999
0,999
Ex09
c.9015G>T (het)
p.G301C
Bunge, 1997
GGC,TGC
0,999
0.999
Molecular Characterization on Mucopolysaccharidosis Type IV-A Patients at—Cali, Colombia
A. Sánchez Gómez1, J. Satizábal Soto1, A. Escudero Rodriguez1, L. Moreno Giraldo1
1Genomics, Cali
Introduction: Morquio syndrome (MPS IVA Mucopolysaccharidosis type IV-A) is a lysosomal storage disease autosomal recessive, with a low prevalence in the general population. The down-regulated expression of the GALNS gene encoding Galactosamine 6 - sulfatase involved in the catabolism of two glycosaminoglycans (GAGs): chondroitin-6-sulfate (C6 S) and sulfate keratan (KS) in chondrocytes, is responsible for the onset of clinical manifestations of the disease, in which the most obvious affect the osteoarticular system with appearance of stunting and skeletal dysplasia or dysostosis multiplex. The molecular analysis is performed as an additional confirmation that the different mutations of the affected gene are described, studies in different ethnic population groups have revealed more than 185 different mutations of the gene GALNS. This extensive allelic heterogeneity of mutations of the gene involved is consistent with the broad spectrum of clinical phenotypes observed in patients suffering from MPS IVA. Methodology: Whole blood samples were taken from 12 patients, with a positive enzymatic test for MPS IV-A, on whatman paper. DNA was isolated and GALNS gene was sequenced by next-generation sequencing methodology . mutations present were determined and bioinformatic studies for finding referential background in databases The National Center for Biotechnology (NCBI) and ClinVar, The European Bioinformatics Institute (EMBL-EBI) tool were performed. Genomic analysis was performed with several Software tools to predict the impact of amino acid substitutions: SIFT, Polyphen2 (http://genetics.bwh.harvard.edu/), and Mutation Taster (http://www.mutationtaster.org/). Results: 12 patients were included in this study, 58.3% (7 patients) were female and 41.7% (5 patients) were male. Age ranged between 2 and 28 years. All patients had deficient enzyme activity. In the molecular study of the gene GALNS 5 homozygous missense mutation reports were obtained in exon 11 c.1156C> T p.R386C previously reported to cause disease by Ogawa, 1995, with rs118204437, profiled in The European Bioinformatics Institute (EMBL-EBI). This mutation was also found in a heterozygote complemnt in 4 patients. One patient was found with a heterozygous nonsense mutation in exon 9 c.974G> A p.W325 * HGMD CM105265, reported by Wang in 20107 and mutation of exon 3 c.280C> T missense variant p.R94C reported by Ogawa, 1995. Only one patient had missense mutation homozygous c.901G> T in exon 9 p.G301C reported by Bunge, 1997 rs118204443 considered pathogenic in the 1000 genomes project, multiple observations and genotype-phenotype correlations as EMBL-EBI. Four patients had this mutation in heterozygosity. A female patient 2 years old, presented heterozygous missense mutation type in exon 5 c.425A> T p.H142 L which has not been previously reported, which entered into the system the prediction of protein Mutation Taster (http://www.mutationtaster.org/) shown to cause disease with a probability of 0.999. In Polyphen2 it is predicted to cause disease with a score of 1,000.
Conclusions: The diagnostic algorithm of MPS IV-A established by the working groups in Prague (2011) and San Diego (2012) includes molecular diagnostics. Bioinformatic study of the reported mutations offers the opportunity to conduct research that contribute to the understanding of the disease, as in the case of mutation not described to date in exon 5 in one patient, in which the use of tools predictive defined the pathogenicity of the mutation allowing more accurate and specific genetic counseling, contributing to epidemiological studies.
Novel Mutation on GALNS Gene Registered in a Morquio Syndrome Patient from Colombia
J. Satizábal Soto1, A. Sánchez Gómez1, A. Escudero Rodriguez1, L. Moreno Giraldo1
1Genomics, Cali
Introduction: Morquio syndrome-A (MPS-IV-A OMIM # 253000) is a lysosomal storage disorder that is inherited in an autosomal recessive manner with a very low prevalence. It is characterized by a deficiency of the enzyme N-acetylgalactosamine-6-sulfatase (GALNS), resulting in an accumulation of chondroitin-6-sulfate (C6 S) and keratan (KS) sulfate in many tissues and organs. The most common clinical findings are related to systemic skeletal dysplasia, dwarfism with short trunk, kyphoscoliosis, platiespondilia, instability atlanto-axoid, genus valgus, chest keel, and other alterations such as dental anomalies, ligamentous laxity, corneal opacity, coarse facies, loss hearing, valvular heart disease, respiratory disease. Unlike other MPS, Morquio Syndrome does not exhibit central nervous system involvement, and intelligence ranges on mormal indexes. Molecular analysis of DNA is the gold standrd to confirm clinical results of abnormal enzyme activity. Besides, it facilitates genetic counseling of the family and establishment for the clinical prognosis. The characterization of these patients through molecular diagnostics allows learn more about native mutations, possible genotypic and phenotypic expression. Methodology: DNA from a female patient, at preschool education level from urban area of the municipality of Buga-Valle del Cauca-Colombia, was molecularly analyzed. The patinent has consanguineous parents and she was diagnosed at 2 months from birth with a bilateral hip dysplasia with a radiological follow-up at 2 years. Persistent signs of dysplasia and disostosis lead to a suspition of a lysosomal storage disease (mucopolysaccharidosis). Physical examination registered: genus puffy face, chest carinatum protrusion of the rib cage, umbilical hernia and bilateral valgus. DNA was isolated and sequenced by gene GALNS next-generation sequencing. Results: Patient with phenotypic osteo-muscular alterations confirmed by radiological images that reported signs of disostosis. Measurement of the enzymatic activity showed a value of 0.06 nmol GALNS / mg prot / h (2-35.9 nmol / mg prot / h, normal range) compatible with Morquio disease. Radiographs of full column were analyzed showing hypoplastic vertebral bodies of C3-C4, C5-C6, L2-L3, T10-T11-T12, L4, and bilateral hip dysplasia. The molecular analysis of gene GALNS reported a heterozygous mutation in exon 9 (c.901G> T p. Gly301Cys), (rs118204443), previously described as causing disease by Bunge in 1997 (92 HGMD Professional 2014.2 PMID 9298823). In addition, a novel mutation was found in exon 5 (c425A> T p.His142Leu) located in an area of high conservation of nucleotide and amino acid with moderate physicochemical differences between the amino acid Leucine and Histidine. Software bioinformatic analysis with predicting impact of amino acid substitutions was performed. Polyphen2 (http://genetics.bwh.harvard.edu/) reported exon 5 c.425A> T p.H142 L to cause damage with a score 1,000, and Mutation Taster (http://www.mutationtaster.org/) predicted as a disease disease causing effect with a 0.999 probability this GALNS gene mutation not previously reported in the international databases. Conclusions: Mutations causing Morquio-A disease on GALNS gene are numerous, heterogeneous, and most are missense mutations. We reported a novel mutation on exón 5 for screened gene. Bioinformatic software was helpful to predict pathogenic effect on this mutation. Detection of a new mutation contributes to the International Morquio Syndrome Registry, the Census being conducted by national Health goverment agencies of Colombia on Orphan Diseases. This result would help to build a system of indigenous information to provide more knowledge about the disease, which directly benefits patients in the diagnosis, personalized treatment, monitoring.
Expanding the Genotypic Spectrum of Mucolipidosis II and III Alpha/Beta in Brazil
I. Schwartz1, F. Sperb-Ludwig2, R. Voltolini3, A. Acosta4, E. Ribeiro5, C. Kim6, D. Horowitz7, R. Boy8, S. Phol3, N. Luwig1
1Hospital de Clínicas de Porto Alegre
2Universidade Federal do Rio Grande do Sul, Porto Alegre
3University Medical Center Hamburg
4Universidade Federal da Bahia, Salvador
5Associação do Ceará de Doenças Genéticas, Fortaleza
6Hospital de Clínicas da Faculdade de Medicina da USP, São Paulo
7Instituto Nacional de Saúde da Mulher, Criança e Adolescente, Rio de Janeiro
8Universidade do Estado do Rio de Janeiro
Introduction: Mucolipidosis II (MIM #252500) and III alpha/beta (MIM #252600) are autosomal recessive diseases caused by absence or defect in the manose-6-phosphate markers responsible for the lysosomal hydrolases targeting to lysosomes. Consequently, there is extravasation of enzymes into the extracellular environment and accumulation of macromolecules in the lysosome. The deficient enzyme is N-acetylglucosamine-1-phosphotransferase, encoded by the GNPTAB and GNPTG genes. Purpose: To characterize the profile of the GNPTAB pathogenic mutations in 13 unrelated Brazilian patients with ML II/III alpha/beta. Methodology: Out of the 13 patients, three (and the mother of patient 10) had all the 21 exons of GNPTAB sequenced by the Sanger method. For the remaining patients (n = 9), GNPTAB exons were sequenced by Sanger according to the protocol developed by our group (step 1 = exon 19; step 2 = exons 13 and 10; step 3 = exons 3, 12, 13.2, 14 and 20; step 4 = 1, 2, 4-9, 11, 15-18 and 21). For one ML II patient (pt 13), with no DNA sample available, the DNA of the mother was analyzed. Eight patients were clinically diagnosed as having ML II, and five, as having ML III alpha/beta. Results: The genotype was determined for 12/13 patients, and 1/!3 (pt 13) had only one mutation found. Five novel variations were identified: p.Ser385Leu, c.831delT, c.1763insA, c.1927delAATT and p.Tyr1111*. The c.3503_3504delTC and p.Ile408Thr were the most prevalent mutations, the first one being found in 10/24 alleles, in compound heterozygosity (n = 7 patients) or homozygosity (n = 2 patients); the second one was found in three ML III compound heterozygous patients. The genotype was determined in the first step for 1/9 patient, in the second step for 2/9 patients, in the third step for 3/9 patients and int the fourth step for 3/9 patients. Conclusion: Our data confirm the high heterogeneity of the GNPTAB gene, and increase the knowledge about the understanding of the genotype-phenotype association in the disease.
How to Diagnose Intracranial Hypertension in Mucopolysaccharidosis Patients
A. Dalla-Corte1, C. Souza2, F. Vairo2, M. Anés2, L. Vedolin2, M. Ferreira2, S. Perrone2, A. Federhen2, R. Giugliani1, F. Maeda3
1Universidade Federal do Rio Grande do Sul, Porto Alegre
2Hospital de Clínicas de Porto Alegre
3Hospital Dom Vicente Scherer, Porto Alegre
Purpose: The diagnosis and treatment of hydrocephalus with increased intracranial pressure in mucopolysaccharidosis (MPS) patients lacks a formal, consensus-based definition. Because venous hypertension is a known cause of hydrocephalus, one hypothesis for the ventricular enlargement in these patients could be the reduced venous outflow through bone dysostosis of the skull base. Cerebrospinal fluid (CSF) flow study is a noninvasive imaging technique that is useful for the evaluation of patients with hydrocephalus. Lumbar subarachnoid pressure higher than 20 cm H2O can be considered indicative of hydrocephalus. Our objective is to assess the relationship between ventriculomegaly, hyperdynamic aqueductal CSF flow, high lumbar manometry and variations of the durai venous sinuses in MPS patients. Methodology: We performed a CSF flow study by phase-contrast magnetic resonance imaging (MRI) followed by a standart lumbar puncture with the CSF opening pressure assessment in 33 patients: MPS type I in 9 patients, MPS type II in 13 patients, MPS type III in 3 patients, MPS type IV A in 7 patients and MPS type VI in 1 patient. The age range was 1 to 31 years, 19 males and 14 females. Results: Neurological findings included pyramidal signs in 5 patients and macrocephaly in 10 patients. Twelve patients had no cognitive impairment. The most frequent MRI findings were white matter changes in 21 patients, dilated perivascular spaces in 21 patients, craniovertebral junction stenosis in 22 patients and ventricle enlargement in 12 patients. Of the 12 patients with radiological signs of hydrocephalus, hyperdynamic aqueductal CSF flow was obtained in 5 of them, 6 patients showed CSF lumbar pressure values above 200 mm H2O and 7 presented left cerebral venous drainage. On the other hand, in 13 patients with no typical ventriculomegaly elevated CSF pressure values were obtained and 11 of them had craniovertebral junction stenosis. Conclusions: Ventriculomegaly in MPS patients is not only hypertensive. CSF opening pressure showed better correlation with typical findings of hydrocephalus and with CSF flow restriction across the craniocervical junction. Variations in sinuses of the posterior cranial fossa may be associated. Although this is the first description using these parameters to diagnose high intracranial pressure larger studies must be done for helping diagnosis and best determine which patients will respond positively to shunting.
Restricted Mouth Opening in Patients With Mucopolysaccharidosis—Prevalence and the Role of Mandibular Coronoidectomies
P. Hensman1, I. Bruce2, K. Hood3, A. Kalantzis4, J. Mercer1, S. Jones1
1Willink Biochemical Genetics Unit, Manchester
2Paediatric Otolaryngology, Manchester
3Paediatric Dental Health, Manchester
4Oral & Maxillofacial Surgery, Manchester
This abstract is to discuss and highlight the problems encountered in Manchester within the paediatric mucopolysaccharidosis (MPS) population experiencing limited mouth opening and the difficulties faced when anaesthetising this cohort of patients. At present over 100 MPS patients in Manchester, both on and off treatment (enzyme replacement therapy or haematopoietic stem cell transplantation), are followed up. Difficulty in anaesthetising these patients and significant obstructive airway disease are well recognised in MPS patients, usually this is secondary to multi-level airway obstruction. Recently we have identified an increasing problem complicating general anaesthesia in the form of restricted mouth opening. Following these repeated challenging intubation episodes we decided to obtain further data to monitor mouth opening and explore the impact it has on both every day activities, such as brushing teeth and future surgical interventions. Routine measuring of mouth opening was initiated in children mainly with MPS I, II, IVA and VI with this information being forwarded to the relevant paediatric specialities including metabolic doctors, ENT surgeons, anaesthetists and maxillofacial surgeons. Mouth opening of ≤ 25 mm may limit the insertion of some conventional laryngoscopes blades. Mouth opening of ≤ 20 mm limits the insertion of most conventional laryngoscopes; it is preferable to manage these children in a specialist centre where facilities for fibre optic intubation, ENT support and paediatric intensive care unit backup are all available. We present data on Mouth opening capacity (MOC) in 80 MPS patients. This collected data has resulted in further imaging and we present detailed information on three patients with less than 25 mm of mouth opening capacity (MOC). This led to a novel (in MPS) surgical intervention for these three MPS I patients who underwent bilateral mandibular coronoidectomies. Patient A started on enzyme replacement therapy aged 2, patient B and C underwent haematopoietic stem cell transplantation aged 2 and 1year. Patient A and B underwent bilateral mandibular coronoidectomy surgery performed on the same day by the Oral and Maxillofacial Surgeon alongside the Paediatric Otolaryngologist, whilst patient C had surgery performed by the Oral and Maxillofacial Surgeon alongside the Paediatric dental surgeon. Post-operative issues initially included pain, infection and problems using the passive mouth stretching device. The results, problems and outcomes for the initial 6 months following the procedure are outlined. Significant ongoing storage in the resected coronoid processes was noted histologically. Post-operatively there has been a clinically significant increase in MOC. From the limited results, compliance with the stretching regime appears to be paramount to maintain the MOC. The passive range of MOC increased for patients A, B and C by 16 mm, 3 mm and 8 mm respectively. Currently we have longitudinal measurements for over 75 patients of which a further nine have a MOC of less than 25 mm. MOC is now routinely collected in this cohort of patients to highlight those with restriction and therefore increased risk of complications with anaesthesia. Our standard procedure for patients with MOC of less than 25 mm is to perform a CT scan for review by the Oral and Maxillofacial Surgeon. It remains to be seen if further build-up of glycosaminoglycan (GAG) deposits may continue to develop within the mandibular area.
Anaesthetic Management for Imaging Studies and Diagnostic Procedures in Mucopolysaccharidosis Patients
M. Ferreira1, A. Dalla-Corte2, C. Souza1, R. Giugliani2
1Hospital de Clínicas de Porto Alegre
2Universidade Federal do Rio Grande do Sul, Porto Alegre
Purpose: The high prevalence of airway obstruction and restrictive pulmonary disease in combination with cardiovascular manifestations poses a high anesthetic risk to patients with mucopolysaccharidosis (MPS). The presence of macroglossia, tonsillar hypertrophy and swelling of tissues in laryngopharyngeal challenge the airway management. The anesthesiologist must be prepared to deal with inability to ventilate or intubate and also with sudden airway obstruction during anesthesia. On average 3 anesthetic-surgical procedures will be performed in every MPS patient throughout his life. Our objective is to establish the use of appropriate anesthetic techniques especially in diagnostic tests performed outside the operating room. Methodology: All patients underwent a cerebrospinal fluid (CSF) flow and brain magnetic resonance imaging (MRI) followed by a standard lumbar puncture with the CSF opening pressure assessment in the radiology unit. Patients were assessed before the procedure and the anesthetic technique was individualized for each case due to complexity and rarity of the condition. The difficult airway risk factors were evaluated and an ENT might be called in cases of difficult ventilation and intubation. Results: A total of 20 patients aged 1-34 years old were anesthetized by the same anesthesiologist. In 10 cases it was decided to carry out sedation while in the other 10 cases there was need for general anesthesia. The airway management in cases of general anesthesia was performed with the use of laryngeal mask and tracheal intubation was not necessary. The inducing drugs used included ketamine and propofol, and anesthesia was maintained with sevoflurane. In cases of sedation we choosed the use of diazepam and dexmedetomidine. One patient had an episode of transient desaturation and one patient presented bradycardia which was reversed by atropine. No complications were observed in the other cases. Conclusions: MRI and diagnostic tests in MPS patients can be properly performed under sedation. In cases where general anesthesia becomes necessary, the laryngeal mask is a suitable alternative. The choice of anesthetic technique and the anesthesiologist’s experience in the management of these patients are critical to the success of the procedure and early recovery.
Risk Factors Associated With Spontaneous Subdural Hematoma in Mucopolysaccharidosis Patients
A. Dalla-Corte1, A. A. da Silva2, C. Souza2, I. Schwartz2, D. Horovitz3, A. Barth3, R. Giugliani1
1Universidade Federal do Rio Grande do Sul, Porto Alegre
2Hospital de Clínicas de Porto Alegre
3FIOCRUZ, Rio de Janeiro
Purpose: The mucopolysaccharidoses (MPS) represent a group of inheritable, clinically heterogeneous lysosomal storage disorders, in which progressive accumulation of glycosaminoglycans (GAGs) can affect organs and tissues all over the body, including blood vessels. Spontaneous subdural hematoma (SDH) is a very rare complication seen in all types of MPS and may also develop after a ventriculoperitoneal shunt (VPS) placement. Although not used in diagnosis of MPS, neuroimaging of brain and spine are critical for evaluating the complications of MPS and the need for neurosurgical treatment. Our objective is to identify the possible relationship between the occurrence of subdural hematoma and mucopolysaccharidosis types, molecular analysis and shunting. Methodology: This is a case series study which included six MPS patients with spontaneous SDH: MPS type I in 1 patient and MPS type II in 5 patients, and all with the severe, neuropathic form of the disease. The age range was 2 to 10 years, 5 males and 1 female. Results: MPS II patients presented inversion or complex rearrangement in IDS gene. In four patients the SDH was followed by a VPS placement. There were no new neurological symptoms or focal signs associated with the SDH. Five patients presented massive SDH and three patients required surgical drainage. Three patients were in enzyme replacement therapy. Four patients experienced own complications of natural history of MPS and three of them died. Because only three other cases have been reported, this is the largest series of cases already described. One explanation would be the high GAGs concentration having a “heparin-like” effect, decreasing blood viscosity and enhancing bleeding tendencies. In the other hand, some authors hypothesized that the more vessels vulnerability in MPS characterized by smooth muscle hyperplasia and degenerative changes in the intima and media were responsible for the vasculopathy and central hemorrhages. Due the occurrence of SDH after VPS placement in four of our patients, abnormal cerebral blood vessel architecture secondary to the ventricular enlargement leading to rupture could be considered. SDH may also occur after a VPS insertion due to the siphoning effect and overdrainage of cerebrospinal fluid, producing negative pressure inside the skull and causing rupture of veins in subdural space. Conclusions: The pathophysiology of the SDH in MPS patients is not clear established. Because the spontaneous SDH are usually found without clinical manifestations, neuroimaging should become part of the routine assessment, especially in MPS type II. Molecular analysis can help identify a potential risk group. Moreover, a pressure-programmable valve should be considered by the neurosurgeon to prevent this serious complication in MPS patients.
Conventional Measures of Body Composition may be Less Predictive in Adult Mucopolysaccharidosis Patients—A Small Case Series
G. Wilcox1, B. Strauss2, C. Hendriksz1
1Salford Royal NHS Foundation Trust, Salford
2Monash University, Clayton
Purpose: The mucopolyscaccaridoses (MPS) represent a group of disorders, typically associated with accumulation of specific glycosaminoglycans during growth and development, with multisystem manifestations which may affect musculoskeletal, cardiovascular, respiratory, gastrointestinal and nervous systems. Therefore they can be considered disorders associated with alterations in body composition. Measuring body composition may better define the natural history of these conditions, monitor complications and effects of therapy, such as Enzyme Replacement Therapy (ERT). Methodology: Five adult patients 3 male (m) 2 female (f) with MPS (#1) MPS1 f 54yo late diagnosed Hurler-Scheie, #2) MPS3 m 21yo advanced disease, #3) MPS6 m 19yo young adult with rapidly progressive disease followed serially after commencement of ERT and 2 siblings #4) m 27yo & #5) f 30yo with attenuated disease) were referred for body composition measurements at a dedicated center. Body composition measurements available included Anthropometry (A), Single Frequency Bioelectrical Impedance (BIA), total body nitrogen (TBN) by in vitro neutron activation analysis (IVNAA), Whole Body Displacement Plethysmography by Bod Pod®, and whole body dual energy X-ray absorptiometry (DEXA). Results: The current cohort size does not permit more than a descriptive summary of the findings. Patients ranged in height from 0.965 m (patient #3) to 1.746 m (patient #2). Body composition in Patient #2, with MPS3, measured by A & BIA was not significantly different from the Laboratory’s age matched population. Patient #3 with severe MPS 6 showed the greatest aberration in body composition measurements and the most discrepant results across body composition methods especially relating to percent body fat measurement. GAG accumulation confounding even gold standard body composition methods, as well as extreme short stature, may be contributory. Beyond total body BMD, whole body DEXA informed percent body fat in attenuated patients. Vertebral height Z-score was decreased disproportionately to height in all MPS 1 & 6 patients. Improvement in vertebral height Z-score, associated with overall height increase was seen in patient 3 in response to 2 years’ ERT. Conclusion: MPS disorders warrant more sophisticated measures of body composition, particularly in those patients with more progressive or advanced disease. Many conventional methods, using formulae derived from measurements performed in the general population may not be applicable to MPS patients, particularly those with very short stature. In this small case series whole body DEXA seems to provide useful information regarding vertebral height in interpreting spinal bone mineral density results, assessing disease severity and response to therapy (ERT). This warrants further evaluation in a larger cohort of MPS patients.
Proposal for Diagnostic Suspicion of Mucopolysacharidosis (MPS) in Patients Who Present With Pathological Short Stature
M. Liliana1, H. Hussein2
1Foundation Club noel, Cali
2Santa Fe Family Health, Joliet
Purpose: Short stature represents the main reason for consultation in Pediatrics Endocrinology. 20% of short stature is considered pathological and warrants further extensive studies. A rare disease is one that have an incidence of less than 0.05% of the general population (1 in 20,000 people).Within which this the mucopolysaccharidosis. Severe cases may result in “Dwarfism” or significant growth impairment that may present after a period of normal development resulting in final heights below 3 to 6 standard deviations, hence the importance of an early diagnosis. Materials and Methods: Literature review for clinical clues leading to consider bone dysplasias secondary to MPs in patients with pathological short stature. Results: Consider MPs in patients with pathological short stature associated with: 1-Clinical presentation that compromise bone or cartilage normal growth and development (osteochondrodysplasia, dysostosis), bone dysplasias as in skeletal deformity of the spine and/or extremities. Spinal curvature severe abnormalities as kyphoscoliosis, genu valgum present after 18-24 months, pathological fractures, short extremities in the presence of a normal thorax. Hand and feet distal epiphysis compromise, hands deformity, prominent and slightly wide knuckles. Flattening of Femur and tibia upper proximal epiphyses. Spasticity and non-congenital contractures, claw hand, nerve entrapment, restricted mobility and limited ambulation, hip dysplasia.2- Progressive joint compromise without evidence of clinical or laboratory markers for inflammation. 3- Progressive or degenerative character, marked slowing or loss of prior acquired skills, milestones. 4- Evidence of visceromegaly or abnormal ocular signs.5-History of hydrops fetalis or neonatal ascites.6- Chromosomal abnormalities leading to primary calcium and phosphorus metabolic dysfunction (Rickets), and complex carbohydrates. Analysis and Conclusions: Bone involvement leading to pathological short stature is due to underlying chondrocyte maturation disruption and location in the growth plate secondary to accumulation of MPs generating more compromise in axial growth than appendicular. Short stature in MPs is secondary to a combination of metabolic, endocrine and skeletal structural abnormalities that restrict growth and final adult height. Patients with attenuated forms might have normal or near normal lineal growth. Other deposit diseases may share clinical features as those seen in MPs. Specific enzyme deficiency in leukocytes or cultured cutaneous fibroblasts will provide definite diagnosis. Detailed clinical and radiological evaluation along with type of GAG eliminated in urine and leukocyte activity will narrow the differential diagnosis. We proposed to look diagnostics MPS at patients with commitment to simultaneously of low sature, bone and facial.
Guidelines for the Diagnosis and Management of Infants With MPS I Identified Through Newborn Screening
D. Stockton1, L. Clarke2, A. Atherton3, B. Burton4, D. Day-Salvatore5, P. Kaplan6, N. Leslie7, C. Scott8, J. Thomas9, J. Muenzer10
1Wayne State University, Detroit
2University of British Columbia, Vancouver
3Children’s Mercy Hospital, Kansas City
4Northwestern University Feinberg School of Medicine, Chicago
5Saint Peter’s University Hospital, New Brunswick
6The Children’s Hospital of Philadelphia
7Cincinnati Children’s Hospital, Cincinnati
8University of Washington, Seattle
9University of Colorado, Aurora
10University of North Carolina, Chapel Hill
Mucopolysaccharidosis type I (MPS I) is a progressive multisystem disease with considerable clinical heterogeneity. Available treatment options include hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT) with laronidase. HSCT has been demonstrated to stabilize neurocognitive function in patients with MPS I when performed at an early age, and is therefore currently recommended as standard care for patients who are predicted to have severe disease. ERT is recommended for either severe clinical diagnostic patients in the peri-transplantation period or for attenuated MPS I patients. Since MPS I is a progressive disorder early initiation of treatment ensures optimal long-term outcome. Therefore, early identification of patients is critical. Newborn screening (NBS) for MPS I via determination of IDUA activity in dried blood spots (DBS) is currently underway in two states in the United States and in Taiwan and imminent in several other states. Given the variable manifestation of the disease and the potential difficulty in accurately distinguishing disease subtypes early in the natural course of disease, as well as the existence of pseudodeficiency alleles, there are significant challenges in implementing NBS strategies for this disorder. Hence, it is imperative to establish clear, detailed, and flexible guidelines for interpreting NBS results. Recently, a group of North American physicians, clinician scientists, and genetic counselors with experience in MPS I and NBS convened to develop such guidelines. It was recommended that confirmatory IDUA enzyme analysis from fresh blood be performed in all newborns with screens flagged as abnormal based on a low DBS IDUA activity. If IDUA deficiency is confirmed, urinary GAG determination and complete IDUA gene sequencing should be arranged in addition to a clinical assessment. Infants whose genotype and/or phenotype is predictive of severe MPS I should be referred for HSCT without delay, and ERT should be considered for those infants whose genotype and/or phenotype is predictive of attenuated disease and considered for those awaiting transplantation. Patients who cannot be conclusively classified at this early age should have additional clinical follow-up and laboratory evaluations, and treatment options should be discussed. Genetic counseling should be provided to all families, including reproductive counseling for parents, identification of at-risk older siblings, and a testing plan for subsequent siblings. Support for the advisory board and medical writing were provided by Sanofi Genzyme. AMA is an employee of Shire, is on the speaker’s bureau for Genzyme and Shire, and has participated in advisory boards and received honoraria and travel assistance from Genzyme, Shire; BKB has received clinical trial funding from Genzyme, Biomarin, Shire, Ultragenyx, and Synageva, and consulting fees or honoraria from Genzyme, Biomarin, Shire, Hyperion, and ReGenX Bio; LC has received travel support and honoraria from Genzyme and BioMarin; PK has received honoraria from Genzyme, Pfizer, Synageva, and Actelion; DLD-S, NDL, JM, CRS, DWS, and JAT have no conflicts of interest related to this work.
Natural History of Cardiac Disease and Outcomes With ERT in Patients With MPS I Enrolled in the MPS I Registry
L. Bay1, A. Villa1, K. Moy2, M. Munoz-Rojas2, L. Clarke3
1Hospital Juan P. Garrahan, Buenos Aires
2Sanofi Genzyme, Cambridge
3University of British Columbia, Vancouver
Purpose: Cardiac signs and symptomatology are prominent features in patients with mucopolysaccharidosis type I (MPS I). The goal of this study is to describe the natural history of cardiac disease in MPS I, and to examine the factors which correlate with cardiac findings in patients receiving enzyme replacement therapy (ERT). Methodology: The MPS I Registry is a global, observational database initiated in April 2003 that tracks patients’ clinical outcomes, regardless of treatment status. In the present analyses, a natural history population was defined as untreated patients, or patient observations prior to treatment initiation. Data for the natural history period of patients prior to treatment were selected using the following criteria: a) known ERT status, b) known date of first ERT infusion, c) known hematopoietic stem cell transplantation (HSCT) status, and d) known date of transplant. Descriptive statistics were used to examine cardiac findings and conditions by phenotype (Hurler, Hurler-Scheie, Scheie). Among ERT patients (including those who have received HSCT prior to or following the commencement of ERT), descriptive statistics and multivariate logistic regression were used to evaluate predictors of cardiomyopathy and cardiac valve abnormalities in Hurler patients and in attenuated (Hurler-Scheie and Scheie) patients. Results: Among the ERT-treated population, cardiac symptoms were reported for 615 patients. The majority of these patients (60.3%) had cardiac valve abnormalities. Cardiomyopathy was reported in 20.5% of patients. Cardiac valve abnormalities were more prevalent in patients with attenuated (Hurler-Scheie and Scheie) MPS I (69.4%) than severe (Hurler) patients (52.4%), possibly a consequence of augmentation of cardiac disease in patients who had received HSCT prior to ERT (13.3% of Hurler patients in this study). The opposite was true for cardiomyopathy (25.7% severe vs. 13.7% attenuated). Additional data to be presented include risk factors of cardiac symptoms among ERT-treated patients by phenotype (Hurler and attenuated). Predictors examined include sex, age at treatment initiation, and time between diagnosis and treatment initiation. Conclusion: Cardiac disease is common in MPS I. A better understanding of the cardiac signs and symptoms and response to ERT in MPS I patients may help manage physicians’ and patients’ expectations of treatment.
Supported by Sanofi Genzyme. LB, AV, and LC have no competing interests. KAM and VM are employees of Sanofi Genzyme.
Five Cases of Mucopolysaccharidosis Type I. Classifying the Same Disease
M. Maldonado-Millan1
1Hospital General Regional No.1, Tijuana
Purpose: Mucopolyssacharidosis type I (MPS I) is traditionally divided into three phenotypes: The severe Hurler (MPSI-H), the intermediate Hurler-Sheie (MPS I-H/S) and attenuated Scheie (MPSI-S) phenotypes.(1) Because the predictive value of genotyping is still limited, the phenotypic severity often needs to be assessed solely on the basis of clinical signs and symptoms. The aim of this report is classify the phenotype severity, of five patients, with diagnostic of MPS I, according to the International Consensus Procedure (1), adapted, and validated by biomarkers available in our hospital (2) in order to know the subtype and prognosis of the disease. Methodology: We gather retrospective information by indirect questioning and medical record of five MPSI patients (three females, 3 yr, 13 yr and 15 yr old) two males (5 yr and 11 yr) who receive weekly enzymatic replacement therapy with laronidase (0.58mg/kg), since September 2011. We gather the criteria items (1) according to the six key items for Kingma et al.ing phenotypic severity in MPSI, of a group at the University Hospital of Amsterdam, and introduced three biomarkers; one biochemical (residual IDUA activity in fibroblast) and two clinical (inguinal hernia (bi-or unilateral) and upper airway obstruction) according to an Algorithm for assessment of phenotypic severity in MPSI patients.(2). We mixed and adapted the scales in a table of nine items and correlated with the findings of patients. Results: We match nine clinical features and a residual enzymatic activity of five patients with a previous diagnosis of MPSI. We summarized the number of positive items from total of nine: Patient No.1/MPSI-H,= 8/9 No.2/MPSI-H/S,=4/9 No.3/MPSI-H,=8/9 No.4/MPSI-H/S,= 6/9 and No.5/MPSIH/ S=5/9. We confirm that these indicators of phenotypic severity in MPSI were helpful to classify the five patients with the same disease. Conclusion: Even this scales was made for classifying newly diagnosed MPSI patients, it is also useful for patients already diagnosis, but to classified them in a scale of severity of the same disease, so we can predict the prognosis and maybe, the possibility of being candidates of innovative therapies. It is an effective option when we have the clinical suspicion of MPSI patient and limited resources for molecular studies.
References
de RuMHTeunissenQGvan der LeeJH. Capturing phenotypic heterogeneity in MPS I: results of an International consensus procedure. Orphanet J Rare Dis. 2012;7:22.Kingma. An Algorithm to predict phenotypic severity in mucopolyssacharidosis type I in the first month of life. Orphanet J Rare Dis. 2013;8:99.Langford-Smith. Heparin cofactor II-Thrombin complex and dermatan sulfate: chondroitin sulfate ratio are biomarkers of short-and long term treatment effects in mucopolyssacharide diseases. J Inherit Metab Dis. 2011;34:499–508.
Awareness of MPS I Among Pediatric Endocrinologists
N. Thibault1, J. Cabral2, M. Munoz-Rojas1, S. Bruni3
1Sanofi Genzyme, Cambridge
2FHAJ, Universidade Federal do Amazonas, Manaus
3Sanofi Genzyme, Modena
Purpose: Short stature, one of the key features of MPS I, may lead to early identification, diagnosis, and initiation of treatment of patients with MPS I if it is timely and properly recognized as a symptom of the disease. The goal of this study was to determine the level of awareness of MPS I among pediatric endocrinologists, and the likelihood of these physicians encountering a patient with MPS I. Methodology: Physicians from the US, Canada, Italy, Germany, Spain, Mexico, and Brazil completed an anonymous online survey with questions related to their backgrounds, current knowledge of MPS I, suspected diagnoses and next steps for two blinded MPS I cases, and interest in learning more about the disease. Eligible to participate were pediatricians with a focus on endocrinology, or endocrinologists who see greater than 50% of patients under the age of 18 and saw 50 or more patients for delayed growth or growth failure in the previous 12 months. Results: Of the 135 physicians who responded to the survey, 71.1% indicated that they have seen a patient similar to one of the two patients described in the blinded cases; 68.1% of surveyed physicians have seen such a patient within the last 12 months. Depending on the patient case and information they were presented with, 42.2%-77.8% of physicians failed to take a correct step towards a diagnosis of MPS I; 49.1%-86.8% of these physicians would retain the patient for diagnostics rather than referring. 49.2% of physicians surveyed had not seen information on MPS I in the last 12 months, and 44.8% were unaware of treatment for MPS I. A vast majority (71.1%) of surveyed physicians were very interested in learning more about MPS I after receiving brief information on the disease. Of the physicians who had received some education on MPS I in the past 12 months, 66.1%-69.5% took a correct step towards diagnosing MPS I after being provided with more information about the disease. In contrast, only 35.1%-47.4% of physicians who had not received education about MPS I in the last 12 months took this step. Conclusion: Growth is often affected in patients with MPS I, therefore, endocrinologists may be among the first physicians to evaluate these patients. The majority of surveyed pediatric endocrinologists have seen a patient resembling a patient with MPS I, yet half of the physicians fail to progress to a correct diagnosis when presented with two blinded cases of MPS I. Pediatric endocrinologists are receptive to receiving education on MPS I, but there is a great unmet need for disease awareness among this physician population. Improved recognition of MPS I disease manifestations among pediatric endocrinologists may lead to early diagnosis and treatment, and improved patients outcomes.
Supported by Sanofi Genzyme. NT, VM, and SB are employees of Sanofi Genzyme. JMC has received honoraria for consultancy from Sanofi Genzyme
A Patient With Pyridoxine Responsive Homocystinuria and MPS 1 Hurler Syndrome—An Unlikely Combination?
Y. Yuan1, D. Ketteridge1, D. Bratkovic1, M. Baluyot1, J. Fletcher1
1SA Pathology at The WCH, North Adelaide
Background: Mucopolysaccharidoses (MPS) are rare disorders with an estimated total prevalence of all types of MPS of 1 in 20,000, with MPS 1 (Hurler syndrome) the most common enzyme deficiency, affecting 1 in 88,000 Australians. The chance of an individual having homocystinuria is between 1 in 58000 and 1 in 1,000,000.The chance of an individual being born with both MPS 1 Hurler syndrome and homocystinuria is between 1 in 1 in 5,100,000,000 and 1 in 88,000,000,000, i.e. extremely rare! Case report: A child diagnosed with MPS 1 Hurler syndrome at age of 10 month old, who received a bone marrow transplant at 13 months of age. Because of ongoing issues with development, he was further investigated for an inborn error of metabolism and subsequently diagnosed with pyridoxine responsive homocystinuria at age of 15 years. This second diagnosis was confirmed by DNA mutation studies using stored fibroblasts collected prior to his transplant. Discussion: When patients are diagnosed with one rare disorder we tend to discount the chance of them having a second rare disorder because of these odds. However an individual who has MPS has the same chance as any other individual of having another rare inborn error of metabolism and we should be alert to the possibility. Next generation sequencing testing Conclusion: Although the chance of an individual having two rare metabolic conditions on initial calculation is extremely small, it should not be dismissed.
Ten years of the Hunter Outcome Survey (HOS): A Decade of Improving Our Understanding of Hunter Syndrome
J. Muenzer1, R. Giugliani2, M. Scarpa3, A. Tylki-Szymanska4, M. Beck5, M. Paabøl Larsen6, D. Whiteman6
1UNC Hospitals Children’s Specialty Clinic, Chapel Hill
2Hospital de Clinicas de Porto Alegre, Porto Alegre / RS
3HELIOS Dr. Horst Schmidt Kliniken, Wiesbaden
4The Children’s Memorial Health Institute, Warszawa
5University Medical Center, Mainz
6Shire, Lexington
Purpose: Hunter syndrome (mucopolysaccharidosis type II [MPS II]) is a rare, X-linked disorder caused by deficiency of iduronate-2-sulfatase. The Hunter Outcome Survey (HOS; a Shire-sponsored, global, observational registry initiated in 2005) collects real-world, long-term data on the natural history of this disease and the safety of treatment with idursulfase (Elaprase®, Shire). Methodology: Individuals with a confirmed diagnosis of Hunter syndrome are eligible for enrolment. Prospective clinical data are collected during routine visits, according to local standard practice; historical data may also be recorded. Results: More than 1000 patients have been enrolled at 124 clinics in 29 countries, with 83.2% (788/947) of prospective patients having received idursulfase treatment (January 2016 data; median age at treatment start, 7.2 years). The data collected have contributed to understanding the onset and prevalence of clinical manifestations (including cardiac involvement and hearing loss), growth patterns, and identification of characteristic surgical histories (including hernia repair and ENT procedures). Important insights have also been gained about the tolerability of idursulfase treatment in different age groups. Conclusion: As the only registry for Hunter syndrome, HOS is a unique source of valuable data that facilitates wider goals of informing physicians, improving clinical outcomes and quality of life for patients.
Shire sponsors the Hunter Outcome Survey and funds medical writing support.
Severe Tracheal Collapse in Type 2 Mucopolysaccharidosis
M. Langeveld1, M. Rutten1, P. Ciet1, R. van den Biggelaar1, E. Oussoren1, J. Langendonk1, A. van der Ploeg1
1Erasmus University Medical Center, Rotterdam
Purpose: Mucopolysaccharidosis type 2 (MPS2) is a rare X-linked lysosomal storage disease characterized by accumulation of glycosaminoglycans (GAGs) in various organs and tissues1. Deposition of CAGs in tissue of the throat and trachea is thought to be responsible for progressive airway dysfunction and laryngeal and tracheal obstruction in this disease. Dyspnea is a frequent symptom in MPS2 with partially unknown etiology. The occurrence of tracheal narrowing and changes of shape of the trachea have previously been described in children with MPS.2,3 We studied the severity and prevalence of tracheobronchomalacia in adult MPS2 patients. Methods: All five adult MPS2 patients (mean age 40 years) treated at the Erasmus Medical Center were included. Repeated routine pulmonary function tests were obtained during several years of follow-up. Analyzing respiratory symptoms, we performed in- and expiratory chest CT scans in all patients. The cross-sectional area of trachea and main bronchi were measured at end-inspiration and -expiration and % collapse was calculated. Results: There was a diffuse narrowing of the entire intra-thoracic trachea and the main bronchi and a severe expiratory collapse of the trachea in all patients. At 1 cm above the aortic arch the median % collapse of the trachea was 68% (range 60 to 77%), at the level of the aortic arch 64% (21-93%) and at 2 cm above the carina 49% (0-83%). The collapse of the main bronchi, measured at 1 cm below carina was 58% (26-66%) on the left side and 44% (9-76%) on the right side. Pulmonary function tests showed obstructive airway disease in all but one patient (FEV1 ranged from 18-62%). The fifth patient classified as restrictive airway disease with a very small total lung volume (TLCHe 49%). Conclusion: In adult MPS 2 patients central airway caliber is strikingly reduced and upon expiration there is a severe collapse of the trachea and main bronchi. This is the first study to show dramatic narrowing of the trachea and main bronchi in adult MPS2 patients during expiration. Clinical Implications: Severe respiratory symptoms in MPS2 patients can be explained by the observed tracheal and bronchial collapse and abnormal lung function results. Because of the very specific nature of the airway pathology it requires tailored therapy, with for example modified non-invasive ventilation. We recommend to include dynamic airway imaging in the assessment of pulmonary symptoms in MPS2 patients.
References
MartinRBeckMEngC. Recognition and diagnosis of mucopolysaccharidosis II (hunter syndrome). Pediatrics. 2008;121:377–386.MorimotoNKitamuraMKosugaMOkuyamaT. CT and endoscopic evaluation of larynx and trachea in mucopolysaccharidoses. Mol Genet Metab. 2014;112:154–159.ShihSLLeeYJLinSPSheuCYBlickmanJG. Airway changes in children with mucopolysaccharidoses. Acta Radiol. 2002;43:40–43.
Attenuated MPS II Patient With IGF1 Deficiency and Improved Growth on Growth Hormone and Enzyme Replacement Therapy
K. Kim1, D. Zimmerman1, B. Burton1
1Lurie Children’s, Chicago
Introduction: Short stature is a prominent feature of all the MPS disorders and current therapies are limited in their ability to significantly improve growth and final adult height. Observations of growth pattern from the Hunter Outcome Survey (HOS) revealed that most MPS II males have short stature (<-2SD) by 8 years of age and do not experience the typical pubertal growth spurt. Enzyme replacement therapy with idursulfase may improve growth velocity in treated MPS II patients, most significantly in those who started treatment at a younger age. Data analyzed from patients in HOS noted that the change in growth rate in the first 24 months post ERT was similar to reported growth rates in those treated with growth hormone therapy for idiopathic short stature. However long term impact of ERT on final adult height is yet unclear. There have been a few case reports of MPS I and II patients treated with growth hormone therapy and given the small numbers it is unclear if growth hormone is effective in improving growth in MPS patients. Case Report: We report our experience of treating an adolescent male with attenuated MPS II with growth hormone. Our patient initiated ERT with idursulfase at 7 years of age at the standard dose of 0.5 mg/kg IV weekly. His growth at the time was normal; height increased along the 25th percentile on CDC growth chart for boys. Between age 7 and 11.8 years, he continued to grow in height along the 25th percentile at a velocity of 5.5 cm/year. During this time, IGF1 levels were persistently low, while IGFBP3, T4 and TSH levels were normal. At his 12.5 year follow up visit, growth velocity had decreased, with only a gain of 0.3 cm within the interval 8 months. Given his history of low IGF1 levels and slow growth, the patient was referred to Endocrinology for further evaluation. Additional testing revealed normal growth hormone levels following stimulation testing with insulin and arginine. He had persistently low IGF1 levels. Bone age was slightly delayed at 11 years 6 months at chronological age of 12 years 10 months but within 2 SD. He started recombinant IGF1 replacement therapy with mecasemin at 13.1 years of age; however, due to lack of product availability, the patient was switched to growth hormone treatment with somatropin after 8 months of treatment. Growth between 13.1 years of age and 15.3 years of age proceeded along the 10th percentile at an average growth velocity of 6 cm/year. IGF1 levels increased to the normal range for age and Tanner stage. Bone age was estimated at 13 years at chronological age of 15 years 3 months, which is delayed. Between 15.3 years and 16.4 years, the patient’s growth velocity increased to 9.6 cm/year with height percentile increasing from 10th percentile to 25th percentile. The patient’s height at 16.4 years was 170 cm and his genitalia were not yet Tanner stage 5. Due to his delayed bone age, the decision was to continue somatropin treatment for additional potential growth. Conclusion: Our attenuated MPS II patient with IGF1 deficiency had maintained normal growth velocity on ERT until 12 years of age when he was noted to have slowing of his growth velocity from 25th percentile to 10th percentile. Recombinant IGF1 and then growth hormone treatment was initiated and the patient was able to increase his growth rate and maintain normal stature. While this is only one patient’s experience of growth on ERT and growth hormone therapy, we feel that treatment with growth hormone in patients with MPS II and growth factor deficiencies should be considered and tailored to the individual. Further studies with additional patients are needed to determine if indeed growth hormone treatment improves height in certain MPS II patients.
Bone Metabolism Profile in Mucopolysaccharidosis IVA Patients
C. Gondim1, G. Charara2, A. Guedes1, F. Bandeira1, P. Medeiros1
1Hospital Universitário Alcides Carneiro, Campina Grande
2Hospital Universitário Alcides Carneiro, Campina Grande Pb
Purpose: This study aimed to analyze the bone metabolic profile of patients with mucopolysaccharidosis IVA (MPSIV-A). Methodology: An observational cross-sectional study to evaluate the bone metabolic profile of patients with MPSIV-A. None of them had received enzyme replacement therapy (ERT), hematopoietic stem cell transplantation or medications that might affect Bone mineral density (BMD) prior to this study. BMD of the whole body (WB) and 33% radius were acquired by dual-energy X-ray absorptiometry (DXA). Biomarkers of bone turnover were assessed: alkaline phosphatase, Collagen Type I C-Telopeptide (CTx), 25OH D vitamin, PTH, and 24 h urine calcium. Information of clinical data were collected: age, genre, height, weight, IMC, ambulatory capacity, calcium intake, bone fracture. Results: Participants included 15 patients (7 males and 8 females) with a mean age of 29.33 ± 8.77 years (16 to 46 years). The bone mineral density (BMD) of the whole body (WB) was normal in 15/15 patients. The cortical bone (33% radius) assessed in 10/15 patients showed osteoporosis in 8/10 patients and osteopenia in 2/10 patients. Among the patients able to walk, the mean of z-score 33% radius was −2.48 (range −2 to −2.9) while in non-ambulatory patients, it was −3.78 (range −2.7 to −4.4). The z-score of 33% radius was indirectly proportional to the height and age of the patients. Two factors impaired the assessment of BMD of 33% radius in all patients: bone deformation or lack of reference of the z-score for those younger than 20 years old. Alkaline phosphatase, as a marker of bone formation, was normal in all patients. The CTx, a bone reabsorption marker, was over the upper limit of normality in 6/15 patients (0.560 ± 0.310 ng/mL). In 15/15 patients, the calcium intake was lower than the one recommended, but the 25OH D vitamin and PTH were normal. The high 24 h urine calcium in 15/15 patients could not be valued because it was associated with a low diuresis in all patients whose etiology has not been determined. No patient reported bone fracture. Conclusion: In 10/15 Morquio A patients, osteoporosis or osteopenia were found in DXA 33% radius. Despite the high levels of CTX, an important marker of bone reabsorption, no patients presented fractures probably due to their low mobility. Paradoxically for the Morquio A disease, the z score of 33% radius was better in older patients still capable of walking. The findings of this study can provide foundation for assessing bone metabolism after ERT
Guided Growth may not be the Best Option in Knee Valgus Deformity in Patients With Morquio A
D. Colmenares-Bonilla1
1Hospital Regional De Alta Especialidad Del Bajio, Leon
Valgus deformity on the knees is a common clinical finding that precedes walking disability in patients with Morquio A syndrome. This progressive deformity worsens the walking capacity, and is aggravated by the very hypermobility joint disease, which increases as does the size and weight of the patient, also accelerating the hip dysplasia and disturbed biomechanical axis. This leads to premature aging (arthrosis) of both joints and confining the patient to use a wheelchair for transportation and autonomy. It has been suggested guided growth for the treatment of this progressive deformity, however, due to the characteristics of patients with Morquio A, the remaining growth may not be enough to alleviate so severe valgus knee deformity. Since the study population Morquio A has an average size ranging from 101 to 143 centimeters adulthood, it is not possible to reliably predict the long-term effect of physeal arrest, used for the correction of angular deformities in skeletally immature patients. Purpose: Describe the physeal osteotomy as a viable and effective option for acute correction of deformity in valgus knees in patients with Morquio A syndrome. Compare this osteotomy with the traditionally recommended epiphysiodesis for guided growth, and show this osteotomy as greater in effectivity for acute deformity correction along with its easy reproducibility. Methodology: Prior informed consent and safe surgery protocol, we perform a closing wedge osteotomy at the distal femoral physis level in two Morquio A patients (four knees) The deformity average angle was 38 degrees. This osteotomy was fixed with percutaneous k-wires that were removed at 6 weeks after surgery. Both limbs were protected with fiberglass petrie cast from thigh to ankle to allow early weight bearing with these devices. As well as in the epiphyseal injuries, immobilization was retired at 6 weeks postoperatively and autonomous ambulation was allowed. Results: Correction of valgus angulation on the knees was achieved in all four limbs. There were no adverse reactions in the postoperative period. Short-term follow-up showed no recurrence of the deformity. No epiphyseal lesion was shown in any of the physis after six months postoperatively. The satisfaction of patients showed improvement immediately after surgery and removal of immobilization. The questionnaire Pediatric Outcomes Data Collection Instruments (PODCI) improved compared to before surgery in the area of transfers, mobility, physical role and global levels by an average of 10 points in the short term. The size of patients in the standing position decreased 5 mm in one patient and the other remained unchanged versus preoperatively. Conclusion: The epiphyseal osteotomy in the distal femur is a viable option, easy to reproduce and effective for acute correction of the valgus knee deformity. It is managed as an epiphyseal injury and not as a planned fracture, which offers advantages such as less immobilization time, relative stability, prompt reinstatement to activities and early weight bearing. It allows sudden correction, without waiting for the remaining limb growth, especially in patient populations whose final height is not easily predictable, such as Morquio A patients. As an epiphyseal injury (type I Salter and Harris) there is very low possibility of growth arrest and thus the long-term residual deformity. It is a report with only two pediatric patients with open physis, so more studies are needed with larger number of cases to validate the usefulness of this procedure as the one of choice. A long-term monitoring of these patients is necessary to confirm the absence of recurrence of the deformity in valgus knees. The aesthetic result is encouraging in the short term, as good as the functional outcome.
Assessment of the Angulation of the Knees and Ankles in the Sagittal Plane During the Gait of Patients With Mucopolysaccharidosis Type VI
B. Rinaldo Da Silva1, M. Sales Lins2, C. De Vasconcelos2, K. Lacerda2
1Associação Pernambucana de Mucopolissacaridoses (AMPS-PE), Olinda, PE - Brasil
2Universidade Federal de Pernambuco, Recife-PE
Purpose: The purpose of this study is to assess the angulations of the knee and the ankle joints during the sub phases of initial contact, midstance, and pre swing of the stance phase, in the sagittal plane, during the gait of patients with mucopolysaccharidosis type VI (MPS VI). Methodology: A total of 19 patients that fulfilled the inclusion criteria agreed to participate in the study. The study was conducted at an Inborn Errors of Metabolism Treatment Center of a hospital in Pernambuco (Northeast of Brazil). The participants were classified into three groups according to their ages: children (n = 11), adolescents (n = 4) and adults (n = 4). Questionnaires were used to collect sociodemographic and clinical biologics data. Anatomic points of the lower members were marked and the left and right profiles of the patients were recorded during the gait. The images taken at the first moment for each subphase of the gait were analyzed via Tracker Software—an analysis and physical modeling tool for videos. Results: Alterations in joint angulation during the three sub phases of gait were identified in all groups. However, the highest alteration values were presented in the adult group. Higher alteration values were seen in both plantar flexion of the ankles and knee flexion when compared to standard values. Knee angulations during the initial contact phase of gait, in which the expected angulation is 5º bending, ranged from 12,36º ± 6,8º among the children group, 8.5º ± 7,42º among the adolescent group, and 8.5º ± 7,42º among the adult group. For the ankle analysis, in which the expected normality was 5º of plantar flexion (−5°), the following results were obtained: −23,73º ± 8,53º among the children group, −25º ± 11,22º among the adolescent group, and −27,75º ± 3,3º among the adult group. Conclusion: Angulation alterations during the three sub phases of gait were found in all groups, with higher values identified for plantar flexion of the ankles and knee flexion. The results of this study provide a better understanding of the alterations in angulations that happen during the gait that are associated with MPS VI. Knowledge of this information is relevant when considering the use of better therapeutic approaches.
Markers of Oxidative Damage and Antioxidant Defense in MPS IV A and B Cuban Patients
T. Acosta Sánchez1, E. Morales1, A. La O1, L. Martinez1, L. Marin1, C. Lopez1, Y. Castillo1, R. Gutierrez1, O. Martinez1, M. Miranda1
1National Center of Medical Genetics, Havana
Mucopolysaccharidosis-IV (MPS IV) or Morquio syndrome is a group of diseases that are caused by mutations of 2 different genes but with similar clinical manifestations and morphological changes. MPS IVA is characterized by a loss of activity of the N-acetyl galactosamine 6-sulfate sulfatase that leads to accumulation of the keratan sulfate and chondroitin 6-sulfate in body fluids and in lysosomes. Morquio B is caused by mutations of beta-galactosidase and leads to the storage of keratan sulfate. Clinically, the deficient activity of both hydrolases is manifested by corneal clouding and skeletal abnormalities. These metabolic disorders are generally considered caused by lysosomal accumulation of partially or totally undegraded GAGs, leading to organelle impairment and cytotoxicity. However, the pathophysiology of this lysosomal storage disorder is not completely understood. The idea that other early cellular and developmental defects may contribute to patients ‘clinical phenotypes is clearly emerging. Recent evidence strongly suggests that mitochondrial dysfunction and alterations of intracellular calcium homeostasis have place in MPS. Its conditions have been associated with oxidative stress. Purpose: The aim of this study was to investigate biochemical and oxidative stress parameters in a MPS IV A and B Cuban patients. Methodology: Blood samples for one Morquio A and one Morquio B Cuban children were obtain by venipuncture. Samples were evaluated for markers of oxidative damage to lipids and proteins, thiobarbituric acid reactive substance and advanced oxidation products of proteins. We also determined the activities of intraerythrocytic antioxidant enzymes: Cu-Zn superoxide dismutase, catalase, glutathione peroxidase, and glutathione transferase, total antioxidant capacity and plasmatic levels of free thiol groups, as markers of antioxidant capacity. All techniques were performed using spectrophotometric methods. The patient’s results were compared with reference values of our laboratory. Comet assay was used in this study to evaluate the basal levels of single stranded breaks, DNA repair capacity against oxidation induced by H2O2 repair capacity and endogenous oxidized guanines damage in lymphocyte DNA of the patients compared to healthy subjects Results. Patients showed elevated plasma levels of MDA, advanced oxidation products of proteins, calcium and increased activity of intraerythrocytic Glutathione S-transferase. Also a decrement in the concentrations of thiol groups, transferrin and enzyme activity of catalase were observed as well. No significant differences in DNA damage and the DNA repair capacity were observed between controls and patients (p>0,05), Conclusion: Patients showed an increment in oxidative damage to lipids and proteins, with a decrease in the ability of antioxidant response. The evaluated indicators could be extrapolated to routine clinical analysis and contribute to an integral overview of the redox environment in patients with these diseases and could also be used as indices of treatment efficacy.
Oxidative Stress and Inflammation in Patients with Mucopolysaccharidosis Type IVA Under Enzyme Replacement Therapy
B. Donida1, D. Marchetti1, C. Jacques1, C. Mescka2, R. Giugliani2, C. Vargas1
1Universidade Federal do Rio Grande do Sul, Porto Alegre
2Hospital de Clínicas de Porto Alegre
Mucopolysaccharidosis type IVA (MPS IVA), is a rare autosomal recessive lysosomal storage disorder (LSD) caused by a deficiency in the enzyme N-acetylgalactosamine-6-sulfatase. As a consequence of the enzyme deficiency, the substrates (glycosaminoglycans-GAGs- keratan sulfate and chondroitin-6-sulfate) are retained in body fluids and lysosomes of many cell types of the organism. Considering the involvement of oxidative stress and inflammation in other MPSs and the lack of reports about MPS IVA, the aim of this study was to evaluate oxidative damage to biomolecules, antioxidant defenses, pro-inflammatory cytokine and GAG levels in patients with MPS IVA under enzyme replacement therapy (ERT). Occasional urine and heparinized blood samples were obtained from patients immediately before the session of ERT (n = 17) and from healthy age-matched controls (n = 10-15). All MPS IVA patients were receiving ERT treatment (elosulfase alfa- Vimizim® 2 mg/kg) every week by intravenous infusion, for about 32 weeks. Reduced glutathione (GSH) levels, the main non-enzymatic intracellular antioxidant, were measured by a fluorescence assay in erythrocytes. The both antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured by commercially available kit from Randox Lab (RANSOD® and RANSEL®, respectively). To determine oxidative damage to proteins, plasmatic concentration of sulfhydryl groups was measured by a spectrophotometric technique and urinary di-tyrosine was detected by fluorescence. Damage to lipids was evaluated by urinary isoprostanes, which were quantified by competitive ELISA commercial kit. Interleukin 6 (IL-6), a pro-inflammatory marker, was measured by ELISA commercial kit and urinary GAGs were detected by spectrophotometric technique. MPS IVA patients presented a reduction of antioxidant defense levels, assessed by a decrease in GSH content when compared to control group. SOD activity was significantly increased in patients, while there was no significant difference in GPx activity between patients and controls. Concerning lipid and protein damage, it was verified increased urine isoprostanes and di-tyrosine levels, and decreased plasma sulfhydryl groups in MPS IVA patients compared to controls. Interleukin 6 was increased in patients and presented an inverse correlation with GSH levels, showing a possible link between inflammation and oxidative stress in MPS IVA disease. GAGs urinary levels were still high in patients compared to the control group, which suggest that GAGs are related, at least in part, with the oxidative damage found in MPS IVA patients. The data presented propose that pro-inflammatory and pro-oxidant states occur in MPS IVA patients even under ERT. Taking these results into account, supplementation of antioxidants in combination with ERT can be a tentative therapeutic approach with the purpose of improving the patient’s quality of life. Further researches and clinical trials are needed to reveal how safe and effective would be the supplementation of antioxidants in combination with ERT in MPS IVA disease.
Coexistence of Autosomal Recessive Diseases: Cystic Fibrosis and Mucopolysaccharidosis Type IVA. Case Report
G. Porras Hurtado1
1comfamiliar Risaralda, Pereira
Purpose: Analyze and report a case of a girl with two autosomal recessive diseases: IVA mucopolysaccharidosis (IVA MPS) and cystic fibrosis (QF). Two diseases that can be confused when diagnosed in an early life by the pulmonary compromise that both produce. Methodology: It presents a case of a girl of 7 years with consanguineous parents. She beginning with several episodes of bronchial obstruction syndrome from 6 months of age with long hospitalizations, steatorrhea and rectal prolapse. At 2 years old pediatrics pulmonology makes a diagnostic of cystic fibrosis by steatorrhea, bronchiectasis and history of maternal cousin died of QF. Sweat test was negative, the patient improved with QF treatment but continued with respiratory and gastrointestinal exacerbations and pseudomonas aeruginosa infection. One year later the girl was sent to geneticist for QF molecular confirmation and she finds, corneal opacity, epigastric hernia, umbilical hernia, lumbar kyphosis, genu valgus, short stature, generalized ligamentous laxity, moderate deformity of the wrist, hip dysplasia with severe hypoplasia head of femur, odontoideo hypoplasia with compression of the medulla. It found congenital fusion of T11-T12, L1- L2 and L2- L3 that produce dural sac compression. Physical therapy and handling with elosulfase alfa is initiated. A year later seen improvement but continued rectal prolapse and steatorrhea. Molecular tests for both diseases is requested. Results: By complete sequencing of 14 exons of the gene N-acetyl-galactosamine-6-sulfatase sulfate (GALNS) it is confirmed homozygous mutation (c.901G to> T); which generates a change in the GALNS protein in the 301 amino acid position producing a change of glycine for cysteine. This result indicates that the patient is positive for IVA MPS, which can produce a severe phenotype. In complete sequencing of Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) homozygous deletion was identified deltaF508 (p.F508del (Phe508del)) with classification Pathogenic. Comorbidity cystic fibrosis and IVA MPS is confirmed. Conclusion: IVA MPS is an autosomal recessive disease. Affects approximately 1 in 200,000 live births and is characterized by a wide variety of symptoms, including severe skeletal abnormalities, hearing loss, corneal clouding, heart valve disease, and respiratory symptoms.(1-4) Homozygous mutation (c.901 to G> T) founded in the patient is considered severe phenotype. QF is also an autosomal recessive disease caused by mutations altering the it is found primarily in the apical surface of epithelial cells in the lung, pancreas, intestine liver and male reproductive system, altering the secretions of the major glands of the body(5). CFTR mutation found in the patient is common in European population. It is considered that this mutation causes a severe phenotype. In patients with QF, lysosomal enzymes function is higher as compensation of accumulated mucus clearance by poor hydration that CFTR alteration produces. Comorbidity of both diseases worsens clinical symptoms. In patient treatment alpha elosulfase and dornase alpha has improved respiratory symptoms and quality of life.
Diagnosis of Morquio A patients in Mexico. Where far are We from Prompt Diagnosis?
D. Colmenares-Bonilla1, N. Esquitin-Garduño1
1Hospital Regional De Alta Especialidad Del Bajio, Leon
Purpose: The aim of the present work is to set data and find out how is the clinical scenario in Morquio A patients in Mexico, since there is nor any official data of this patients in our country. This will serve as a base to improve prompt diagnosis, sequel, quality of life, as well as verify the possible interventions to improve early detection of this disease. Methodology: Through a self-administered or family helped questionnaire, we review clinical characteristics and general data of 50 Mexican patients with confirmed diagnosis of Morquio A syndrome. We describe and analyze results with mean tendency measures. Results: Results were obtained from 50 patients with confirmed diagnosis of Mucopolysaccharidosis type IVA, whose ages ranged from 3 to 41 years (mean 15.2 years). The average age at diagnosis was 5 years, ranging from the month of life and up to 15 years. Two patients were diagnosed before one year of age (one month and nine months, respectively) due to have affected relatives. Twenty-one patients were diagnosed between one and three years (five of them had family history); eight patients were diagnosed between 3 and 5 years (three with familiars affected). Twelve patients aged 5 to 10 years and seven patients were diagnosed after 10 years. Thirty patients (60%) mention not having any previous diagnosis, while 9 patients (18%) did not remember the initial diagnosis. In the other 18% of patients (n = 9) the initial diagnosis ranged from achondroplasia, epiphyseal dysplasia, espondilodysplasia and Sotos syndrome. More than half of patients (26 patients) had already been subjected to any surgical procedure, with an average of 2.03 surgeries per patient (from 1 to 8). Of the 53 procedures they have been divided into: 19 alignment knees (36%), followed by 11 cervical spine (20.1%), 6 hernioplasties (11.3%) and 10 other orthopedic procedures (hip dysplasia, bone lengthening, mechanical alignment axis) corresponding to 18%. Significantly revision surgery represents 21 of the 53 procedures (39%), predominating 11 knee surgeries and 6 primary spinal decompression. Of these patients, nine were operated without yet having had the definitive diagnosis of Morquio A syndrome. Conclusion: The results show that the diagnosis of Morquio syndrome is delayed, however it remains within global descriptions. more research and greater diffusion of the clinical features as well as therapies and alternatives therapeutic are required for the physician treating the patient with syndrome of Morquio and avoid as much as possible surgeries knowing the high rate revision, mainly in the field of orthopedic surgery.
Morquio Type B Syndrome. Case Report
E. Morales Peralta1, L. Martinez1, T. Acosta1, L. Larrinaga1, M. Miranda1, L. Fariñas1, A. Alles1, I. Fuentes1, Y. Valdes1, I. Camayd1
1National Center of Medical Genetics, Havana
Introduction: Morquio syndrome or Mucopolysaccharidosis (MPS) IV is an autosomal recessive inherited metabolic disease that affects the lysosomal metabolism. There are two clinical forms (IV A and IV B), with indistinguishable clinical pictures. The most characteristic features of this syndrome are skeletal deformities and shortness of stature. The β galactosidase enzyme is defective in MPS type IV B. This disease is diagnosed through the detection of high keratan sulfate levels in urine and deficient β galactosidase activity in blood. Purpose: To present clinical and biochemical findings in a patient diagnosed with Morquio B syndrome. Methodology: The diagnosis was made by qualitative chemical tests in urine and Thin Layer Chromatography (TLC) for the determination of mucopolysaccharidos in urine. The diagnosis was confirmed by enzymatic methods. Results: The patient was evaluated by the orthopedics service when she was a year old because of her feet and legs abnormalities. Main symptoms included pectus carinatum, dorsolumbar kyphosis, genu valgum, broad ankles and feet, forearm ulnar deviation, anthropometric alterations without short stature and dermatoglyphic alterations. The clinical picture suggested a mucopolysaccharidosis type IV. Biochemical tests for the screening of mucopolysaccharides (cetyl trimethyl ammonium bromide and toloudine tests) were positive and mucopolysaccharides TLC showed high keratan sulfate levels. The β galactosidase enzymatic activity was lower than 2% of negative controls. Conclusions: Suggestive clinical findings and laboratory results allowed the patient diagnosis as a mucopolysaccharidosis IVB or Morquio B syndrome. The interdisciplinary collaboration between different medical services resulted in the rapid diagnosis of this case, and will permit the appropriated palliative treatment and parents’ genetic counseling.
Gait Pattern and its Parameters in Children With Mucopolysaccharidosis Type VI (MPS VI)
M. L’Herminez1, E. Oussoren1, A. Vollebregt1, I. Plug1, A. van der Ploeg1, L. van der Giessen1
1Erasmus MC Sophia, Rotterdam
Purpose: Mucopolysaccharidosis type VI (MPS VI, Maroteaux-Lamy Syndrome) is a clinically heterogeneous disease in terms of the extent and rate of progression of organ impairment in affected individuals. Clinical manifestations include coarse facial features, cardiopulmonary impairments, hepatosplenomegaly, dysostosis multiplex and joint stiffness resulting in growth retardation. In order to improve physiotherapeutical treatment for these patients we studied gait patterns in combination with muscle strength and mobility. We performed a cross sectional study in which gait, hip abnormalities, joint mobility, muscle strength and endurance were assessed within a short time span. Methodology: This study included ten children diagnosed with MPS VI, treated with enzyme-replacement therapy. Gait analysis was performed in a gait analysis laboratory. Hip radiographs were obtained to investigate hip abnormalities. Joint mobility, muscle strength and endurance were measured by respectively goniometer, hand-held dynamometer and 6-minute walk test (6MWT). In order to assess the cause of limited mobility pulmonary and cardiac functions were tested. Patients characteristics were retrieved from medical charts. Results: Ten MPS VI children participated, of whom 6 males, age at assessments ranged between 5.8 and 13.5 years. Nine children have lumbar lordosis, valgus knees and eight of them had a waddling gait. All participants had hip abnormalities on radiographs. In general joint mobility was reduced, especially hip extension. Median scores on endurance (6MWT), and muscle strength of several muscle groups were all below normal values. Conclusions: The majority of Dutch children with MPS VI walked with a waddling gait, characterized by lumbar lordosis and valgus knees. In order to support mobility of patients, specific physiotherapeutic therapy should focus on the improvement or stabilization of joint mobility, muscle strength and restricted hip extension.
Assessment of Gravity Center of Patients Siblings With Attenuated Form for Mucopolysaccharidosis VI: A Case Report
B. Rinaldo Da Silva1, K. Lacerda2, P. Freire Magalhães3, M. Almeida4
1Associação Pernambucana de Mucopolissacaridoses (AMPS-PE), Olinda, PE - Brasil
2Instituto de Desenvolvimento Educacional, Recife/PE
3Instituto de Medicina Integral Prof. Fernando Figueira, Recife-PE
4Universidade Federal de Pernambuco, Recife-PE
Purpose: to evaluate the center of gravity (vector application point is that the body weight) siblings patients with the milder form of mucopolysaccharidosis type VI (MPS VI). Methodology: two patients were selected brothers maternal and paternal with the attenuated form of the disease, its clinical and sociodemographic characteristics were evaluated using a structured questionnaire, patients were asked about balance changes, and then were evaluated through postural analysis software (SAPO), where spherical markers were attached to the skin of patients in anatomical reference points, ex: tibial tuberosity, lateral epicondyle of the knee, among others. Participants were instructed to adopt the standing position, with feet in parallel, using a digital camera (Sony® DSC-HX60) positioned on a tripod, to 2 meters of the participants. A plumb line was positioned in the same body plan participants with a known measure (50 cm) in order to calibrate the scanned images. Four images were captured: front view, rear view, right and left of the body. From the points, the following body alignments were calculated: the center of gravity of the asymmetry in the frontal plane (front CG) and center of gravity of the asymmetry in the sagittal plane (sagittal CG). Results: male siblings, live in different cities, both are in the post-pubertal development stage and perform weekly enzyme replacement therapy (ERT). The older brother C.V., sedentary, has 38 years of age, weight of 62.8 kg, height of 1.52 cm, was diagnosed to 9 years old and began ERT to 35 years old. The younger brother J. V., 32 years old, also sedentary, weight of 53.35, height = 1,49 cm, had his first diagnosis with 6 years, and only eight months weekly performs ERT. The center of gravity (CG) showed asymmetrical in the two planes for the two brothers. In older brother C.V., the asymmetry in the frontal plane was −10.5%, and the asymmetry in the sagittal plane was 40.2%. In younger brother K.V. the asymmetry in the frontal plane was −2.7%, and the asymmetry in the sagittal plane was 31.0%. When asked about balance deficits both reported changes resulting in stumbles, but older brother reported two falls in the last 6 months. This could be explained in biomechanical terms, since motion can be viewed as the body of CG shift through space, this displacement occurs up and down and from side to side, and if the CG was altered in the static phase, in the gait phase (dynamic) this alteration could result in greater balance deficits. Conclusion: the data showed that both brothers showed asymmetries in CG, the eldest brother had more alterations. With this study it is possible to show the importance of study and work CG of a patient with MPS VI. The CG of the body is modified at any time during the gait and if this center is already changed in the static phase, the rehabilitation and treatment should be started.
Evaluation of Pain, Articulation Stiffness and Range of Motion of the Upper Body Limbs Among Patients With Mucopolysaccharidosis Type VI
B. Rinaldo Da Silva1, J. Silva Medeiros2, K. Soares3, D. Queiroz2, P. Freire Magalhães4
1Associação Pernambucana de Mucopolissacaridoses (AMPS-PE), Olinda, PE - Brasil
2Universidade Federal de Pernambuco, Recife-PE
3Faculdade Estácio do Recife, Recife-PE
4Instituto de Medicina Integral Prof. Fernando Figueira, Recife-PE
Purpose: The purpose of this study is to evaluate the presence of pain, articulation stiffness and range of motion of the upper body limbs of patients with Mucopolysaccharidosis VI in order to observe how these factors are likely to affect and to correlate with the activities of daily living. Methodology: The study was conducted in the state of Pernambuco in the Inborn Errors of Metabolism Treatment Center in Brazil. All range of motion (ROM) were measured of the joints of the upper limbs through manual goniometer. A total of 30 upper limbs were evaluated in three steps by two different researchers and an average was taken. Was applied a structured questionnaire with questions related to the presence of pain through the Borg scale, articulation stiffness was assessed by specific movements and the structured questionnaire as well as difficulties in performing activities of daily living (ADL’s). Results: The sample consisted of 16 patients, 9 male and 7 female with a mean age = 17,76 years and average weight and height 31,30kg and 1,17 cm respectively. The flexion of the shoulder joint in the sagittal plane showed the commitment of all participants, ROM middle joint equal to 90.38º and 93.38º to the left member in the right limb (normal value of 180º). ROM below the predicted average found in the study was fist extension in both left and right member, the presence of pain was reported in 12 patients (75.0%) and patients attending school and/or college reported pain hands to write and loading weight. When asked about the presence of stiffness in articulations that achieved greater account was the shoulder articulation for 6 (37.5%) of individuals; 15.4% had pain in wrists and 15.4% in the metacarpophalangeal articulations and proximal and distal interphalangeal. Carpal Tunnel Syndrome has been diagnosed in only 15.4% of the patients. When asked about the use of medication for pain in the upper limbs 53.8% said they make use of painkillers in times of intense pain. With regard to the difficulties in performing the ADL’s activities, various activities have been compromised due to limited articulation. The most affected (76.9%) were spending soap in the body, carrying a heavy object and put the shoes, and the lowest percentage of activity was to hold a toothbrush 7.7%. The activities that required greater range of motion and muscle strength showed up probably affected due to articulation stiffness caused by the deposit of glycosaminoglycans. Conclusion: Through this study it was observed as the limitation of the arms of these individuals tend to influence negatively the performance of ADL’s. It’s expected that the study calls the attention of the scientific society to the importance of the subject and to serve as a means of monitoring and evolution of MPS VI, encouraging more and more the role of physical therapy for alleviating the symptoms and thus improve quality of life.
A 10 Year Old Girl Had MPS VI (Morateux-Lamy syndrome) With Symptomatic Cord Compression had a Falsely Reassuring Normal Supine MRI. An Erect Spinal X-ray Revealed Thoracolumbar Subluxation
A. Inwood1, G. Askin2, J. McGill1
1Queensland Lifespan Metabolic Medicine Service, Brisbane
2Department of Orthopaedics, South Brisbane
Purpose: To alert clinicians to a potential limitation of MRI to detect cord compression. Methodology: A 10-year-old girl from a non-consanguineous family was diagnosed with Morateux-Lamy syndrome (MPS VI) at the age of 13 months. There was no arylsulfatase B activity detected on leucocytes. She had presented with symptoms of spinal cord compression at C1 and C2 due to atlanto-axial instability. A cervical posterior occiput to C5 fusion stabilized her cervical spine. Enzyme replacement therapy was commenced as soon as federal government funding permitted. Infusions of Naglazyme at a dose of 1mg/kg every week was given via an infusaport from the age of 2 years and 8 months. Routine 6 monthly clinical assessments of physical examination with annual spinal imaging from diagnosis had remained stable since the cervical spine fusion. At the age of 10 years and 2 months, neurological examination showed deterioration in her gait and sustained clonus bilaterally. She was referred to her orthopedic surgeon who arranged an early MRI. Results: MRI demonstrated a stable spine, compared with the previous film 6 months previously and no significant cord compression. Her orthopedic surgeon then ordered an erect lateral spinal x-ray that showed marked subluxation at T12/LI junction. This subluxation was not visible on the MRI as in the supine position it corrected. The patient developed urinary incontinence a few days before surgery. Conclusion: This patient demonstrates the importance of further investigation, including erect plain radiography of the whole spine, when an MRI fails to reveal the cause of a patient’s symptoms. In this case an erect spinal X-ray revealed the pathogenic subluxation.
Pertinence of Using Urinary Glycosaminoglycan Determination in the Diagnostic Approach for Mucopolysaccharidosis in Colombia
N. Pulido1, J. Guevara2, O. Echeverri2, C. J. Alméciga-Díaz2, L. A. Barrera1
1Hospital Universitario San Ignacio, Bogota
2Pontificia Universidad Javeriana, Bogota
Purpose: To analyze the biochemical approach to MPS diagnosis in Colombia. Methodology: Retrospective analysis of samples processed for MPS urinary test during the last four years in the Institute for The Study of Inborn Errors of Metabolism in Bogotá - Colombia. Results: In Colombia, the incidence of mucopolysaccharidoses (MPS) is still unknown. However, in our laboratory, a national reference center for diagnosis of inborn errors of metabolism, we have received around 18000 samples for testing during the last 4 years, among which 6% corresponded to preliminary urinary analysis for MPS. In countries like Colombia, initial qualitative turbidimetric tests are frequently used as the first biochemical approximation for MPS diagnosis. This results in a high number of false positive tests, causing additional costs, delays in the diagnostic process and discomfort for the patient that must be submitted to further testing. In our laboratory, we implemented the 1,9-dimethylmethylene blue (DMB) quantitative assay as an alternative to turbidimetric tests by mid-2014. After one and a half years, DMB implementation, lead to a reduction in false positive rate from 37.98% to 12.30%. In addition, improvement in the process for glycosaminoglycan (GAG) extraction from urine, prior unidimensional electrophoresis for identification, allowed a better patient selection for enzyme analysis. Thus, specific enzyme activity is only performed for patients with truly increased GAG excretion and a well-supported clinical suspicion of MPS. Conclusion: In spite of the limitations associated to the usage of urine analysis for GAG detection, it is important to take into account that in the Colombian context, these analyses are covered by the health system, therefore allowing the clinician to start a diagnostic approach. Our laboratory is constantly improving the technical procedures to enhance the detection and identification specificity of urinary GAG determinations in order to offer better biochemical tools for diagnosis. With the implementation of the above-mentioned improvements and the higher availability of specific enzymatic tests in our country, we hope the diagnosis of MPS will be more and more efficient.
Clinical, Biochemical and Molecular Diagnosis of Lysosomal Storage Disease in Cuban in the Last Decade
T. Acosta Sánchez1, L. Larrinaga1, D. Campos Hernandez1, M. Menendez2, V. Tamayo1, N. de Leon1, L. Martinez1, E. Morales1, K. Lavaut1, A. Garcia1
1National Center of Medical Genetics, Havana
2Cuban Neurological Institute, Havana
Introduction: Lysosomal storage disorders (LSDs) are a large group of more than 50 different inherited metabolic diseases. Most cases are a result of the defective function of specific lysosomal enzymes and, in few cases, of non-enzymatic lysosomal proteins or non-lysosomal proteins involved in lysosomal biogenesis. The progressive lysosomal accumulation of undegraded metabolites results in generalized cell and tissue dysfunction, and, therefore, multi-systemic pathology. In Cuba, the biochemical and molecular confirmation of these diseases takes place in the National Center of Medical Genetics. Purpose: the objective of this work is to characterize the diagnosis of LSD in Cuba during the period 2006-2015, taking into account clinical, biochemical and molecular findings. Methodology: We included 325 post-natal studies under clinical suspicion of LSD. Clinical genetics services requested the genetic tests in all cases. Each LSD was confirmed by an analysis of specific enzymatic activity (nmol/mgprotein/h) by spectrophotometric and spectrofluorometric tests. It was considered as a deficit enzymatic individual when the enzymatic activity relative to healthy control was less than 30%, and other lysosomal enzymatic activity was greater than 75% in order to ensure the reliability of the biochemical diagnosis. Confirmatory tests were carried out for patients Diagnosed with Pompe disease in the Chamoles Neurochemistry Laboratory in Argentina and the Biophysics Department- UNIFESP in Brazil. Those cases with low fucosidase and glucocerebrosidase enzymatic activities were confirmed by molecular genetic testing. Results: The facial and skeletal dysmorphia, hepatosplenomegaly, progressive psychomotor deterioration and growth retardation were the most common clinical findings. Samples were received from all Cuban provinces. In this period, 21 patients were biochemical diagnosis with mucopolisaccharidosis, 13 with sphingolipidoses, 9 with Glycoprotein Storage Diseases and 4 with Pompe Disease. The Hurler syndrome (10) and fucosidosis disease (9) were the most frequent. All fucosidosis patients were from the province of Holguin and have the Q422X mutation. A Gaucher patient was homozygous for the N370 S mutation and three patients were homozygous for the L444P mutation. Conclusions: In this period, 47 LSD cases were diagnosed (14.5% of the total of tested patients). A previous study reported a frequency of LSD detected in symptomatic Cuban patients of 8.2%. Our results are a consequence of the Cuban Network of Medical Genetics, which ensure a close collaboration between geneticists, clinicians and laboratory specialists.
Mucopolyssacharidosis in Portugal—Where do we stand?
I. Ribeiro1, C. Caseiro1, F. Pinto1, E. Pinto1, E. Silva1, S. Rocha1, C. Ferreira1, D. Sousa1, H. Ribeiro1, D. Quelhas1, F. Laranjeira1, L. Lacerda1
1Centro de Genética Médica Jacinto Magalhães, CHPorto
Purpose: Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases (LSD) caused by deficiency of enzymes required for the stepwise breakdown of glycosaminoglycans (GAGs). Deficiency in each of these enzymes originates GAGs accumulation in the lysosome in various organs, leading to cellular dysfunction and clinical abnormalities (1). MPSs have been classified according to enzyme defect and systemic manifestations and include MPS I-H (Hurler), MPS I-S (Scheie), MPS I-H/S (Hurler-Sheie), MPS II (Hunter), MPS IIIA (Sanfilippo A), MPS IIIB (Sanfilippo B), MPS IIIC (Sanfilippo C), MPS IIID (Sanfilippo D), MPS IVA(Morquio A), MPS IVB (Morquio B), MPS VI (Maroteaux-Lamy), MPS VII (Sly) and MPS IX (Hyaluronidase deficiency). These LSD are rare with an estimated overall incidence of approximately 1:20.000 live births (1). MPS phenotype covers a broad spectrum ranging from mild to severe forms. Patients usually seem normal at birth, but during early childhood they develop clinical disease, including skeletal deformities, joint stiffness, facial coarse features, cardiac complications, hearing loss and vision impairment. Most patients display severe central nervous system involvement. Generally severity relates to residual enzyme activity, which usually is linked to the genotype of the affected individuals. MPSs have an autosomal recessive transmission mode, with the exception of MPSII (X-linked). The importance of this group of disorders among LSD led us to make a retrospective analysis of MPS patients diagnosed at Centro de Genética Médica Doutor Jacinto Magalhães from 1984 until present. Methodology: Between 1984 and 2015 laboratory diagnosis of 160 MPS patients was made according to biochemical and molecular results. Patient’s biochemical evaluation includes determination of urinary GAGs quantification, fractionation by electrophoresis and enzyme activity determination, usually in peripheral blood leukocytes and/or plasma. Mutation analysis of the affected gene was also performed. Results: Mucopolysaccharidosis account for 19% of the total LSD diagnosed in the laboratory of Bioquímica Genética (Portuguese Reference Laboratory in Portugal). Taking into account the 13 subtypes of MPS all together, over 160 patients have been diagnosed so far in our laboratorydespite no diagnosed patients with MPS IIID and MPS IX. MPS I, MPS II and MPS III are the most frequent mucopolysaccharidosis diagnosed in our laboratory. Conclusion: This analysis may be considered representative of the MPS spectrum in Portugal, since our center is the Portuguese reference Laboratory for LSD and therefore most patients are referred to us. Detection of excess urinary GAGs provides a useful preliminary orientation towards a MPS disease, however diagnosis has to be confirmed by lysosomal enzyme testing and genotype analysis. Molecular characterization of these patients becomes important for genotype-phenotype correlation, and essential to offer genetic counselling and prenatal diagnosis to affected families. Multidisciplinary team approach contributes to an early suspicion and laboratory confirmation is crucial for a final accurate diagnosis as well as indicating precocious treatment initiation with enzyme replacement therapy, currently available for MPS I, MPS II, MPS IV A, MPS VI and MPS VII and underway for MPS III.
The two first authors contributed equally to the work.
Three Years of Biochemical Diagnosis of MPS in a Mexican Reference Center
J. Garcia-Ortiz1, T. Da Silva-Jose1, J. Juarez-Osuna1, S. Mendoza-Ruvalcaba1
1CIBO-IMSS, Guadalajara
Mucopolysaccharidosis are a complex and heterogeneous group of lysosomal disorders that block the degradation of glycosaminoglycans (GAG) and condition their accumulation in several organs and tissues. The diagnosis of MPS is based on the clinical features and it can be confirmed by biochemical (DBS or leucocytes) and/or molecular analysis. Here we report the analysis of residual enzymatic activity in 8 types of MPS in almost 600 patients with clinical suspicion of MPS. Methods: Peripheral blood was drawn from patients with clinical diagnosis of MPS. Leucocytes were extracted and homogenized by sonication. Proteins were measured by the Lowry method in a Beckman Coulter DU 730 spectrophotometer. Residual activities of 8 enzymes were measured using modification of methods published elsewhere. Each reaction was performed with the addition of proteins to specific 4-MU flurogenic substrate with an incubation period at 37°C to continue adding, in some cases, potentiators and /or inhibitors, and if required a second incubation; then reactions were stopped with a pH 10.6 buffer. Readings were carried out in a 450 Turner fluorometer with a 360 nm excitation filter and a 415 nm emission filter. Additionally, in each reaction a 4-MU curve was performed as quality control. Results: 592 samples of equal number of patients with clinical suspicion of MPS were included in this study; samples were received from August 1, 2012 to December 31, 2105. Almost all the samples were referred to our laboratory by a trained clinician on the diagnosis of MPS. Enzymatic activity measurements were standardized for MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IVA, MPS IV B, MPS VI and MPS VII. 80% of the requested enzymatic test were for MPS IV (38%), VI (28%) and I (13%); 20% were for the rest of available tests (MPS II, IIIA, IIIB, IVA and VII). 26.52% of all the samples gave a positive result, 73.48% gave a normal result. The MPS with the highest positivity was MPS IVA (44.5%) and the lowest was MPS IVB (0%). Positive samples were observed in 7 of 8 MPS test. Conclusions: These results show an epidemiological view of the MPS in Mexico, from a reference center; however it also shows that a sub-diagnosis is still evident in this sample, probably indicating that MPS with a more evident clinical phenotype was the most commonly referred for confirmation, and a major educational effort is necessary to improve the MPS diagnosis in our country. Finally, molecular confirmation of the positive cases by biochemical results is still incomplete.
Prenatal Counseling and Diagnosis of Mucopolysaccharidoses in Egypt: 15 Years Experience
A. Aboulnasr1, E. Fateen2
1Faculty of Medicine, Cairo University, Cairo
2National Research Centre, Cairo
Purpose: Prenatal Counseling for Mucopolysaccharidoses (MPS) diagnosis. Methodology: 127 pregnancies among 96 females counseled from June 2000 to January 2016.All have one or more affected sibling with MPS except four cases (2 MPS IVa,1 MPS I and 1MPS II)who have positive family history in a close relative . MPS types were 47 (37%) type I, 18(14.2%) type II, 16 (12.6%) type III,23(18.1%) type IVa and 23(18.1%) type VI. 73 couples counseled in one pregnancy and 23 couples counseled 54 times in successive pregnancies, 17 couples in 2, five in 3 and one in 5 pregnancies. Consanguineous marriage was present in 110 (86.6%) pregnancies. In 45 (35.4%) pregnancies, couples have no normal children. However 87 (68.5%) couples have no normal boys. Prenatal diagnosis (PD) was performed in 100(78.7%) pregnancies. Amniocentesis done at 14-15 weeks gestational age in 46 (46%) to withdraw 10 ml for glycosaminoglycans (GAGS) analysis by 2-dimensional electrophoresis. In one case we failed to take enough amniotic fluid. Chorionic Villus Sampling (CVS) done at 11-12 weeks in 54(54%) pregnancies to measure enzyme activity fluorimetrically. In 8 (14.8%) cases, Amniocentesis was needed to verify diagnosis of borderline result (5 cases) or because CVS did not yield enough villi (3 cases).27(21.3%) of 127 counseled were not subjected to PD. 17 (63%) did not show up at scheduled time for PD, 4(14.8%) had spontaneous abortion before PD, 4(14.8%)) came late, one (3.7%) had vesicular mole and one refused. Results: 99 cases were successfully diagnosed .71(71.7%) had normal fetus and 28(28.3%) had affected fetus. Conclusion: All types of MPS could be diagnosed prenatally. Analysis of glycosaminoglycans by 2-DEP of amniotic fluid is sensitive and accurate. Measuring enzyme activity in chorionic villi is recommended as CVS is done 3-4 weeks earlier than amniocentesis which is more favorable medically, ethically and psychologically. High consanguinity rate in Egypt leads to more genetic diseases. To have a normal boy is a motive for repeated pregnancies.
GM1-Gangliosidosis: Late Diagnosis due to Clinical Heterogeneity
L. Arash-Kaps1, M. Seegräber1, A. Dieckmann2, M. Smitka3, S. Gökce1, Y. Amraoui1, J. Reinke1, E. Paschke4, K. Mengel1, J. Hennermann1
1Children Hospital at the University Medical Center Mainz
2Universitätsklinikum Jena
3Universitätsklinikum Carl Gustav Carus, Dresden
4Medical University of Graz
Purpose: Deficiency of the lysosomal enzyme β-galactosidase is an essential biochemical feature of 3 different diseases: GM1-gangliosidosis, mucopolysaccharidosis IV B (Morquio B) and galactosialidosis. ß-galactosidase deficiency results in an accumulation of GM1-gangliosides and/or keratin sulfate. Patients with GM1-gangliosidosis show progressive neurodegeneration and skeletal abnormalities. There are 3 main clinical variants classified by severity. Patients with the infantile form show infantile dementia, facial dysmorphism, macular cherry-red spots, skeletal dysplasia, hepatosplenomegaly and early death. Patients with the juvenile form show mental impairment, seizures and skeletal involvement. Patients with the adult form are characterized by skeletal involvement and dystonia, gait, or speech disturbance. The purpose of this study was the evaluation of the clinical course in patients affected from GM1-gangliosidosis. Methodology: We analyzed retrospectively the clinical data of all patients consistent with the diagnosis of with GM1-gangliosidosis who were recognized in our biochemical laboratory within the last 20 years or known in a cooperating clinic. Out of 19 patients 13 were male and 6 female. 6 patients were classified as infantile, 9 patients as juvenile, and 3 patients as adult form of GM1-gangliosidosis. Evaluation of the patients included medical history, clinical and neurological examinations, ophthalmological evaluations and radiological examinations. Results: Patients with the infantile form presented with first symptoms at age 2.5 months (median), patients with the juvenile form at age 1.83 years and patients with the adult form at age 3 years. Time between first symptom and diagnosis was 5 months in patients with the infantile form, 2 years in the juvenile form and 14 years in the adult form. During the study 5 patients with the infantile form and 1 patient with the juvenile form died. Time between first symptom and death in infantile patients was 15 months (median). All patients with GM1-gangliosidosis had neurological involvement such as cognitive deterioration, spasticity and pyramidal signs. 9 patients suffered from epileptic seizures. Among them 3 were of infantile, 5 of juvenile and 1 of adult form. Only 50% of patients with the infantile form revealed a cherry-red spot and none of the patients with juvenile and/or adult form. Dysostosis multiplex was diagnosed in 2/3 patients with infantile, 2/6 with juvenile and 3/3 with adult form. Patients with the infantile form showed developmental delay and hypotonia as first symptoms already within their first 6 months of live. Patients with the juvenile form presented with diverse first symptoms. Patient with the adult form of GM1-gangliosidosis suffered from walking difficulties and hip pain as first symptoms. Conclusion: The diagnostic delay of patients with GM1-gangliosidosis may be caused by the broad variability of the clinical course of the disease, especially in the attenuated forms. ß-galactosidase deficiency should be considered in all patients presenting with progressive neurodegeneration and spastic-dystonic movement disorders of unknown origin. Early diagnosis is important as new therapeutic options are being developed.
Acid Ceramidase Deficiency: The Largest Cohort of Patients Yet Assembled Reveals a Broad Clinical Spectrum and Insights into the Phenotypes of Farber Disease
A. Solyom1, C. Simonaro2, X. He2, E. Schuchman2
1Plexcera Therapeutics LLC, New York
2Icahn School of Medicine at Mt. Sinai, New York
Purpose: We present new information on the Farber disease phenotypes associated with acid ceramidase deficiency. Mutations in the ASAH1 gene lead to acid ceramidase deficiency, the resultant accumulation of the lipid ceramide, and a distinct variety of disease phenotypes, culminating in two recognized diseases: Farber disease and Spinal Muscular Atrophy with Progressive Myoclonic Epilepsy (SMA-PME). Farber disease represents a broad clinical spectrum presenting from infancy through late childhood, associated with the pro-inflammatory and pro-apoptotic characteristics of ceramide. SMA-PME, a late childhood onset, primarily neurologic disease, has been less well characterized to date, and the pathophysiologic mechanisms underlying the clinical manifestations are the subject of ongoing study. Methodology: Information was collected from around the world, on the phenotypes of patients with Farber disease cared for by lysosomal storage disease specialists, pediatric rheumatologists and other physicians. When possible, genetic and biochemical data was also recorded. Results: Our findings, based on a cohort of 40 patients, reinforce the validity of the characteristic symptoms of Farber disease: early-onset polyarticular arthritis, subcutaneous nodules and dysphonia. However, it also reveals that there are patients who present with only one or two of these symptoms, and that the spectrum of disease includes remarkably attenuated forms with relatively little associated disability. 52% of patients were considered to have a moderate phenotype, 31% a severe phenotype and 17% a mild or attenuated phenotype. Over 50% of patients were initially diagnosed with some form of childhood inflammatory joint disease, such as juvenile idiopathic arthritis. Conclusion: The prevalence of Farber disease is currently unknown, but it is likely underdiagnosed do to a lack of awareness of the disease itself and the broad spectrum of associated phenotypes. We feel there is an indication for the diligent screening of certain pediatric (and even young adult) polyarticular arthritis patients for Farber disease. The initiation of such studies is underway and a natural history study is planned. Acid ceramidase enzyme therapy is currently under development and is expected to be available for clinical trials in the near future.
Systematic Review and Metanalysis of Enzyme Replacement With Laronidase for MPS I Patients
A. Dornelles1, O. Artigalas2, A. da Silva1, D. Ardila1, T. Pereira3, T. Alegra1, I. Schwartz1
1Hospital de Clínicas de Porto Alegre
2Children's Hospital, Grupo Hospitalar Conceição/GHC, Porto Alegre
3Hospital Alemão Oswaldo Cruz, São Paulo
Introduction: Mucopolysaccharidosis type I (Hurler, Hurler-Scheie and Scheie syndromes)—MPS I—is an autosomal recessive lysosomal storage disorder caused by deficiency of α-L-iduronidase. It has multisystemic involvement and a therapeutic option is enzyme replacement therapy (ERT) with laronidase (0.58mg/kg, IV, weekly). There are only one phase II (Kakkis et al., 2001) and one phase III clinical trials published in the literature (Wraith et al., 2004). The only meta-analysis available (Jameson E, 2013) did not take in consideration the fact that MPS I is a rare disorder, and included only the phase III trial. Purpose: To evaluate the efficacy and safety of ERT with Laronidase for treatment of MPS I patients. Methodology: A systematic literature review was conducted using MEDLINE/PubMed, EMBASE, LILACS, and the Cochrane Library. The search was limited to clinical trials published until March 31, 2015. The inclusion criteria were: randomized controlled trial (RCT), open-label trials, nonrandomized controlled trials, and prospective case series (≥ 5 patients) evaluating relevant outcomes. Relevant outcomes were previously defined by survival, quality of life, cost-effectiveness, clinical outcomes, and adverse events (AE). The systematic review was conducted according to the method proposed by the Cochrane Collaboration and will be reported in accordance with PRISMA guidelines. The systematic review has been registered at PROSPERO (CRD42015032570). Results: Our search identified 600 articles; 381 were excluded after reviewing the abstract. We reviewed the full-texts of 153 articles. Twenty met inclusion criteria, but only 7 were included in the quantitative synthesis. Among the 7 studies with complete data for meta-analysis, only two studies were RCTs; 2/7 evaluated the six-minute-walking test; 2/7 evaluated the liver volume; 5/7 evaluated adverse effects; 3/7 evaluated shoulder flexion; and 3/7 evaluated urinary GAGs. Conclusions: Our results suggest, as expected for rare disorders, that: 1) there are few studies on this issue published in the literature; 2) available studies display substantial methodological diversity, preventing most variables from being meta-analyzed. With the advancement of technology, new treatment options are emerging, including those aimed at rare diseases. We put forward the notion that, particularly for rare diseases such as MPS1, the combination of multiple sources of information (e.g. observational and randomized studies) may be crucial not only for a more robust assessment of new and existing interventions, but also to stratify those patients who are more likely to benefit from the treatment.
Management of Infusion-Related Hypersensitivity Reactions to Enzyme Replacement Therapy for Mucopolysaccharidosis
C. Aranda1, L. Ensina1, C. Mendes1, D. Solé1, A. Martins1
1Universidade Federal de São Paulo
Purpose: Enzyme replacement therapy (ERT) has been used in the treatment of mucopolysaccharidosis (MPS). ERT with human recombinant enzymes has shown to delay disease progression and improve the quality of life. Infusion-related reactions (IRR) to ERT can occur and be severe, including hypersensitivity reactions (HSRs) such as anaphylaxis. We have used a standardized rapid desensitization protocol for achieving temporary tolerance to ERT. In this study we evaluate the safety of this protocol in MPS Brazilian patients. Methodology: MPS patients under ERT in 4 centers from Brazil were assessed from January 2011 through January 2016. In the presence of suggestive symptoms of an adverse reaction, ERT was stopped and skin tests (ST) were performed. In patients with symptoms of acute infection after infusion and with negative ST, ERT was maintained at the same rate. In patients without a history of infection and with negative tests, ERT was maintained with an increased infusion rate. For patients with positive ST and for those patients who continue to react after the adjustments in the infusion rate, a 3 bags 12-steps desensitization protocol was generated. Results: Six patients presented a suggestive HSR, during treatment with laronidase (n = 3), galsulfase (n = 2), idursulfase (n = 1). Urticaria was the most common symptom observed (50%), followed by fever (20%), cough (20%) and anaphylaxis (10%). One of the patients, beyond the normally used premedications, required omalizumab. Of the 460 desensitization protocol performed, 20% induced mild reactions, but all patients received their full target dose. No severe, life-threatening HSRs or deaths occurred during the procedure. Conclusion: Considering the importance of ERT in the treatment of MPS, a standardized diagnostic protocol for IRR allows us to establish a correct diagnosis and propose an efficient treatment algorithm, which includes infusion rate modification and desensitization.
to show the 12 MPSII Patient Cohorts.
age
Total Infusion time
access
comments
1
29
3-4hrs
cannula
Reactions++
2
27
90mins
cannula
3
26
1hr
cannula
4
27
2hr
cannula
5
24
90mins
cannula
6
22
90mins
cannula
7
25
2hrs
cannula
8
18
3hrs
cannula
9
21
90mins
cannula
10
21
1hr
cannula
11
20
2hrs
cannula
Port not active
12
22
2hrs
cannula
A Review of the Total Times for the Administration of ERT for MPS1 MPSII & MPSVI, Together With a Review of Compliance to Home Infusions
L. Thompson1, C. Hendriksz1, M. McLoughlin1, B. McNelly1
1The Mark Holland Metabolic Unit, Manchester, Salford Royal NHS Foundation Trust, Manchester, ENGLAND United Kingdom
Purpose: to review the experience of this cohort of patients and what is the minimum time these therapies can be administered in combined with a review of home infusions and barriers to compliance Methodology: we looked at our cohort of patients on enzyme replacement therapy. 13 MPS1, 12 MPS II and 5 MPSVI. We looked at the administration times for each and reviewed the infusion guidelines. Once established all patients transferred to home care. We reviewed the reasons around missed infusions when on home care. Results: Although these are not licensed times for infusion in our experience Elaprase can be safely administered in 90 mins. Aldurazyme can be safely administered in 150 mins and Naglazyme 180 mins.
Conclusion: Compliance to home infusions was very good and patients missed when hospitalized, poorly or due to access problems. This and the clinical outcome measures will be reported in poster with greater detail. Young people have very busy lives and when a clinician is faced with the option of infusing an ERT at unlicensed infusion rate compared to stop ERT this issue raises many issues. Further work is needed to review risks and benefits or not of rapid infusions in both short and long term. Explore management of infusion related reactions and its correlation with the rate of infusion and pre medications. Find new IV access routes for adults with difficulties for home access.
The Review of the Management of Port-A-Caths in Adult Patients With Lysosomal Storage Disorders Receiving Enzyme Replacement Therapy
M. McLoughlin1, B. McNelly1, L. Thompson1, C. Hendriksz1, K. Stepien1
1The Mark Holland Metabolic Unit, Salford
Purpose: Patients with Lysosomal Storage Diseases (LSDs) often require recurrent courses of Enzyme Replacement Therapy (ERT). Over time these patients may lose peripheral access, and indwelling venous catheters are used. There are currently no available guidelines on the management of port-a-caths (also called Totally Implantable Vascular Access Devices, TIVAD) in patients with Inherited Metabolic Disorders. Furthermore, port-a-caths are not frequently used in adult patients with these conditions and there is also no clear policy in place for the current homecare companies that are managing these devices in patients on ERT. We aimed to review patients with port-a-caths in situ and determine any risks associated with them. This was to enable patients with LSDs to be fully informed of the risks prior to placement of these devices. Methodology: We reviewed patients that are using port-a-caths and are also currently attending our Adult Inherited Metabolic Disorders clinics at the Salford Royal NHS Foundation Trust (SRFT). Demographic information was obtained along with the type of catheter and duration of use, type and number of complications, and the use of anticoagulant medication. We explored how many port-a-caths each patient had and how long each one lasted. We also reviewed the missed home infusion reports for the patients who have active ports. Results: Among 229 of patients who were treated with ERT, 15 patients (6.55%) had a port-a-cath inserted due to poor venous access. Of the 15 patient, 9 were known to have Mucopolysaccharidosis, (3 type I, 5 type II and 1 type VI) 3 patients had Fabry disease, 1 patient had Gaucher disease and 2 patients had Pompe disease. All 15 patients had port-a-caths inserted in their childhood. Importantly, in most cases the management of the port-a-cath was not handed over during the transition from Children’s Hospital to the Adult Inherited Metabolic Medicine Centre and there was no supporting documentation available. Of the 15 patients, 3 were on their first port, 5 were using their second port and 3 patients were unsure how many devices they had throughout their ERT period or when they were inserted. The remaining 4 patients had inactive port-a-caths. The majority of patients with active port-a-caths never missed more than one consecutive infusion, although one patient missed 2 consecutive infusions whilst on holiday. We identified significant gaps in patients’ and their families’ understanding of the management of port-a-caths and risks associated with them. Conclusion: The management of long-term venous catheters in adult patients with Inherited Metabolic Disorders requires particular care to avoid complications. The risk could be reduced by improving the level of documentation during the transition process from Children’s Hospital to the Adult Inherited Metabolic Disorders unit. As a result, we hope to produce local guidelines on the management of port-a-caths in patients on ERT and risks associated with them. This should include alternative methods of long-term venous access in those patients and frequency of them being flushed when unused. A standard approach to removing a port-a-cath needs to be established to ensure safe removal of these devices in our patients. Finally, the education of patients and their families is essential to port-a-cath management when ERT is administered at home.
Enzyme Replacement Therapy (ERT) in 27 Brazilian Patients With Mucopolysaccharidosis (MPS) Types I, II and VI
J. F. S. Franco1, D. C. Q. Soares1, L. M. J. Albano1, R. S. Honjo1, D. R. Bertola1, G. N. Leal1, M. T. Cunha1, A. C. Kim1
1Instituto da Criança, São Paulo
Mucopolysaccharidosis (MPS) are multisystemic disorders caused by lysosomal enzyme deficiencies. Currently there are specific treatments with for MPS I, II, IV and VI. ERT is effective in the control of some of the clinical manifestations. Objective: The aim of this study is to report the clinical evaluation before, during and after ERT in MPS I, II, and VI. Methods: We have studied 27 MPS patients (13 MPS I; 8 MPS II; 6 MPS VI) diagnosed by enzymatic and urinary GAGs analysis, followed in three reference centers for ERT. Results: First symptoms appeared at about 6mo to 8y (MPS IH—mean 7mo; MPS IHS—mean 2y; MPS IS—mean 6y10mo; MPS II—mean 3y6mo; MPS VI—mean 1y). The mean ages at diagnosis were: MPS IH—1y6mo; MPS IHS—4y8mo; MPS IS—13y7mo; MPS II and VI—5y. There were five familial cases. The age of onset of ERT ranged from 1y2mo to 31y9mo. The duration of ERT ranged from 40wk-556wk (mean 259wk). Adverse infusion reactions were reported in 55% (15/27) of the patients: skin rash, high blood pressure, fever, and bronchospasm that improved with antihistamines, antipyretics and/or reduction of infusion rate. Severe reactions were noticed in two patients. Before ERT, the main clinical complications were high blood pressure (25%), hypoacusis (37%), and hydrocephalus (15%). After ERT these same findings worsened: 37%, 59%, and 22%, respectively. Among 27 patients, 22 (81%) were submitted to surgical procedures, with a mean of 2.9 surgeries/patient. Five patients presented anesthetic complications (23%) and two of them deceased during the procedures due to orotracheal intubation. The percentage of missed ERT infusions ranged from 0%-32% (mean 17.8%) due to: not available enzyme (problems with purchase by the government), holydays, clinical problems, such as upper respiratory tract infections, hospitalizations, surgical procedures, and parents/caregiver problems. Six patients died (22%), although they had received ERT for a minimum period of 136wk (2.6y) and a maximum of 317wk (6y). Conclusions: ERT ameliorates some clinical findings, but it was not able to stop the progression of the disease that remains with a high mortality rate. Early diagnosis and ERT are critical for a better outcome and for enhancing the quality of life of these patients and their families.
Home-Based Therapy for Children and Adults With Mucopolysaccharidosis I, II and VI in the Netherlands
J. Hardon1, T. Oskam1, A. van der Ploeg1
1ErasmusMC, Rotterdam
Purpose: To share our experiences with weekly enzyme replacement therapy at home or at school for children and adults with mucopolysaccharidosis and their families. Introduction: Enzyme replacement therapy (ERT) was registered as treatment for mucopolysaccharidosis (MPS) I, II and VI in 2007 Currently our expertise center takes care of 33 MPS I, II and VI patients, of whom 24 children and nine adults on ERT. Patients travel between nine and 253 km to receive therapy in the hospital, which is very time-consuming and exhausting for these patients. It is preferable that ERT can be implemented in the patients daily life, both at home and in school. Therefore our center provides home infusion therapy. We evaluated the safety, compliance and comfort of home-based therapy in the Netherlands. Methods: Since 2007 patients receive ERT at home assisted by local home care teams or parents (the latter at their specific request). Home based therapy occurs under responsibility of the expert center ErasmusMC Rotterdam. Training days are organized for home care nurses to instruct them on the application of ERT and to learn more about MPS. Both parents and nurses are trained and certified for use of the venous-port catheter to infuse ERT. During the first six months of ERT infusions with ERT are given within our outpatient clinic in the hospital. Patients are monitored closely for potential infusion reactions. All children receive premedication, which is stopped in week 28 of treatment. At week 29 patients are transmitted to home-based ERT with a specialized home care team. The first infusion at home is supervised by a specialized nurse of our center. During ERT the home-care nurses keep a log on vital signs and details of each infusion and potential Infusion Associated Reactions (IAR’s). These logs are checked by our center nurses. If an IAR occurs at home, a nurse and a medical specialist are available for consultation. For safety reasons the following ERT application is then given within the hospital. After one year ERT at home, children over the age of six years may get there ERT at school, with the home care nurse in or just out of the classroom. Results: Currently 24 children and nine adults receive ERT at home, five of these children also receive ERT at school. On a yearly basis approximately 1600 infusions are given at home in the population with MPS. In six years we were contacted for problems on access to the venous-port-catheter or to the veins, subcutaneous infusions, problems with the ambulatory device and fever or malaise of the patient. IAR’s appeared in two children, one with a mild rash after fever the day before infusion and the other patient had three venous-port-catheter infections. These infections were treated with antibiotics at home. No IAR’s were noticed in adult patients. The observed complications in the home situation are infrequent and do not differ from those experienced in the hospital. The hospital is contacted by the home-care-teams about twice a week. Conclusion: Home treatment with ERT at home is safe, comfortable and patients and their families are very satisfied with this opportunity. They have more time and energy left for daily activities.
Home Treatment With Aldurazyme in Italy. The Experience in the Italian Regions Allowing This Procedure
R. Parini1, M. Di Rocco2, C. Galimberti1, S. Gasperini1, F. Menni3, F. Papadia4, M. Rigoldi5
1Fondazione MBBM- San Gerardo Hospital, Monza
2Gaslini Institute, Genova
3Fondazione Policlinico, Milano
4Ospedale Pediatrico Giovanni XXIII, Bari
5San Gerardo Hospital, Monza
Introduction: Home treatment with intravenous enzyme replacement therapy (ERT) has been reported to be safe and successful for Gaucher and Fabry disease and for Mucopolysaccharidosis type I and II. It is a common practice mainly in Northern Europe where more than 50% of patients are home treated at present. In Countries where it is not feasible through the Public Health Service it is generally economically supported by the Company producing the drug through contracts with providers in respect of patient’s privacy and all the other limitations related to “conflict of interest”. In Italy this practice has been developed slowly, although many patients were favorable, since in some Regions there has been the willing for years to organize the whole service through the Health Care System, however without reaching satisfactory results. We report here our positive experience with MPS I patients treated at home with ERT. Methods and Results: Aldurazyme was infused at home at a dosage of 100U/kg/week to 7 MPS I (2 Scheie, 3 Hurler-Scheie, 2 Hurler) patients from three different centers, in the last 5 years. The provider (Allianz Global Assitance, sponsored by Sanofi Genzyme) organized the pick up of the drug at the hospital ensuring a safe transport and guaranteed the correct treatment and the assistance during the entire period of infusion and one hour immediately after. Patients are at present 3-42 (mean 18, median 11) years old and are home treated since 16-59 months (mean 39, median 44). Before starting home treatment they were all treated in the hospital for 6 months or longer and had had no reactions for at least 6 months before. One of them, a 37 year old man, while still treated in hospital, had drug related adverse reactions like severe urticaria and dyspnea successfully managed with antihistaminics (chlorphenamine maleate and later hoxatomide hydrate), cortisonics (hydrocortisone or methylprednisolone) and temporary reduction of the dosage. The management was successful and he could be sent home after 6 months without reactions at full dosage. Aldurazyme infusions at home were 68-248 per subject with a total of 1117 administrations and a compliance of 95% (range 88-100%, mean 95%, median 96%). No adverse reactions related to the drug were registered during home treatment. The patients and their families reported essential benefits derived from the ERT home-setting because, they said, it allows the patient having more independence and control of the disease and, reducing the time spent in hospital and for the travels to and from the hospital, they gain time for their private life. Furthermore, all the patients and their families said that they liked the particular personal relationship established week by week with the same nurse going to their home. From the point of view of the clinical teams it also allowed to release hospital resources for alternative use by other patients, thereby improving the efficiency of the hospital services. Conclusions: Considering our positive experience we hope that the number of MPS I patients shifted to home treatment will increase in Italy in the next years with the probable result of improving their quality of life.
References
- Cox-Brinkman J, Timmermans RG, Wijburg FA, et al. Home treatment with enzyme replacement therapy is feasible and safe. J Inherit Metab Dis. 2007;30(6):984.
- Hughes DA, Mlilligan A, Mehta A. Home therapy for lysosomal storage disorders. Br J Nurs. 2007, 2008;16(22):1384, 1386-1389.
- Parini R, Pozzi K, Di Mauro S, Furlan F, Rigoldi M. Intravenous enzyme replacement therapy: hospital vs home. Br J Nurs. 2010;19(14):892-894, 896-898.
Home Infusion Therapy for Patients Living With Mucopolysaccharidosis Type I (MPSI) in Canada
M. Mackrell1, M. Inbar-Feigenberg1, J. Gilles2, A. Khan3
1Sickkids Hospital, Toronto
2British Columbia Children’s Hospital, Vancouver
3Alberta Children’s Hospital, Calgary
Purpose: To describe the Canadian Experience with home infusion therapy for patients receiving weekly infusion of Laronidase. In Canada, with a land area of more than 9 million square kilometers, the availability of home enzyme infusions since January 2011 provide a more convenient and economical alternative to institutional infusions. Methodology: We did an informal survey of metabolic centers administering enzyme infusion for type I mucopolysaccharidosis comparing home versus institutional enzyme infusion therapy. Three centers (Toronto, Calgary, and Halifax) responded. Results: Data on travel distance was available on 11 patients and showed the mean distance of the home residence from the medical center was 166 km and the furthest patient was 642 km away. All patients had the option of home or institutional infusion. Available data from 10 patients 2369 prescribed home infusions showed 2269 infusions (95.8%) were successfully completed. No severe reactions adverse events were recorded. The estimated costs of an institutional enzyme infusion ranged from $ 375 to $ 442 per day. The cost of a home infusion was estimated at $ 178 per day. Patients and their families reported that home infusion required less time, reduced travel, and provided cost savings compared to institutional infusions. Conclusions: This preliminary survey showed that home infusion therapy with Laronidase has proven to be a safe, efficient and cost effective option for Canadian patients with acceptable compliance rates comparable to those of hospital infusions.
Home Therapy With Idursulfase in Italian Patients With Mucopolysaccharidosis II (MPSII)
S. Sestito1, F. Brambillasca1, M. Battistini1, L. Dizione1, G. Belletti1, B. Bembi2, D. Concolino3, F. Falvo3, F. Deodato4, S. Fecarotta5, G. Parenti5, P. Strisciuglio5, A. Fiumara6, R. Parini7, P. Pilia8, F. Lilliu8, G. Zampino9, M. Cellini10, A. Tagliaferri1, T. Marcacci1
1Caregiving Italia srl, Milano
2University Hospital Santa Maria della Misericordia, Udine
3University Magna Graecia, Catanzaro
4Children’s Hospital Bambin Gesù, Roma
5University Hospital Federico II, Naples
6University Hospital Policlinico Vittorio Emanuele, Catania
7San Gerardo Hospital, Monza
8University Hospital of Microcytic Anemia, Cagliari
9Sacro Cuore University, Roma
10University Hospital - Policlinico, Modena
Purpose: Enzyme replacement therapy (ERT) with Idursulfase, commercially available since 2007, has been proven to be effective in improving some of the clinical manifestations of Mucopolysaccharidosis II (MPSII). The treatment, however, involving weekly intravenous infusions over a period of 3-4 hours, is onerous for both patients and their families. Since 2010, in Italy, a homecare therapy is possible for selected patients. We evaluated the feasibility and safety of home therapy in Italian patients with MPSII, and improvement in patients’ and their families’ quality of life, resulting from transition to home care. Methodology: all patients were allowed to transfer to home treatment if they showed stable disease, had experienced a minimum period of 6 months of in-hospital administration of ERT, and had not reported adverse drug reactions during the last 3 months of therapy. Each patient was assigned to a specific nurse who had received adequate training in lysosomal storage disorders, in particular regards to MPS II, had experience with enzyme infusion treatment and was also able to recognize and manage any possible adverse event, having specific competence in pediatric and adult basic life support (PBLSD and BLSD). Compliance to ERT was assessed and the number of infusions missed during home treatment was compared to those missed during hospital management. The same was for the number of adverse events occurred during the two different periods. In order to assess how and if home transition resulted in any improvement on patients’ and their families’ quality of life, KIDSCREEN-27 questionnaires translated and adapted by experienced psychologists have been administered to both patients and/or their relatives. Results: 20 Italian patients with MPSII started home therapy after an average of 58 months of Idursulfase. 25% (5/20) of patients were under 5 years of age when starting home therapy. Age range of the other patients was 6 to 45 years with an average of 13.5 years. All patients showed typical disease manifestations; over two-third (14/20) had cognitive impairment, in nine cases severe; one patient had tracheostomy due to a worsening of respiratory involvement. For two out of the patients ERT was administered by vein catheter, central for one patient, peripheral for the other. After a mean of 27 months of home treatment (2078 infusions administered) we observed that four infusion-related reactions occurred in two out of the twenty patients who had received home therapy, compared to twelve reactions in seven patients reported during hospital management. All reactions occurred at home were mild in severity and easily managed by the nurse. Only one patient stopped home therapy because he died at age of 18 years for septic complications of endocarditis. Compliance to ERT reached 99.5% after transition to home therapy compared to a mean of 91% at hospital for the thirteen patients for which this data was available. Based on questionnaires administered we can show an improvement in the way to experience ERT and to cope with the burdens of the disease for both patients and their relatives. Conclusion: Home treatment with Idursulfase should be considered feasible and safe for patients with MPS II, including those who are severely affected so as those patients with a very young age. An almost perfect compliance with the treatment schedule was observed after transition, so as an improvement in quality of life for both patients and their families, suggesting that home care might be able to alleviate the burden of life-long intravenous treatment in these patients.
Pregnancy in a Patient with Mucopolysaccharidosis Type I (MPS I) Treated with Enzyme Replacement Therapy—Case Report
C. de Souza1, A. Silva1, M. Sanseverino1, F. Vairo1, S. Fagondes1, D. Manica1, J. Magalhaes1, R. Giugliani1
1Hospital de Clinicas de Porto Alegre
Objective: Describe the case of a MPS I Scheie patient in her first pregnancy, treated with laronidase, every other week, in double dose during throughout pregnancy. Methods: Chart review and description of gestational and postnatal follow-up data. Results: The mother is a 28-year-old with Scheie MPS I (genotype R89Q/W402X), no consanguineous couple. The patient remained in ERT every other week and monitored by a multidisciplinary team throughout pregnancy (including obstetrician, pulmonologist, cardiologist, otorhinolaryngologist, neurosurgeon, geneticist, fetal medicine group and nutritionist). On physical examination, the patient had height = 136 cm. Echocardiogram shows mild to moderate valve insufficiency and espirometry suggest moderate restrictive respiratory disorder. Morphological ultrasound and fetal echocardiogram were normal. A healthy 1,7 kg female was delivered by cesarean section at 31 weeks. There are no complications or congenital malformations. No adverse event related to ERT was reported. The IDUA enzymatic dosage of the baby was normal. Conclusions: There are still few reports of pregnancies in patients with MPS. No teratogenic effects are expected of ERT. Reports of pregnancies treated with ERT available for other lysosomal diseases also showed pregnancies without adverse events to the fetus. The strict monitoring of patients for possible complications is important and multidisciplinary management was critical to the successful outcome of this case.
Growth Patterns in Attenuated MPS I Patients Started ERT in Childhood or Not: 4 Clinical Cases
N. GUFFON1, D. AZZI1, A. Fouilhoux1
1Hôpital Femme Mère Enfant, LYON
Purpose: Short stature and growth retardation are very important features in MPS I patients, but to date, the growth profiles of patients with attenuated MPS I (Hurler-Scheie HS and Scheie S phenotypes) have not been described. In this study, we recorded the growth curve evolution from birth to adulthood in 4 female MPS I patients; two (Hurler-Scheie, 42 years old and Scheie, 35 years old) were naïve to replacement therapy (ERT) during childhood and received treatment in adulthood, and for the other two patients (both Hurler-Scheie, both 18 years old of aged) ERT was initiated during childhood. Methodology: Data on height measurements from birth to adulthood were retrospectively collected from all 4 patients’ files. Growth curves were generated and compared to the healthy population and to patient’s clinical status. Results: For the two patients who received ERT in the adulthood period only (at 21 years old for the S patient and 30 years old for the HS patient), growth was normal or near normal during the first year of life, then progressively declined to −3 SD at 17 years old for the S patient (height 146.5 cm) and −4 SD at 10 years old for the HS patient (adult height 133 cm). For the two patients for whom ERT was initiated in childhood (at 10 years old for one and 5years old and 8 months for the second) the initial growth follows the same trend during birth to childhood, but the final height was higher (155 cm and 156.5 cm) than for the patients who did not have early ERT initiation. There was a pubertal delay in the females who initiated ERT later in life and their gain in growth during puberty was very low compared to the females who were treated earlier (2 and 3 cm) and proceeded with normal puberty. It appears that the final height was greater when ERT is initiated earlier and before the puberty period. Conclusion: The cases presented here show that growth of attenuated MPS I patients appears normal or near normal between 1 to 3 years of age then progressively decreases until puberty where the peak of growth is very modest. Starting ERT before puberty appears to be beneficial on the height of patients with MPS I.
Does Enzyme Replacement Therapy or Bone Marrow Transplantation Improve Corneal Clouding in MPS?
A. Javed1, J. Ashworth1, T. Aslam1
1Manchester Eye Hospital, Manchester
Purpose: To determine if corneal clouding improves over time in patients treated with enzyme replacement therapy (ERT) or following bone marrow transplantation (BMT). Methodology: Patients with mucopolysaccharidoses (MPS) types I and IV who attended specialist pediatric ophthalmology clinics at Manchester Royal Eye Hospital were recruited. Corneal opacification was measured with the iris recognition camera (Irisguard model IG-AD100®, Irisguard Ltd, Buckinghamshire, UK) to determine the objective corneal opacification measure (COM) score over time. In addition, all patients had extensive ophthalmic examination performed including digital imaging of cornea, anterior segment and retina. The corneal opacity (COM) score was measured using software developed and previously validated to quantify corneal opacity objectively. Results: We report on 10 eyes from 5 patients with MPS types I and IV; in 4 of the 5 patients corneal opacity score worsened, and in 1 patient there was an improvement during mean follow-up of 60 +/− 4 months. Patients 1, 2 and 3 all had higher corneal opacity scores over time in the right eye indicating worsening of corneal opacity. Patient 4 had a significantly higher COM score in both eyes over the follow-up period. One patient 5 had an improvement in his COM score in both eyes from a score of 14 to 4.8 in the left eye and 18 to 11.2 in the right. Patients 1-4 were on ERT and patient 5 had received BMT. Conclusion: This is the first time that change in corneal clouding over time has been objectively measured in MPS patients; previous studies have used subjective grading or unreliable imaging to assess corneal clouding. COM score measurements show no improvement in corneal clouding over time in patients on ERT, but demonstrate improvement in the patient who had a previous BMT.
References
PitzSOgunOArashLMiebachEBeckM. Does enzyme replacement therapy influence the ocular changes in type VI mucopolysaccharidosis?Graefes Arch Clin Exp Ophthalmol. 2009;247(7):975–980.AslamTMShakirSWongJAuLAshworthJ. Use of iris recognition camera technology for the quantification of corneal opacification in mucopolysaccharidoses. Br J Ophthalmol. 2012;96(12):1466–1468.
Preliminary Results of a Phase I/II Safety and Dose-Finding Study of AGT-182 in Adult Patients with Mucopolysaccharidosis II (Hunter syndrome)
J. Enriquez1, P. Rioux1
1ArmaGen, Calabasas
Purpose: To investigate the 1.0 mg/kg dose of AGT-182 in patients with Mucopolysaccharidosis Type II (MPS II), to assess the safety and tolerability, and to evaluate the biologic effect of AGT-182. AGT-182 is a genetically engineered fusion protein comprised of a chimeric monoclonal antibody (MAb) to the human insulin receptor (HIR) and a recombinant human iduronate 2-sulphatase (IDS), or is alternatively designated as the HIRMAb-IDS fusion protein. Methods: In this open label Phase 1/2 study, adult patients with MPS II were enrolled. AGT-182 was administered via an IV route given weekly for 8 weeks. Samples were collected for PK, antibody, and biomarkers (glycosaminoglycans [GAGs]). Plasma AGT-182 and serum antibody levels were assessed by ELISA. Urinary heparan sulfate and dermatan sulfate were assessed using an LC/MS assay. Total urinary GAGs were assessed using a dye-binding assay and were normalized to urine creatinine. Safety, including infusion reactions and blood glucose during and after IV infusion, was monitored throughout. MRI scans of the liver and spleen volumes were obtained. Results: Five (5) patients were enrolled and treated in Cohort 1 at 1mg/kg: Two (2) patients completed 8 doses One (1) patient withdrew after 5 doses due to worsening of a pre-existing asthma One (1) patient withdrew after 4 doses due to personal reasons One (1) patient withdrew after 1 dose due to personal reasons and was subsequently replaced. Liver and Spleen Volume Liver (Figure 1) and spleen (Figure 2) volumes were measured via MRI scans. Marked volume increases were observed for patients during the washout period, and volume decreases were observed after AGT-182 infusion. Patient 07-001 liver and spleen volumes decreased at Week 4 when compared to Baseline. However, the liver and spleen volumes increased when measured at Week 8, with no clear explanation for the moment. Urinary GAGs During untreated periods (washout and discontinuation), uGAGs show marked increases (Figure 3). During treated period, uGAGs showed a decrease. Patient 13-001 was ERT-naïve and, thus, did not participate in the 6-Week washout period. Furthermore, this patient was treated for 5 weeks prior to withdrawing the consent. Safety Blood glucose level was monitored at regular intervals using a glucometer (Figures 4 and 5). None of these glycemic changes were considered as clinically significant by the Investigators. Transient blood glucose levels of less than 70mg/dL were observed in all patients and were managed with a light snack. In addition to hypoglycemia, AEs were unlikely related to AGT-182 (Mild bilateral leg cramps, bilateral leg pain, Sleep disturbances, Hip pain, Decreased endurance, Fatigue, Maculopapular abdominal rash, Emesis prior to dose start, Cough, Headache, Back pain, Neck Pain, and Migraine) or possibly related to AGT-182 (Hypoglycemia, Anxiety, Headache, Throat itchiness, Light headedness, Nausea, Jitters, Nausea, Clammy hands, Weak) as also seen while under treatment with Elaprase. Conclusions: Weekly IV infusion of AGT-182 was well tolerated and appeared to have a similar somatic effect as well as safety profile as Elaprase®. Per an independent Data Monitoring Committee (DMC) review of the available safety, clinical, and bioanalytical data, the DMC recommended to proceed to Cohort 2 at 3mg/kg.
Design and Rationale of the Clinical Study Programs for BMN 250, a Novel Investigational Enzyme Replacement Therapy (ERT) for Sanfilippo B Syndrome
A. Shaywitz1, M. Oh1, S. Kent1
1BioMarin Pharmaceutical Inc., Novato
Sanfilippo B syndrome (mucopolysaccharidosis IIIB; MPS IIIB) is a devastating lysosomal storage disease characterized by rapid and progressive neurological decline, due to deficiency of the human alpha-N-acetylglucosaminidase (NAGLU) enzyme. Children affected with the severe form of disease are initially symptom-free but between ages 1-4 years begin to manifest developmental delay, progressing to severe behavioral problems, sleep-wake disturbance, and intellectual decline. Severe decline of all motor functions ensues and death usually occurs in the second or third decade of life. BMN 250 is a novel enzyme replacement therapy (ERT) designed to restore functional NAGLU activity to the brain. To determine whether the effects of treatment are clinically meaningful, it is critical to understand the nature of MPS IIIB disease progression, and in particular cognitive decline, in untreated patients. Development quotient (DQ) derived from scores on either one of two cognitive tests (BSID or KABC) has been validated as a cognitive measure in children with MPS IIIA. While studies have demonstrated marked changes in DQ in younger MPS IIIA patients, information regarding DQ trajectory in young MPS IIIB patients is scarce. BMN 250-901 (www.clinicaltrials.gov; NCT02493998) is an observational study designed to quantify the progression of MPS IIIB over time in affected children primarily aged 1-5 years at baseline and to correlate changes in clinical features of the disease, in particular changes in DQ, with both MRI characteristics and biochemical markers of disease burden. Concurrently, a treatment study (BMN 250-201) is also planned using BMN 250. Part 1 of this treatment study is a dose-escalation period to establish safety and Part 2 is a dose-expansion period. Eligible patients from the 250-901 baseline observational study may be able to roll over into Part 2. Efficacy will be assessed by comparing changes in disease progression in the observational study vs changes observed in Part 2 of the treatment study.
Initial, 24 Week Results of Heparan Sulfate Levels in Cerebrospinal Fluid (CSF) and Serum, Brain Structural MRI and Neurocognitive Evaluations in a Phase I/II, First-in-Human Clinical Trial of Intravenous SBC-103 in Mucopolysaccharidosis IIIB
S. Rojas-Caro1, C. Whitley2, M. Escolar3, S. Vijay4, G. Parker5, M. Leavitt1, A. Rossomando1, X. Zhang1, A. Cinar1, K. Patki1
1Alexion Pharmaceuticals, Inc., New Haven
2Department of Pediatrics, University of Minnesota, Minneapolis
3Children’s Hospital of Pittsburgh
4Birmingham Children’s Hospital, Birmingham
5Bioxydyn Limited, Manchester
Purpose: Mucopolysaccharidosis IIIB (MPS IIIB; Sanfilippo syndrome type B) is caused by a marked decrease in alpha-N-acetyl-glucosaminidase (NAGLU) enzyme activity which leads to the accumulation of heparan sulfate (HS) in the brain and other organs, progressive brain atrophy, neurocognitive decline and behavioral disturbances. At present there is no treatment for this disorder. We report initial results from the ongoing first-in-human trial (NCT02324049) of recombinant human NAGLU enzyme (SBC-103) after 24 weeks of intravenous (IV) therapy. Methodology: This was an open label study with 3 parallel dose groups. SBC-103 (0.3 mg/kg (n = 3), 1 mg/kg (n = 4), and 3 mg/kg (n = 4)) was administered as an IV infusion every other week in patients aged 2-12 years (developmental age ≥1 year) for 24 weeks. Primary objective: assessment of safety and tolerability of IV SBC-103. Secondary objectives: effect of SBC-103 on total HS levels in cerebrospinal fluid (CSF) and serum, brain structures (MRI) and neurocognitive status, and pharmacokinetic (PK) profile of SBC-103. Results: Eleven patients were enrolled (median age 4 years; range 2-10 years) and received IV SBC-103. During the 24 weeks, there were 3 treatment-emergent serious adverse events (SAEs) in a single patient and 6 infusion-associated reactions in 3 patients. No SAEs were considered related to SBC-103. At week 24, total HS percent change from baseline (mean [SD]) in the CSF was 10.9 [14.7], −0.43 [11.9] and −26.2 [20.9] for 0.3 mg/kg, 1 mg/kg, and 3 mg/kg groups, respectively. Total HS percent change from baseline (mean [SD]) in the serum was −39.6 [15.4], −53.9 [19.7] and −40.5 [23.9] for 0.3 mg/kg, 1 mg/kg, and 3 mg/kg groups, respectively. HS reduction in CSF was linearly correlated with SBC-103 serum PK exposures (maximum concentration [Cmax] and area under the curve [AUC]). Preliminary volumetric MRI (cortical gray matter volume) and neurocognitive outcomes (age equivalent/developmental quotient) suggested potential for disease stabilization in subjects receiving the 3 mg/kg QOW for 24 weeks compared to their baseline. Overall, response profiles among the 3 mg/kg treatment groups suggest a potential dose effect as compared to the 0.3 mg/kg and 1 mg/kg groups. Conclusions: These initial observations suggest that IV SBC-103 was biologically active and well tolerated in MPS IIIB patients. Potential blood brain barrier penetration of IV SBC-103 was illustrated by PK/PD correlation for HS reduction in CSF. Preliminary evidence of disease stabilization in pharmacodynamics (PD)/efficacy was suggested by changes in brain structures and neurocognitive assessments after 24 weeks of IV SBC-103 in some patients. These data support plans to test higher doses in the study.
Impact of Elosulfase Alfa in Patients with Morquio Syndrome Type A Who have Limited Ambulation: An Open-Label, Phase 2 Study
P. Harmatz1, A. Jester2, E. Mengel3, M. Treadwell1, B. Burton4, C. Hendriksz5, T. Geberhiwot6, Z. Sisic7, C. Decker8
1UCSF Benioff Children’s Hospital Oakland
2Birmingham Children’s Hospital, Birmingham
3Mainz Medical University, Mainz
4Lurie Children’s Hospital & NWU Feinberg, Chicago
5Salford Royal Foundation NHS Trust, Salford
6New Queen Elizabeth Hospital, Birmingham
7BioMarin Europe Limited, London
8BioMarin Pharmaceutical Inc, Novato
Purpose: An open-label, phase 2, multinational study was performed to evaluate efficacy and safety of elosulfase alfa enzyme replacement therapy (ERT) in patients with Morquio syndrome type A aged ≥ 5 years who have limited ambulation (inability to walk ≥30 meters in the 6-minute walk test). Methodology: Patients received elosulfase alfa 2.0 mg/kg/week for 48 weeks. Primary efficacy measures were functional dexterity, pinch/grip strength, mobility in a modified timed 25-foot walk (mT25FW, in which patients were allowed to crawl, roll, walk on knees), pain, and quality of life. Safety/tolerability of elosulfase alfa was also assessed. Results: Sixteen patients were enrolled; 15 (9.8-42.4 years) received elosulfase alfa, and 12 completed treatment through 48 weeks. All patients had very poor upper extremity function and mobility. Seven patients were unable or refused (e.g. due to pain) to perform the mT25FW. The mT25FW results improved versus baseline in 6 patients and remained stable in 1 patient; 1 patient refused to repeat the test. Other test outcomes varied considerably between patients. This was most likely due to a variety of factors including clinical heterogeneity among patients, difficulties associated with travel to and from testing sites, and challenges with test execution in this severely affected population. Nevertheless, patients who did not show improvements in physical tests mostly indicated improvements in other disease aspects, such as increased energy or better ability to type, lift a cup, comb hair, or stand up without braces. The nature of adverse events (AEs) was generally similar to other studies with elosulfase alfa, with the majority of subjects experiencing AEs limited to mild or moderate severity. Conclusion: Overall, this study demonstrates the considerable challenges in objectively measuring impact of ERT in this very disabled patient population. However, despite their severe impairments, most patients reported subjective benefits from elosulfase alfa ERT.
Impact of Long-Term Elosulfase Alfa Treatment on Pulmonary Function in Patients With Morquio Syndrome Type A
C. Hendriksz1, B. Burton2, R. Giugliani3, N. Guelbert4, D. Hughes5, J. Mitchell6, R. Parini7, J. Raiman8, A. Shaywitz9, P. Harmatz10, M. AlSayed11, M. Mealiffe9, P. Slasor9, M. Solano Villareal12
1Salford Royal Foundation NHS Trust, Salford
2Lurie Children’s Hospital & NWU Feinberg, Chicago
3Hospital de Clinicas de Porto Alegre
4Hospital de Ninos de Cordoba
5Royal Free London NHS Foundation Trust & UC, London
6Montreal Children’s Hospital, Montreal
7Azienda Ospedaliera San Gerardo, Monza
8Belfast City Hospital, Belfast
9BioMarin Pharmaceutical Inc, Novato
10UCSF Benioff Children’s Hospital Oakland
11King Faisal Specialist Hospital & Research Center, Riyadh
12Fundacion Cardioinfantil, Bogotá
Purpose: We present long-term pulmonary function test outcomes from an ongoing, open-label, multi-center, phase 3 extension study assessing the long-term safety and efficacy of elosulfase alfa enzyme replacement therapy (ERT) in patients with Morquio syndrome type A. Methodology: In part 1 of the extension study, patients who were initially randomized to ERT in the original placebo-controlled 24-week study [1] remained on their regimen (2.0 mg/kg/week or every other week); placebo patients were re randomized to one of the two treatment regimens. During part 2, all patients received ERT 2.0 mg/kg/week. Pulmonary function was evaluated as a secondary efficacy endpoint. Results: Changes from the original 24-week study [1] baseline to 72 and 120 weeks are presented. In the 24-week study, non-statistical increases were seen in each dosing group for forced vital capacity (FVC) and maximum voluntary ventilation (MVV) versus placebo [1] and both endpoints continued to improve for the combined patient population during the extension study for up to 120 weeks. MVV increased from baseline by a mean (SE) of 1.78 (0.74) L/min by week 72 and 1.80 (1.04) L/min or 11.04 (4.55) % by week 120. FVC increased from baseline by a mean (SE) of 0.05 (0.01) L by week 72 and 0.08 (0.02) L or 8.6 (1.8) % by week 120. In contrast, comparable untreated patients from the MorCAP natural history study [2] showed mean decreases in MVV and FVC over 2 years. Conclusion: Ultimately, long-term ERT was associated with continued or sustained improvements in pulmonary function measures in Morquio A patients over 2 years, suggesting that elosulfase alfa slows down the natural progression of pulmonary dysfunction in Morquio A.
New Biomarker Response in Replacement Therapy With Enzyme Elosulfase Alfa for Morquio A Syndrome: Large and Scarce Glicosaminoglycans Granulations in Peripheral Neutrophils
M. Melo1, P. Medeiros2
1Laboratório Marcelo Magalhâes, Recife Pe
2Hospital Universitário Alcides carneiro, Campina Grande
This study aims to demonstrate the association between the enzyme replacement therapy (ERT) with elosulfase alfa for patients with mucopolysaccharidosis (MPS) type IVA or Morquio A syndrome and the removal of inclusions of glycosaminoglycans (GAG), chondroitin-6 sulfate (C6 S) and keratan sulfate (KS) in the form of large and sparse granules in peripheral blood neutrophils observed only in this type of lysosomal storage diseaseThe study involved 13 patients with Morquio A syndrome before and after five months of the beginning of ERT with elosulfase alpha. The patients were treated at the Hospital Alcides Carneiro, Campina Grande, PB, Brazil and the Treatment Center for Inborn Errors of Metabolism IMIP—Pernambuco—Brazil. Blood counts were conducted on all patients before and after five months from the beginning of ERT for neutrophil counts with large and sparse granules in optical microscopy through immersion objective (100X). The tests were conducted in the Laboratory Marcelo Magalhães, Recife—Pernambuco—Brazil. The 13 patients with Morquio A syndrome had an average age of 23.5 years (range 3 years to 38 years), being 6 of them male and 7 female. There were about 56.7% of the neutrophils with inclusions before therapy. After over five months of ERT, a total absence of neutrophils with large and sparse granules was observed, proving biomarker to be a new and effective response to this new therapy
Multi Stakeholder Engagement Leading to Access to Treatment for MPS IVA (Morquio A)—A Model for the Ultra Rare Disease Community
C. Roberts1, C. Lavery1, N. Nicholls2, M. Jain2, C. Hendriksz3, S. Upadhyaya4, E. Jessop5
1The Society for Mucopolysaccharide Diseases (MPS Society), Amersham
2BioMarin Europe Ltd, London
3Salford Royal NHS Foundation Trust, Salford
4National Institute for Health Care Excellence (NICE), London
5National Health Service (NHS) England, London
Objectives: To achieve reimbursement for elosulfase alfa for MPS IVA patients resident in England. Methods: MPS IVA is an ultra-rare disease affecting less than 130 patients in the UK. The only treatment currently available, elosulfase alfa, was licensed by the European Medicines Agency on 28th April 2014. The UK had been a major contributor to the phase III clinical trial with 35 patients being enrolled out of the 176 recruited worldwide. As a consequence there was a high degree of interest and concern in continuing access to treatment in the UK. Changes in the reimbursement decision making process at this time meant that NICE had created a special process for highly specialized technologies (including ultra rare diseases) to replace the previous Advisory Group for National Specialized Services process. Patients together with the patient organization MPS Society UK, members of Parliament and clinicians canvassed NHS England and the Department of Health for a fair process with equal access to therapies as for common disorders. This resulted in elosulfase for MPS IVA being referred to NICE for full evidence review and decision. During the NICE process, the MPS Society suggested a robust procedure whereby all patients that met a set of criteria would be able to access treatment. Stopping criteria were also included for the first time ever. This was incorporated by NICE and announced in their draft guidance in September 2015. The development of the Managed Access Agreement (MAA) became a working partnership between NHS England, NICE, the MPS Society, BioMarin and a clinical expert. The MAA was designed to be inclusive for patients, ensuring response to treatment in a minimum of 4 out of 5 criteria through consistent clinical and quality of life monitoring. An intensive follow up program and multi domain assessments would be required and treatment would stop for those not meeting treatment targets. Results: On 16th December 2015 NICE guidance recommended elosulfase alfa for patients in England via the MAA. At the time of writing, just 12 weeks since the MAA was approved, a total of 40 patients have been recruited to the MAA through 7 hospitals in England. This represents 43% of the 93 patients known to have MPS IVA in England. Of these, 26 patients previously took part in the clinical trials for elosulfase alfa, and 14 patients are receiving this new treatment for the first time. Conclusions: In an environment where health systems are having to choose between high cost drugs and the funding of other health resources, the MAA, with a confidential negotiated pricing scheme, offers all patients meeting the treatment criteria access to reimbursed therapy in the first 12 months. The MAA will be subject to annual review under the chairmanship of NICE and the data collected will be used to assess whether NICE will continue to fund the treatment after the 5 year term of the MAA. While we are in the first year of this new initiative, patients have embraced the MAA and recognized that this is the only way forward to ensure access to treatment. Only time will tell if the stopping criteria are fair and if patients affected by common disorders will become subject to similar requirements in the future to ensure fairness and equality.
Effect of Galsulfase on the Cardiac Problems in Mucopolysaccharidosis Type VI
E. Fateen1, M. Sabry1, S. Metwally1
1National Research Centre, Cairo
Purpose: Monitoring the effect of galsulfase (Naglazyme) on severe cardiac problems in mucopolysacharidosis (MPS) type VI. Methodology: diagnosis of MPS VI was confirmed in three Egyptian children by quantitative determination of GAGS in urine, two dimensional electrophoresis of the GAGS in urine and by fluourometric assay of Arylsulfatase B .All were under enzyme replacement therapy (ERT) by galsulfase. Follow up of the heart response to treatment was done clinically and by Echocardiography. Results: Three MPS type VI patients were given ERT weekly according to their weight. The first patient was seven years old boy; has mitral and tricuspid valves regurgitation in addition to heart hypertrophy .He received ERT for 40 weeks. The treatment was interrupted twice for two weeks because the child developed dyspnea, tachycardia and heart failure. ERT was resumed in both times after treating the heart failure. The second patient was nine years old girl with tricuspid and aortic valves stenosis. She was tachycardiac. Echocardiography showed uniform thickening of the cusps of all valves with restricted movement and diminished cardiac contractility. ERT was stopped after two weeks due to tachycardia. Treatment was resumed after one week. Her condition improved after ten weeks of ERT. The third patient was eleven years old boy. His mitral valve was stenotic, thickened with restricted mobility and regurgitation. He received ERT for 48 weeks without interruption. Echocardiography showed improvement of heart function and decrease of the valve thickening .Clinically his general condition was improving. Conclusion: The heart condition and the general condition in our three MPS VI patients were improving under galsulfase treatment although we have stopped the treatment twice for the first patient due to heart failure and once in the second patient due to tachycardia. Close monitoring of the patients during infusion specially the cardiac condition is recommended. ERT is promising in improving the cardiac problems associated with MPSVI on the long term.
A Novel, Randomized, Placebo-Controlled, Blind-Start, Single-Crossover Phase 3 Study to Assess the Efficacy and Safety of UX003 (rhGUS) Enzyme Replacement Therapy in Patients With MPS VII
P. Harmatz1, R. Wang2, C. Whitley3, M. Bauer4, S. Chesler5, S. Agarwal5, W. Song5, C. Haller5, E. Kakkis5
1Children’s Hospital & Research Center Oakland
2Childrens Hospital of Orange County, Orange
3University of Minnesota Childrens Hospital, Minneapolis
4Miami Children’s Hospital, Miami
5Ultragenyx Pharmaceutical Inc., Novato
Purpose: To report a novel clinical trial design that utilizes unique patient-specific endpoints to investigate a new therapy (recombinant human beta-glucuronidase; rhGUS, UX003) for mucopolysaccharidosis type VII (MPS VII, Sly syndrome). MPS VII is an ultra-rare disease with fewer than 200 known patients, and has a highly heterogeneous clinical presentation. A blind-start design enabled all subjects to contribute to assessment of treatment effect, reduced potential bias, maintained objectivity in clinical assessments and overcame the challenge posed by the low prevalence of MPS VII. Methodology: A Phase III, multicenter, randomized, placebo-controlled, blind-start, single crossover study was conducted in the US to assess the efficacy and safety of UX003 in 12 subjects with MPS VII aged 5 to 35 years. Subjects were blindly randomized 1:1:1:1 to one of 4 treatment sequence groups (Group A, B, C or D; 3 subjects per group) to start treatment with UX003 (Group A) or placebo (Groups B, C, and D) (Figure 1). Placebo-initiated groups crossed over to treatment with UX003 at 8, 16 or 24 weeks for Groups B, C, and D, respectively. During active treatment, subjects received IV infusions of 4 mg/kg UX003 every other week through Week 46, with all subjects receiving at least 24 weeks of UX003. Subjects had the option to continue into an open-label extension study at the completion of the trial. Endpoints included urinary levels of glycosaminoglycans (uGAG), and clinical assessments such as walking distance, pulmonary function, shoulder flexion, fine and gross motor function, visual acuity, and questionnaires assessing fatigue and health. Notably, a multi-domain responder index (MDRI) was defined, and a unique Individual Clinical Response (ICR) endpoint was selected for each subject based on a major disease impact reported by the patient. Clinically meaningful threshold changes in these endpoints were pre-specified. The efficacy of UX003 will be assessed by the percent reduction of uGAG excretion, the MDRI, and the proportion of subjects achieving a positive ICR outcome after 24 weeks of UX003 treatment relative to the pre-UX003 baseline. Results: Enrollment was completed in June of 2015 with 12 subjects from 4 countries. At the time of writing, four subjects have completed the study and enrolled into the long-term extension study. All of the other subjects remain on the study with the last subject visit scheduled for May 2016. Efficacy and safety results will be presented at the meeting. Conclusion: A rigorous, placebo-controlled Phase 3 study of an ultra-rare disease can be conducted with a small sample of patients utilizing a novel blind-start single crossover design that maximizes subject exposure to active treatment, while reducing the potential bias of an open-label design.
UX003-CL301 study schema.
Enzyme Replacement Therapy for Alpha-Mannosidosis: Long-Term Safety Data in Patients Treated With Velmanase Alfa (Recombinant Human Alpha Mannosidase)
D. Ardigò1, L. Borgwardt2, C. Dali2, M. Gil Campos3, N. Guffon4, E. Jameson5, S. Jones5, F. Boukef1, F. Cattaneo1, A. Lund2
1Chiesi Farmaceutici S.p.A., Parma
2Rigshospitalet, Copenhagen
3Reina Sofia University Hospital, Córdoba
4Edouard Herriott Hospital, Lyon
5Central Manchester University Hospital, Manchester
Purpose: To assess the long-term safety profile of velmanase alfa (Lamazym or rhLAMAN) as replacement treatment of Alpha-Mannosidosis, an ultra-rare, monogeneic, autosomal recessive, multisystem lysosomal storage disorder currently without any approved treatment. Methodology: 33 patients, adults (N = 14, age range 18-35 years) and pediatrics (N = 19, age range 6-17), previously enrolled in phase I/II (n = 9; rhLAMAN-02, -03, -04) and phase III (N = 24; rhLAMAN-05) studies with velmanase alfa were assessed for efficacy and safety in a comprehensive, open-label, uncontrolled, pivotal clinical trial (rhLAMAN-10). Mean (SD) exposure was 890.5 (461.5) days (ranging from 357 to 1625 days). All patients included in the study received the intended clinical dose of 1 mg/kg/week by intravenous infusion for at least 12 months, whereas more than half (N = 19, 57.6%) for at least 24 months. The duration of exposure was longer in subjects who participated in the phase I/II studies (48 months for all) than in subjects enrolled in the phase III trial rhLAMAN-05 (N = 14 for at least 24 months; N = 10 for at least 12). Since all phase I/II patients were younger than 18 years at study start, safety data in the pediatric population are characterized by a significantly greater exposure. Results: A total of 536 adverse events (AEs) were reported in 29 (87.9%) subjects. The proportion of patients experiencing AEs was similar across age groups, but—as anticipated by a greater exposure—the majority of the events was reported in the pediatric population (416 vs 120 events in subjects aged ≥18 years). The most frequently reported AEs (in >25% of subjects overall) were nasopharyngitis, pyrexia, vomiting, diarrhea, and cough. Approximately half of the subjects (N = 17, 51.5%; for a total of 84 events) experienced adverse drug reactions (ADRs; i.e. AEs judged by the investigator as reasonably correlated with the treatment), which included increased weight, pyrexia, diarrhea, headache, arthralgia, chills, vomiting, hypersensitivity, increased appetite, and pain in extremities (all reported in >1 subject). All ADRs were mild or moderate in severity, except for a case of pyrexia and tremor in one subject and loss of consciousness in another, all of which completely resolved. Approximately one third of patients (N = 12, 36.5%) experienced serious AEs (14 events), and these were reported in a similar proportion in subjects aged <18 years (36.8%) and adults (35.7%). Only 2 subjects experienced serious AEs which were judged as ADRs: loss of consciousness in one subject and acute renal failure in another (who was concomitantly receiving a long-term therapy with high dose ibuprofen), both of which recovered. No deaths occurred during the clinical trials and only one patient discontinued due to reaction 3 months since treatment beginning during an early phase study (rhLAMAN03). The patient was then enrolled in the Phase III trial and is still receiving velmanase-alfa after 36 months of drug-exposure. Infusion related reactions (IRR’s) were defined as those ADRs occurring during or up to one hour after the infusion of velmanase alfa and assessed by the Investigator as being related to the infusion. A total of 19 IRRs were reported in 3 patients (9%), 14 of which were experienced by a single subject. IRRs included: anaphylactoid reaction (1 patient, corresponding to the 3% of the overall population), hypersensitivity, chills, hyperhidrosis, nausea, vomiting, pyrexia, and feeling hot. The IRRs were characterized by a rapid onset of symptoms and were of mild-moderate severity. All reported events were manageable by stopping the infusion and resuming at a reduced infusion rate. Treatments or pre-treatments with antihistamines, antipyretics, and/or corticosteroids were not routinely required. Only 8 subjects (24.2%) developed anti-drug antibodies (ADA) at any time, but ADA+ status was confirmed (i.e. present at more than one time point) in only 6 subjects (18.1%), 4 of them with ADA titer <10 U/mL for the whole follow-up. The remaining 2 subjects with higher ADA values experienced manageable IRRs of mild or moderate intensity. Conclusions: Safety and immunogenicity profile of long-term treatment with velmanase alfa was favorable and compatible with the chronic administration of the product, confirming it as a viable option for adult and pediatric patients affected by Alpha-Mannosidosis.
Long-Term Follow-Up of Patients Treated With Velmanase Alfa (Recombinant Human Alpha-Mannosidase): A Comprehensive Evaluation of Efficacy of Enzyme Replacement Therapy in Alpha Mannosidosis
L. Borgwardt1, F. Cattaneo2, C. Dali1, Y. Amraoui3, O. Andersen4, M. Beck5, J. Fogh6, S. Geraci2, D. Ardigò2, A. Lund1
1Rigshospitalet, Copenhagen
2Chiesi Farmaceutici S.p.A., Parma
3Villa Metabolica, Mainz
4Sahlgrenska University Hospital, Göteborg
5University Medical Center, Mainz
6Zymenex A/S, Hillerød
Purpose: To investigate the long-term efficacy of velmanase alfa (Lamazym or rhLAMAN), a recombinant form of the human enzyme alpha-mannosidase, as replacement therapy in the treatment of Alpha-Mannosidosis. Alpha-Mannosidosis is an ultra-rare, monogenic disorder caused by the deficient activity of alpha-mannosidase, a lysosomal enzyme critically involved in the catabolism of glycoproteins. Lack of glycoprotein degradation leads to systemic cellular accumulation of undigested mannose-rich oligosaccharides, which clinically manifests as motor function impairment, physical disability, immunodeficiency, recurrent infections, psychiatric symptoms, intellectual disability, and skeletal abnormalities. At present, no licensed therapeutic options are available. Methodology: Long-term efficacy of velmanase alfa 1 mg/kg once a week intravenous infusion was investigated in an open-label, uncontrolled, pivotal clinical study (rhLAMAN-10), including all patients previously enrolled in the clinical development of the enzyme. A total of 33 subjects (19 pediatrics and 14 adults, aged 6-35 years) were assessed and in integrated database created by pooling databases from all phase I-II and III studies and after-trial extensions. Patients from phase I-II studies rhLAMAN-02, -03, and -04 (N = 9, aged 6-17 years) provided data up to 48 months, while patients from the phase III study rhLAMAN-05 (N = 24, aged 7-35 years) contributed for 12-36 months of treatment data. Change from baseline in serum oligosaccharide concentration and 3-Minute Stair Climb Test (3MSCT) were the co-primary endpoints of the study. 6-Minute Walking Test (6MWT) and Bruininks-Oseretsky test of motor proficiency, second edition (BOT-2) were investigated as main secondary efficacy parameters. Clinical assessments were centralized. Results: A significant clearance of serum oligosaccharides was observed over time and across age groups, with a mean change from baseline of −64.6% at 6 months (n = 24), −72.7% at 12 (n = 31), −76.0% at 18 (n = 11), −77.7% at 24 (n = 10), and −81.8% at 48 months (n = 9) [p<0.001 from baseline to last observation]. In addition, a consistent increase in serum IgG concentrations was also observed (+3.33 g/dL from baseline to last observation, corresponding to +44.0%, p<0.001), reverting clinically-relevant hypo-gammaglobulinemia when present. Results for both biomarkers were similar across age groups. The co-primary endpoint 3MSCT showed a consistent improvement from baseline to 6 months (+8.3%), 12 (+9.3%), 18 (+24.5%), 36 (+30.9%), and 48 months (+39.1%) [p≤0.004]. Efficacy was more evident in pediatric patients (+13.15%, +15.3%, +26.1%, +11.6%, +33.9%, and +39.1% respectively reflecting an increase of 69.7 m from baseline at 48 months). For secondary measures of motor function, a progressive improvement from baseline was also evident in the 6MWT (+6.1% at 6 months, +7.3% at 12, +16.4% at 18, +24.4% at 36, and +22.5% at 48 months [p=0.05 from baseline to last observation]). A trend for a greater improvement emerged in subjects below 18 years, who benefited for a mean change from baseline to the last observation of 39.1 m compared with no change (−0.3 m) in adults. In addition, the mean age-equivalent values for all seven BOT-2 subtests of motor proficiency increased from baseline to the last observation in the pediatric group, indicating clinically-relevant fine motor and gross motor developmental skill acquisition. Similar improvements were detected for pediatric scale scores, whereas no change in BOT-2 results were observed in adults. Conclusions: The biological activity of velmanase alfa in correcting alpha-mannosidase deficiency was demonstrated by a significant and sustained clearance of serum oligosaccharide accumulation. A statistically significant benefit on patients’ motor function was also documented as progressive improvement from baseline in the 3MSCT, in the overall treated population. In addition, clear trends for improvement in clinically relevant secondary endpoints including 6MWT and BOT-2 motor proficiency tests were observed in the pediatric population. This study provides evidence that the treatment of patients with Alpha-Mannosidosis with velmanase alfa is associated with clinically relevant benefit in a wide range of disease-related manifestations, especially when treatment is started early in the pediatric age.
Hurler Syndrome and Transplantation: Analysis of Mortality Over 3 Decades Following Allogeneic Transplantation at the University of Minnesota
P. Orchard1, N. Rodgers1, A. Kaizer2, K. Rudser2, W. Miller1, T. Lund1, E. Braunlin1
1Department of Pediatrics, Minneapolis
2School of Public Health, Minneapolis
Before the implementation of allogeneic hematopoietic stem cell transplantation (HSCT) for the treatment of Hurler syndrome (MPSIH), death occurred most commonly within the first decade of life, with cardiac and pulmonary causes responsible for the majority of deaths. To determine if HSCT has altered the incidence and causes of death, we determined the 30-year follow-up in all patients with MPSIH undergoing HSCT from inception of our metabolic transplant program. We identified 134 patients (65 female, 69 male) with MPSIH transplanted between 4/30/1984 and 7/25/2013 and determined follow-up status for each up to 12/31/2013. Status was based on chart review, social media and data compiled by the Centers for Disease Control’s National Death Index (NDI); 12 patients (9%) were lost-to-follow-up. The mean age at transplant was 21.8 months. There were 35 patients (26%) transplanted with related grafts, 50 (37%) using adult unrelated donors and 49 (37%) were recipients of cord blood grafts. Overall survival for the entire cohort at one- and 25-years (95% confidence interval) was 70% (62%-78%) and 37% (19%-55%) respectively (Figure). Prior to 2004, there were no patients that received enzyme therapy in the peri-transplant period (0/96), while from 2004 onward all patients (38/38) received enzyme therapy. In addition, prior to 2004 cord blood grafts were infrequent (18/96; 18.9%) while from 2004 onward cord blood was the most common graft source (31/38; 81.6%). Overall survival at one- and 8-years in patients transplanted from 2004 onward was 84% (73%-96%) and 81% (69%-94%) respectively. This compares to 65% (55%-74%) and 57% (47%-67%) at one- and 8-years among those transplanted before 2004. Cumulative incidence frequency of deaths for the entire cohort at 25 years follow-up showed that 12% were cardiac related, 26% from infectious complications and 42% pulmonary associated deaths. The remaining 20% deaths occurred from neurologic, hematologic and gastrointestinal events, as well as unknown causes. HSCT has improved the survival of individuals with MPS IH. These investigations document survival for as long as 30 years after the procedure. Pulmonary complications were the most commonly observed cause of death after HSCT. Cardiac-related deaths have decreased as a cause of mortality in comparison to historical data. There are ongoing deaths observed beyond 10 years after HSCT. The increase in availability of unrelated grafts, improved supportive care and modifications in the preparative regimen to decrease peri-transplant mortality, and peri-transplant enzyme therapy may contribute to better outcomes.
Hematopoietic Stem Cell Transplantation for MPS I: Experience of Three Brazilian Centers
C. de Souza1, A. de Boer1, F. Vairo1, D. Horovitz2, C. Ae Kim3, C. Lourenço4, J. Felloni3, L. Daudt1, J. Franco3, R. Giugliani1
1Hospital de Clinicas de Porto Alegre
2Instituto Fernandes Figueira, Rio de Janeiro
3Hospital das Clinicas de São Paulo, São paulo
4Hospital das Clinicas da Faculdade de Medicina de São Paulo, Ribeirão Preto
Background: Hematopoietic stem cell transplantation (HSCT) has been a successful strategy for the treatment of selected lysosomal diseases, especially Hurler syndrome (Mucopolysaccharidosis I, severe forma, or MPS-IH). It is clear that HSCT addresses the enzyme defect in white blood cells of MPS-IH patients, although it does not provide complete clinical correction. The natural history of the disease is modified, and the fatal complications prevented, so MPS-IH children treated with HSCT generally have an increased lifespan compared to untreated children. Objective: As in Brazil we have a limited experience with BMT for rare disorders, the aim of this communication is to report the experience of two centers with BMT for MPS-IH. Patients and Methods: BMT was carried out in 7 MPS-IH patients over a period of 5 years (2010-2015). The cases included in this report (3 males and 4 females) were transplanted in three different centers in Brazil (Curitiba, São Paulo and Porto Alegre). All were homozygous for p.W402X mutation. The age at diagnosis ranged from 1to 22 months of life. Age ranged at BMT ranged from 8 to 28 months of age. In 6/7 patients ERT with laronidase was provided since 10 to 24 months before the BMT. In 5/7 cases the donor was HLA identical unrelated volunteer. The conditioning regimen consisted in the association of Busulfan (1 mg/kg/dose every 6 hours for 16 doses, from Day-7 to Day-4) and Cyclophosphamide (2 g/m2/dose, Days -3 and -2, with Mesna). Rabbit antithymocyte globulin (from Day-6 to -2) to prevent graft rejection was used in combination with the conditioning regimen only in the unrelated HSCT setting. Results: Primary graft failure was observed in 6/7 patients. Three patients have died, one received a second transplant. The primary cause of death was infection in two cases and disease progression in one case, after primary graft failure. Of the two living cases, one received three transplants and had severe disease progression after graft failure and the other has functional grafts with a favorable long-term outcome after a follow-up of 5 years, having mixed chimerism (30%). Despite the low chimerism, the patient had improvement of motor skills, language and cerebral MRI; however, dysostosis multiplex has progressed. Conclusion: the outcome of HSCT for MPS-IH has not been favorable in the experience of the three Brazilian centers, at least in the group of patients included in this report. The reasons for this probably are: the diagnosis of MPS is still performed late, the waiting time for transplantation still long, there is lack of a unified protocol to indicate the procedure and to perform the follow-up. On the other hand, the patient with favorable outcome stabilized disease progression, normalized biochemical parameters and had a better neurological development, although the bone dysplasia progressed.
Early Enzyme Replacement Therapy and Hematopoietic Cell Transplantation Improves Clinical Outcomes in Patient with Mucopolysaccharidosis Type I
T. Okuyama1, M. Kosuga1, R. Mashima1, H. Yabe2
1National Center for Child Health and Development, Setagaya
2Tokai University School of Medicine, Isehara
Mucopolysaccharidosis type I (MPS I, Hurler syndrome) is caused by a deficiency of the enzyme alpha-L-iduronidase, resulting in accumulation of glycosaminoglycan at the systemic organization. Patient with MPSI is developing coarse facial features, corneal cloudings, Intellectual disability, hydrocephalus, hepatosplenomegaly, dysostosis multiplex and joint contracture gradually. Enzyme Replacement Therapy (ERT) may have effect on the progression of somatic findings. However it does not expected to influence the CNS disease in severe type. Hematopoietic stem cell transplantation (HSCT) should be performed before age two years to maximize benefit and appears to slow the course of cognitive decline We experienced early hematopoietic cell transplantation improved clinical outcomes in patient with MPS type I.18 day old girl was presented to our hospital. She was the fifth child born to non-consanguineous Japanese parents and had a healthy sister and a late sister with severe MPSI. She was suspected MPSI at birth because her older sister was previously diagnosed with MPSI. α-L-iduronidase activity in cord blood was < 0.8nmol/mg protein/4 hr (normal range:28.8-89.8 nmol/mg protein/hr) and urine uronic acid at one month old was 438mg/g creatine. IDUA gene analysis revealed she was compound heterozygote for the pathogenic gene mutations. She was diagnosed with MPSI and started ERT at 34 days after birth. ERT continued until 8 months old. Then she received HSCT as a donor of her healthy sister at 9 months old. After 2ys and 8 ms of HSCT, α-L-iduronidase activity of lymphocytes and urine uronic acid were 47 nmol/mg protein/hr and 24.3mg/g creatinine respectively. While she had mild valvular disease, dysotosis multiplex and joint contracture of fingers, she achieved normal developmental milestone without cognitive impairment. The developmental quotient (DQ) score was 87 at 3 year and 2 months old. HSCT had been successful in reducing the progression of some findings in this patient with severe MPS I while the skeletal and heart valvular manifestations continued. Our case revealed that very early ERT and HSCT appeared to stop or slow the course of MPS I.
A Case Report of a 33-Year-old Female with Hurler Syndrome 30+ Years Post-HSCT
S. Cagle1
1Emory University, Decatur
Purpose: To report the long-term outcomes of a 33-year-old female with Hurler syndrome (mucopolysaccharidosis type I or MPS I) who is 30+ years post a successful hematopoietic stem cell transplant (HSCT). Methodology: Long-term outcomes collected by medical chart review, physical exam, laboratory tests, clinical assessments, and conversations with patient and parents. Results: A 33-year-old female patient with Hurler syndrome presented to the genetics clinic for a follow-up appointment. She had not been evaluated by genetics in about 10 years. The patient received a successful HSCT at 29 months of age. At the age of 33, she has cognitive impairment but is still able to converse on the level of a 4-5 year old. Her parents are her caretakers, and they reported she has been cognitively stable for the past 10 years. Blood was collected for alpha-L-iduronidase enzyme activity, and the level was within the normal range. Urinary glycosaminoglycan (GAG) analysis was performed and was normal quantitatively. An echocardiogram revealed sclerotic changes in the mitral valve and calcifications in the aortic valve. These findings indicate an increased risk for coronary artery disease. The patient’s blood lipid panel was abnormal for high cholesterol, which also contributes to coronary artery disease. The patient is able to ambulate around her home with the assistance of a walker. Conclusions: A change in diet or taking a cholesterol lowering medication is indicated for the patient, especially in light of the sclerosis and calcifications in her cardiac valves. Overall, the patient is doing quite well for being 33-years-old with Hurler syndrome. Long-term outcomes of patients with Hurler syndrome post-HSCT have been reported in the literature. However, the median years post-HSCT are often around 10 years. There is very little information about patients who are 20 or 30 years post-HSCT, and data focusing on this age group is extremely important to help guide the management of middle aged patients with Hurler syndrome as this population grows in numbers.
Incomplete Biomarker Response in Mucopolysaccharidosis Type I Patients After Successful Hematopoietic Stem Cell Transplantation
G. Kuiper1, E. Langereis1, N. van Vlies1, F. Wijburg1
1Emma Children’s Hospital / Academic Medical Centre, Amsterdam
Purpose: The concentration of the biomarkers heparan sulfate (HS) and dermatan sulfate (DS) are considered to reflect disease severity in mucopolysaccharidosis I (MPS I). Previous studies on the long-term biochemical efficacy of enzyme replacement therapy (ERT) failed to show complete normalization of HS and DS in urine and, in blood, a complete correction in < 50% of patients was shown. Since it is assumed that the somatic effects of hematopoietic stem cell transplantation (HSCT) is superior to ERT as a result of the continuous production of enzyme, we investigated the response of biomarkers to HSCT in MPS I. Methodology: Urine samples of 6 MPS I patients (4 Hurler phenotype, 2 Hurler-Scheie phenotype) who had a successful HSCT (full engraftment) were analyzed. Urinary HS and DS were identified by LC-MS/MS after full enzymatic digestion. Results: All Hurler patients still had significantly increased levels of HS and DS at 6-11 years post HSCT. The 2 Hurler-Scheie patients both had normal levels of HS and DS at 2.4 and 2.5 years post HSCT. Conclusion: This preliminary analysis showed that the urinary HS and DS levels do not normalize in MPS I Hurler patients after a successful HSCT, which is in accordance with the extent of the residual musculoskeletal disease observed in these patients. Additional long-term longitudinal data on HS and DS levels in both blood and urine of 12 MPS I Hurler patients after HSCT are now collected to further investigate the consequences of this lack of full biomarker response.
Cerebrospinal Fluid Shunting (CFS) in Children With MPS and Stem Cell Transplantation (SCT). Results of a Monocentric Retrospective Analysis
U. Mücke1, B. Maecker-Kolhoff1, M. Sauer1, K. Sykora1, E. Hermann1, L. Grigull1
1Hannover Medical School, Hannover
Purpose: SCT is the treatment of choice for children with MPS I, controversial results after SCT are reported for children with MPS II and III. Most children with MPS exhibit clinical or radiological signs for elevated intracranial pressure, however guidelines on cerebrospinal shunting in the context of SCT are lacking and clinical data are scarce. Analysis of data might be helpful for developing recommendations for the management of CFS in children with MPS prior to SCT. Methodology: Retrospective monocentric analysis of all consecutively transplanted MPS patients at our institution. Results: 17 children with MPS received SCT between 2001 and 2015. 13 MPS I patients, 2 MPS II, and 2 MPS IIIa. 3 children with MPS I received a ventriculo-peritoneal (VP) shunt prior to SCT. One child required emergency placement of a VP shunt on day +18 after SCT. One of the remaining children received a VP shunt ten years after SCT. VP shunt associated complications were seen in 3 children (bleeding/hematoma (n = 1), infection (n = 1), re-surgery (n = 1)). Two children died due to non VP-shunt-associated complications (multi-organ failure (n = 1), progress of the underlying disease (n = 1)). Discussion: VP shunt requirement before SCT remains controversial. The largest retrospective series from Duke University shows a high rate of VP shunt dysfunction and significant mortality rates in children requiring VP shunts after SCT. In our series, one child needed emergency shunting after SCT. His current neuropsychological sequelae might be associated with this incident. In 5 children with VP shunt, one infection was reported. A register for collecting all data on VP shunts to develop criteria for the management of CFS in the context of SCT would be helpful.
Impact of Hematopoietic Stem Cell Transplantation for Patients With Mucopolysaccharidosis II
F. Kubaski1, Y. Suzuki2, S. Tomatsu1
1Alfred I. duPont Hospital for Children, Wilmington
2Gifu University, Gifu
Purpose: Little has been known about the long-term outcomes of hematopoietic stem cell transplantation (HSCT) for Hunter Syndrome except sporadic cases, the clinical and biochemical consequence was compared between patients with HSCT and enzyme replacement therapy (ERT). Method: The data derived from our 90 cases and 63 reported cases and from the Statistical Center of the Center for International Blood and Marrow Transplant Research (CIBMTR) (56 cases): mean age of 3 years (<1-8 years) and 5.1 years (10 months-19.8 years) respectively, at the time of HSCT. Results: In the CIBMTR 46.4% received transplant from related donors, while 54.3% in Non-CIBMTR patients whose data were available. Bone marrow was the graft source on 64.3% of the patients, followed by cord blood (32.1%) and 3.6% of peripheral blood stem cells (PBSC) (CIBMTR) and 85.7% BM followed by CB 14.3%. The Kaplan-Meier overall survival probability was estimated as 79% (1 year survival) and 74% (3 years survival) using 95% confidence interval in a median follow up of 72 months (3-267) (CIBMTR) and 95.5% (0.2 months), 92.3% (8 months) using 95% confidence interval (Non-CIBMTR). Nine patients died due to transplant associated complications (8.6% mortality rate) (Non-CIBMTR). Among 67 Non-CIBMTR patients, 73.1% patients were associated with severe phenotype and 26.9% attenuated. GVHD occurred in 31%patients. Most patients presented improvement in somatic features, urinary GAGs excretion, joint stiffness, and activity of daily living (ADL). At least 35% of patients stabilized or improved growth. Analysis of heparan sulfate level showed the more reduction in HSCT-treated group compared with ERT-treated group, consistent with the finding of a better activity of daily activity. Conclusion: HSCT is effective for patient with Hunter and a therapeutic option.
Clinical Outcome Following Hematopoietic Stem Cell Transplantation in Two Patients With Hunter Syndrome
A. Köhn1, I. Müller1, K. Ullrich1, N. Muschol1
1University Medical Center Hamburg-Eppendorf, Hamburg
Background: Hematopoietic stem cell transplantation (HSCT) is the treatment of choice for patients affected with Hurler syndrome <2.5 years of age. In Hunter disease there is no proof of efficiency in preserving neuropsychological function. The present study summarizes the clinical outcome following HSCT in two patients with Hunter disease. Methods and Patients: Two boys underwent HSCT at 2.1 years (patient A) and 4.25 years of age (patient B), respectively. A systematic annual follow for at least 3 years was conducted. Patient A carried a frame shift mutation (c.135-138delTGAC), patient B a missense mutation (c.1007-2A>C). Results: Both patients achieved successful engraftment. Iduronate-2-sulphatase activity normalized after HSCT, urine glycosaminoglycan excretion declined significantly. Hepatosplenomegaly disappeared in both patients 2 years after HSCT. Aortic valve thickening was progressive in patient A, resulting in mild insufficiency 4 years following HSCT. Cerebral MRI revealed enlarged Virchow-Robin spaces and abnormal T2 signal intensity in the white matter prior to transplantation in both patients. Three years following HSCT patient A developed a progressive brain atrophy whereas patient B showed a decline in white matter abnormalities. The size of Virchow-Robin spaces did not change. Patient A developed hyperactive and aggressive behavior starting 6 months after HSCT and lost speech after 2 years. Three years after HSCT patient B was developmentally delayed, but language and motor abilities continuously improved. Conclusions: HSCT was effective in reducing non-neuropsychological manifestations of Hunter syndrome. The present data do not give evidence that HSCT has a clear impact on the neuropsychological outcome. The continuous development of patient B might either be a consequence of HSCT or of the natural history of the disease.
Hematopoietic Stem Cell Transplantation in a Young Infant With Hunter Syndrome: Six Year Follow-Up
A. Barth1, D. Horovitz1, A. Reis1, C. Bonfim2, R. Giugliani3, M. Burin3, S. Segal3
1Instituto Fernandes Figueira / Fiocruz, Rio de Janeiro
2Hospital de Clínicas / Universidade Federal do Paraná, Curitiba
3Hospital de Clínicas de Porto Alegre
Purpose: Enzyme replacement therapy (ERT) in Hunter Syndrome (MPS II) does not cross the blood brain barrier, limiting results in neurological forms of the disease. Hematopoietic stem cell transplantation (HSCT) may be effective for the non-neuropsychological symptoms only, although better outcome may be expected in young patients. Methodology: We describe the six-year follow-up of a MPS II child submitted to HSCT with umbilical cord blood cells at 70 days of age preceded by ERT. He was investigated due to positive family history; undetectable iduronate-sulphatase (IDS) activity and the familial p.R88 H mutation were diagnosed in amniotic fluid. At birth slight hyper-reactivity and lumbar gibbus, with corresponding L3-L5 abnormality on X-ray were noted. Abdominal and cerebral ultrasound, echocardiogram, ophthalmology, audiology, pulmonary function, brain & spine MRI were normal. Lysosomal storage in placental cells and pericytes were observed by electronic microscopy. IDS activity in plasma [1,2nmol/4h/ml (ref=122-463)] and leukocytes [4,3nmol/4h/mg/ptn (ref=31-110)] re-confirmed MPS II. He received 6 infusions of Idursulfase from age 10 days without adverse reactions. HSCT with umbilical cord blood from unrelated donor was performed at 70 days of age, without additional ERT. Urinary glycosaminoglycans (GAGs) began to decrease during ERT and were in normal range 3 months post-transplant. Engraftment after 30 days revealed mixed chimerism with 79% donor cells; after 5 years engraftment remained at 80%. IDS activity 30 days post-transplant was low in plasma (22nmol/4h/ml; ref=122-463) and normal in leukocytes (49nmol/4h/mg/ptn; ref=31-110); 270 days, 2, 3, 4 and 5 years after transplant the same pattern was observed. Results: At age 6 years, growth charts are normal and he is very healthy. Very mild lumbar gibbus and signs of dysostosis multiplex in hands and lumbar spine are present. Brain CT and upper airway fiberoptic findings are normal. The patient is very hyperactive. Hearing testing revealed important hearing loss with hearing threshold of 80dB in the right ear and 70dB in the left ear, as measured by auditory brainstem response. His echocardiogram shows minimal mitral regurgitation. Despite good general health and minimal bone MPS-related abnormalities, his intellectual ability, as measured by Wechsler Scale (WPPSI-III), disclosed a total IQ of 50. Conclusion: Follow-up studies after HSCT in MPS II are scarce, lacking information on longer term effects. We decided to go through with the transplant since no reports for children so young were available, and the procedure was the only “possible” option to prevent neurodegeneration, since the family history was compatible with the severe phenotype. ERT was used as an attempt to prevent storage during the elapsed time from birth to transplant / engraftment. Transplantation prior to the onset of neurological symptoms seems to have not modified the neurological deterioration. Longer follow-up and reports of similar cases are recommended.
Case Study of a Child With MPS II With Abnormal MRI Undergoing HSCT
K. Bhattacharya1, P. Owens1, P. Shaw1, S. Keogh1, K. Prelog1
1The Children’s Hospital at Westmead, Sydney
Purpose: To describe the decision making process and outcome to date, of a 4 year old boy with Mucopolysaccharidosis (MPS) Type II and an abnormal brain MRI, who then proceeded to hematopoietic stem cell transplantation (HSCT). Methodology: The subject is the youngest of 4 children. He has 2 older step sisters and one older full sister who has a severe aggressive neurological disorder with an unexplained etiology. The subject presented with an increased head size, increasing abdominal girth and mild speech developmental delay. MRI demonstrated innumerable dilated perivascular spaces in the parietal occipital region—the overlying cortex was thought to be normal but difficult to assess in the context of a honeycombed-like appearance of temporal and frontal lobes of the cerebral white matter. Clinically he had mild hepatomegaly and the spleen was palpable. The urinary glycosaminoglycans were elevated at 51.6μmol/L (Ref. Range 4.9-21.1) White cell enzymology result: Iduronate-2-sulphatase (IDS) level 1.8 pmol/min/mg protein (Ref. Range 10.9-88 pmol/min/mg protein. The patient was hemizygous for a missense variant in the IDS gene not previously reported—c.692C>G (p.Pro231Arg) Neuropsychometric assessments indicated that the subject was broadly within the normal range, but that he had specific deficits of verbal ability possibly due to conductive hearing deficit. Therefore any developmental assessment of the subject underrepresents his verbal ability. Consensus sought nationally and internationally regarding HSCT for this subject given the MRI findings. The family were counselled regarding treatment options for MPS II and opted for HSCT. Adenotonsillectomy, grommet insertion, central line insertion and bone marrow harvest were undertaken pre HSCT. He was admitted to commence conditioning for an unrelated cord HSCT in July 2013. On day-2 of conditioning he developed serum sickness consisting of high fevers and skin rash—a decision was made to abandon the allogeneic transplant and proceed with an autologous rescue transplant. After recovering from his autologous graft, the subject was reconditioned at a later date and successfully underwent a 6/6 unrelated cord HSCT in October 2013. Results: The patient recovered reasonably well post allogeneic graft and Day 30 chimerism was 98%donor. Main problems post-transplant: EBV reactivation, central line infection requiring removal of line and he became Vancomycin Resistant Enterococcus (VRE) positive. IDS level 2 months post-transplant was 16.3 (Ref. Range 10.9-88 pmol/min/mg protein). The patient is about to commence at a main stream school with additional support for language, hearing and physical difficulties. Neuropsychometric assessment in January 2016 showed that the subject continues to perform in the low average intellectually with relative strengths in his working memory and processing speeds. He is making good progress with his literacy and numeracy skills and demonstrates indicators of school readiness. Urinary GAG’s recently were 14mgmmol cr (Ref range 4.4-17.7 mgmmol cr). MRI in February 2016 showed minimal change to the initial MRI pre transplant. Conclusion: MPS II is a rare lysosomal storage disorder. The etiology, natural history and optimal management strategy for these group of patients is still being defined. Comparing treatment options for these patients is difficult given the large number of private mutations in this disorder. Observation of this patient over time will indicate if HSCT was the right course of treatment for this subject. The family were clear after extensive counselling that HSCT remained the best chance they had of averting potential neurological progression in this variable disorder.
Clinical Course in a MPS IIIA Patient Following Hematopoietic Stem Cell Transplantation: An Eight Year Follow Up and Comparison with the Natural History in Two Patients With Identical Mutations in the SGSH-gene
A. Köhn1, L. Grigull2, K. Ullrich1, N. Muschol1
1University Medical Center Hamburg-Eppendorf, Hamburg
2Hannover Medical School, Hannover
Background: Mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo A syndrome) is a progressive glycosaminoglycan storage disorder caused by a deficiency of lysosomal sulfamidase. Clinically the disease is characterized by a progressive decline and loss of cognitive, speech and motor abilities, as well as behavioral and sleep disturbances. Affected children with the classical severe form of the disease show a developmental slowing, which begins in the second year of life and reach a maximum developmental age of approximately 24 months at the age of 30-40 months. Dementia occurs by the age of six and death in the second or third decade of life. Currently there is no cure for the disease. For patients affected with MPS I (Hurler syndrome) <2.5 years of age hematopoietic stem cell transplantation (HSCT) is the treatment of choice. Only a few studies concerning HSCT in MPS IIIA have been published and do not document a clear benefit on neuropsychological function. Methods and Patients: The present study summarizes the clinical outcome in a girl with MPS IIIA who received HSCT at the age of 2.5 years (patient A). The clinical course is compared with the natural history of two untreated MPS IIIA patients, a girl (patient B) and a boy (patient C), who carried the same mutations (p.R74C and p.R245 H) in the SGSH-gene as patient A. Results: Sulfamidase activity normalized after successful engraftment in patient A. At the eight years follow up the girl was ten years old and had full chimerism. She showed a global developmental delay, but development was progressing, albeit very slowly. There were no signs of regression. Patient A was able to talk in short sentences (3-4 words). She could run, climb, jump with both feet and stand on one leg for a few seconds. She was able to perform daily living activities (eating, drinking, getting dressed, going to the toilet, etc.) with only slight supervision. No problems concerning sleep were reported, but severe behavioral abnormalities were present (autistic behavior, aggressiveness, hyperactivity, inadequate laughing and crying). By contrast the two untreated patients with identical mutations in the SGSH-gene showed the typical progressive course of the disease. The first stages of regression became obvious at two years of age in both patients (speech deterioration). By the age of eight years both patients had almost lost speech and were only able to walk with support. Both patients had severe sleeping disturbances and serious behavioral abnormalities (severe hyperactivity, restlessness, crying) from the age of four to five years. Because of frequent choking while eating or drinking patient B received a percutaneous endoscopic gastrostomy tube at age 9. Patient C developed epilepsy. Both patients died in the early second decade of life. Conclusions: All patients carried the well-known mutations p.R74C and p.R245 H in the SGSH-gene, which are associated with a severe clinical phenotype of MPS IIIA. Both untreated children showed the expected severe course of disease with early cognitive decline and death in the second decade of life. By contrast the stem cell transplanted MPS IIIA patient did not show any regression in development by 10 years of age and retained intellectual abilities at a pre-school level. Nevertheless, behavioral abnormalities were profound. The present data suggest HSCT could have some beneficial effect on neuropsychological function in MPS IIIA patients. Further investigations should be conducted to better understand the underlying mechanisms of neurodegeneration and possible impact of HSCT on MPS IIIA.
Hematopietic Stem Cell Transplantation for Morquio A
S. Tomatsu1, H. Yabe2, A. Tanaka3, Y. Chinen4
1Alfred I. duPont Hospital for Children, Wilmington
2Tokai University, Isehara
3Osaka City University, Osaka
4University of the Ryukyus, Okinawa
Purpose: Morquio A syndrome features systemic skeletal dysplasia. To date, there has been no curative therapy for this skeletal dysplasia. No systemic report on a long-term effect of hematopoietic stem cell transplantation (HSCT) for Morquio A has been described. Method: We conducted HSCT for 4 cases with Morquio A (age at HSCT: 4-15 years, mean 10.5 years) and followed them at least 10 years (range 11-28 years; mean 19 years). Current age ranged between 25 and 36 years of age (mean 29.5 years). Results: All cases had a successful full engraftment of allogeneic bone marrow transplantation without serious GVHD. Transplanted bone marrow derived from HLA-identical siblings (three cases) or HLA-identical unrelated donor. The levels of the enzyme activity in the recipient’s lymphocytes reached the levels of donors’ enzyme activities within two years after HSCT. For the successive over 10 years post-BMT, GALNS activity in lymphocytes was maintained at the same level as the donors. Except one case who had osteotomy in both legs one year later post BMT, other three cases had no orthopedic surgical intervention. All cases remained ambulatory, and three of them could walk over 400 m. Activity of daily living (ADL) in patients with HSCT was better than untreated patients. The patient who underwent HSCT at four years of age showed the best ADL score. Conclusion: The long-term study of HSCT has demonstrated therapeutic effect in amelioration of progression of the disease in respiratory function, ADL, and biochemical findings, suggesting that HSCT is a therapeutic option for patients with Morquio A.
Liver Transplant in Niemann Pick B (NPB): Report of 3 Cases
P. Mabe1, M. Acuña2, J. Miquel2, C. Benítez2, N. Jarufe2, J. Roa2, M. Arrese2, J. Martínez2, F. Barriga2, S. Zanlungo2
1Hospital de Niños dr. Exequiel González Cortés, Santiago
2Pontificia Universidad Católica de Chile, Santiago
NPB is a sphyngolipidosis due to acid sphingomyelinase deficiency. Clinical signs are hepatosplenomegaly, pancytopenia, dyslipidemia, bleeding diathesis, interstitial lung damage, cirrhosis. No specific treatment is available. We report 3 NPB Chilean patients, homozygous for p.Ala359Asp mutation, which underwent successful liver transplant due to liver failure. Case 1: Male, 3rd child, non-consanguineous parents. NPB confirmed at age of 2 years. Because antecedent of premature death of a sib due to respiratory failure caused by NPB, a related allogeneic bone marrow transplant at age of 3 years was performed. Normalization of leukocyte sphingomyelinase activity, partial hepatosplenomegaly regression and growth velocity increment were observed. Ten years later portal hypertension was evident, with ascites and large esophageal varices. At age of 16 years and with Child-Pugh score 11 (Child C), a liver transplantation was undertaken. Follow up after transplant (FUAT) 10 years. Case 2: Male, non-consanguineous parents, 2 healthy younger sibs. NPB suspected at age of 22 months due to hepatosplenomegaly, confirmed at age of 12 years. Progressive liver dysfunction noted from age of 15 years. At 22 years he presented a definitive liver decompensation, with ascites and signs of mild encephalopathy. Liver transplantation was performed with Child-Pugh score 11 (Child C). FUAT 4 years. Case 3: Female, unique child, first cousin parents. Hepatosplenomegaly at age of 20 months. NPB confirmed at 8 years old. She presented frequent epistaxis, which reverted after vitamin K and tranexamic acid therapy. Prothrombin and platelets were approximately 40% and 100.000/ml, respectively. At 19 years old, severe asthenia and impairment of life quality were reported. Epistaxis was more frequent; prothrombin descended to 36% and didn’t increase after vitamin K. Liver transplant was performed with Child-Pugh score 11 (Child C), at 20 years old. FUAT 9 months. None of the patients had graft rejection or severe complications after liver transplant. All present normal hepatic function and good quality of life. Conclusions: Liver transplant should be considered in patients with liver failure due to NPB. Despite transient normalization of sphingomyelinase activity after bone marrow transplant, this seems not to avoid progression to cirrhosis in NPB patients.
The Lived Experience of Parents of Children, Adolescents and Young Adults With Mucopolysaccharidoses (MPS)
S. Somanadhan1, P. Larkin2
1Temple Street Children’s Hospital/University College Dublin
2University College Dublin, 4
3Our Lady’s Hospice and Care Services, Harold’s Cross Dublin, Ireland
Background: Mucopolysaccharidoses (MPSs) is one of the many rare inherited metabolic disorders (IMDs) that come under category 3 of life-limiting conditions (Together for Short Lives, 2013). The severity of the disease varies according to the specific type, ranging from very mild symptoms to, in most cases, multisystemic, restricted growth or mental and physical disabilities. Recent developments in treatments for some forms of MPS have made dramatic changes in the quality of life for patients. Other forms of treatment are currently under investigation and development. Very little is known about parents’ experience of living and caring for these children, adolescents and young adults with MPS. The aim of the study: This study aimed to explore and interpret Irish families’ experiences of living and caring for children, adolescents and young adults with MPS. Methodology: A qualitative approach, utilizing hermeneutic phenomenology informed by the philosophical constructs of Heidegger (1962), Gadamer (1960/1998) and Van Manen (2007/2014) was undertaken. Van Manen’s (2007/2014) phenomenological approach was used as a guide for data collection through serial interviewing and phenomenological data analysis. A purposively selected sample of parents’ (n = 8) attending the Irish National Centre of Inherited Metabolic Disorders was invited to participate. The data was collected over a 17 month period (August 2013-December 2014). Findings: Parents spoke about their experience of being the parent of a healthy child to being the parent of a child with a life-limiting condition, and they described as living in a state of liminality within this transition. Parents experienced a range of uncertainties concerning ‘no man’s land’ and ‘future is unknown’ to describe their life world. The findings suggest that parents of children with MPS experienced multiple cyclical movements across all five lived existential themes proposed by Van Manen (2014) and these are Lived Other, Lived Body, Lived Space, Lived Time and Lived Things, and they gradually developed a way of learning to incorporate MPS in their day to day life. Conclusion: Overall, this study provides a voice to the Irish parents of children with MPS, and in doing so make their lives more understandable to the wider audience. The experience and needs of this distinctive population are identified through this study, so that practice can be informed and developed based on actual lived experiences.
The Burden Endured by Caregivers of Metabolic Patients: MPS Compared to Intoxication Disorders
F. Nichelli1, P. Meregalli1, C. Galimberti1, S. Gasperini1, R. Parini1
1Fondazione MBBM-San Gerardo Hospital, Monza
Our purpose was to evaluate the global burden among primary caregivers of patients affected by metabolic diseases with the first aim to find the more adequate way to support them from the psychological point of view. We then administered an anonymized self-reported satisfaction and Quality of Life (QoL) questionnaire (SAT-P-Satisfaction Profile) developed in Italy and validated for adults of both sexes age 20-65 (G.Majani e S. Callegari. SAT-P Satisfaction profile, Edizioni Erickson, Trento, third edition 2006). It consists in five domains (1-psychological functioning, 2-physical well-being, 3-job, 4-sleep and free time, 5-social function), investigated through 32 items which are answered choosing a point from 0 to 100 over a line. The results are differently weighted depending on age and sex and transformed in a zeta-score where a result between −1 and +1 corresponds to normal healthy individuals, −2 to −1 is considered a borderline result needing attention, below −2 is definitely pathological and need psychological assistance. 50 parents of 29 MPS patients and 32 parents of 21 patients with urea cycle disorders, organic acidemia and glycogenosis (overall defined OTHERS) accepted to participate in this survey. Results are reported in the table. Percentages of borderline and pathological individuals were clearly lower among MPS parents compared to OTHERS for item 1 and 4. This trend is confirmed for borderline also for item 2 and 5, while percentages for item 3 (job) are much similar. In the group MPS 66% of caregivers had normal results while only 22% had normal in the group OTHERS. 2 or more borderline/pathological items were present in 22% of MPS and in 32% of OTHERS. We searched an explanation for these surprising results: they could not be attributed to a more recent diagnosis in the group OTHERS nor to different extra support (relatives, baby sitters, social facilities) in the families of the two groups. We hypothesized different psychological effects in the 2 groups due to the disease’s characteristics. MPS parents know that disease is progressively slowly worsening and their major fatigue consists in accepting children’s inexorable loss of abilities and in managing their never ending care. OTHERS parents instead have to learn to cope with steady worries of a permanent risk of sudden decompensation. They need to be always alert, and this necessarily lead to manage a quote of anxiety every day and to be less incline to take care of their personal need. MPS parents require longer adaptation with certainty of death, but OTHERS have to deal everyday with sudden death. Most of MPS parents slowly “metabolized” the disease of their child and re-organized their life in function of it. OTHERS’s parents psychological resources are mostly dedicated to manage anxiety day by day.
Correlation Between Mobility and Quality of Life in Mucopolysaccharidoses
A. Fleming1, N. Cavalcanti1, C. Trevisan1, D. Horovitz1, S. Gomes Júnior1
1Instituto Fernandes Figueira, Rio de Janeiro
Introduction: The Mucopolysaccharidoses (MPS) are characterized by intra-lysosomal accumulation of glycosaminoglycans, leading to the emergence of progressive clinical manifestations and ongoing losses of biological and physiological functions that can lead to disability, reduced mobility and impaired quality of life (QOL). Objective: To study the possible association between mobility and quality of life in children and adolescents with MPS. Methods: A descriptive and cross-sectional study was undertaken at the National Institute of Women’s Child and Adolescent Health Fernandes Figueira (Rio de Janeiro /Brazil), a reference center for MPS and rare diseases. The sample included 14participants (age range2-18years) with MPS I-H (n = 2), MPS II (n = 4), MPS IV-A (n = 2) and MPS VI (n = 6). Held the PEDI questionnaire was applied to the parents, documenting their child’s capabilities in the functional area of mobility, followed by application of the QOL-PedsQL4.0questionnaire, related to physical capacity aspects, emotional, social and school activities. Results: The study hypothesis was confirmed, demonstrating there is significant Pearson correlation of 69% (p = 0.005*) between mobility and quality of life. A strong significant correlation of 78% between the subscale physical capacity with the mobility domain (p = 0.0008*) and of 63% between the subscale school activity with the mobility domain (p = 0.015*) were noted. The study also showed significant negative Pearson correlation of 78% between the physical capacity and degree of caregiver assistance (p = 0.0008*) subscale, 72% between school activity and caregiver assistance (p = 0.003*) subscale and 74% from the total Edsel score and caregiver assistance (p = 0.002*). Conclusion: The results of this study revealed that mobility directly influenced the QoL of children and adolescents with MPS, which demonstrates the importance of early introduction of rehabilitation, aiming at the acquisition of functional benefits and mobility, which will result in better QOL in this population.
Keywords
mucopolysaccharidoses, mobility limitation, quality of life
Items
Psychological functioning
Physical well-being
Job
Sleep and free time
Social functioning
MPS borderline %
4/50 8%
8/50 16%
5/50 10%
7/50 14%
10/50 20%
MPS pathological %
1/50 2%
3/50 6%
2/50 4%
2/50 4%
0/50 0%
OTHERS borderline %
4/32 12.5%
8/32 25%
3/32 9.3%
10/32 31%
4/32 12.5%
OTHERS Pathological %
2/32 6.25%
2/32 6.25%
1/32 3,12%
9/32 28.1%
0/32 0%
Importance of Common Data Elements (CDEs) for Cognitive and Behavioral Measures in Rare Disease Clinical Trials
K. Delaney1, E. Shapiro2
1Shapiro & Delaney, LLC, Mendota Heights
2Shapiro & Delaney, LLC, and University of Minnesota, Portland
Background: .Many new treatments for the mucopolysaccharidoses require neurocognitive assessment as a primary or secondary endpoint. These endpoints must be easy to use, reliable and valid as well as relevant to the age of the patient and the specific disease. For multicenter trials they must be based on standard measurements, that are validated, can be replicated across settings, and applicable in the culture of the setting. Challenges include recruiting young subjects, lack of natural history data, small numbers of patients being recruited for multiple trials, and phenotypic variability. Different test batteries yielding different information across trials confuse comparisons of treatments and leads to difficult choices for patients and providers. Development of common data elements which are sensitive, uniform, and disease/phenotype-specific in clinical trials is crucial to accurate measurement of a treatment’s efficacy. Cognitive and behavioral assessment and patient/parent reported outcomes are needed in diseases affecting the brain, but standard assessment approaches are often insensitive in rare diseases. Generic parent-reported outcomes (PROs) are often irrelevant to the very young and/or severely impaired patient. Consequently, disease-specific PROs and other measures need development. Methods: Improving the rigor, reliability, validity, and applicability of such endpoints in rare disease clinical trials require: 1. Common data elements (CDEs) across studies; note the NINDS project to develop disease-specific CDEs [1]. 2. Natural history studies to develop improved endpoints and CDEs for future trials as well as creating estimates of MCIDs (minimal clinically important differences) linked to rate of development 3. Training of center testers and other personnel to enable consistent data capture 4. Development of PROs specific to each disease Conclusions: It is of critical importance for researchers to combine data across centers using agreed upon CDEs in order to collect sufficient regarding sensitivity and to then utilize this data for treatment trials. CDEs will facilitate multicenter collaboration in data collection, and will provide an impetus to develop sensitive and specific endpoints in natural history studies that can map progression for each disease. In turn, these data can be used to design clinical trials in these rare disease populations.
Health-Related Quality-of-Life of Children With MPS I Hurler Transplanted With Hematopoietic Stem Cells
R. Parini1, R. Ciampichini2, L. Mantovani2, G. Cesana2, A. Rovelli1, S. Gasperini1, C. Galimberti1, L. Scalone2
1San Gerardo Hospital, Monza
2CESP—Research Centre on Public Health, Monza
Purpose: Hematopoietic stem cell transplantation (HSCT) is at present the only available treatment for patients with MPS I Hurler, able to prevent central nervous system deterioration. Its safety and long-term efficacy have recently been shown in a multicenter study on 217 patients [Aldenhoven M et al. Blood 2015]. Few data are available on Health-Related Quality-of-Life (HRQoL) of transplanted children. We aimed to assess HRQoL in patients with MPS I Hurler who underwent successful HSCT. Methodology: We analyzed the answers given on children accessing at our Pediatric Department using the MPS-Health Assessment Questionnaire (MPS-HAQ). MPS-HAQ was administered yearly to successfully transplanted Hurler patients as part of their long-term clinical follow-up. We report results on the assistance required in mobility and self-care at the age between 6 and 10 years. Results: The data refer to 10 patients, 2 males, with a developmental quotient at the time of the data analyzed of 50-110. Their parents completed the questionnaire. All the children were completely independent in their mobility at home and climbing stairs; 2/10 needed minimal assistance while walking outside; 8/10 needed assistance (mostly minimal) in getting into a car. All but one needed assistance in toileting activities (grooming, bathing, tooth brushing, managing toilet seat and toilet paper) and in dressing upper part of the body; all 10 patients needed assistance in dressing lower part of the body. Eating was not independent for 9 of them needing minimal assistance mainly in using the knife. Conclusion: HSCT has completely modified the natural history of MPS I Hurler patients. According to our experience untransplanted patients aged 6-10 years would be much more limited in their activities. However our results show that, despite the beneficial effect of HSCT, most of the patients had joint and musculo-skeletal limitations as shown by their difficulties in mobility, in dressing and in eating. Probably the mild/moderate mental delay of some patients has also a role, but less relevant, in determining these results. We hope that other treatments would be able to further improve in the future the prognosis of Hurler patients.
The Educational Journey of Individuals With MPS II Hunter Disease in the United Kingdom
S. Thomas1, A. Morrison1
1The Society for Mucopolysaccharide Disease (MPS Society), Amersham
Objectives: To determine the cognitive variability in patients with MPS II and to understand their needs and support requirements in an educational setting. Methods: Individuals with MPS II resident in the UK were identified by the MPS Society and invited to take part in the survey via telephone interview. A specifically designed questionnaire was used to assess the individual’s diagnoses, treatment, educational attainment and need for support from primary through to further education. Results for the education section of the survey only are presented here. Interviews took place in December 2015 and January 2016. Results: Forty one patients were recruited to the study, ranging in age from 1 to 36 years (mean 12 years). One patient was too young to have attended nursey/primary school and is excluded from this analysis. The majority of individuals started their education in a mainstream nursery/primary school; mainstream 72% (29/40), special educational needs (SEN) establishment 27% (11/40). One third of individuals (n = 13) moved schools as their learning needs were not being met (average age of move 6.8 years). Individuals with central nervous system (CNS) involvement were more likely to move school than those without (55% vs 7%). Nineteen individuals had attended or were attending secondary school; mainstream 63% and SEN 36%; one individual moved from SEN to mainstream at age 12. Statements of educational need or educational healthcare plans (EHPs) were issued to 73% of individuals in primary school (mean age 4.9 years). Less individuals (68%) had statements or EHPs during their secondary education and 23% of these had been issued at secondary school (mean age 11.3 years). The reasons for issue were; learning needs (15%, 7%), physical needs (5%, 15%) or both (52%, 53%) in primary and secondary education respectively. More individual education plans (73% vs 42%) and flexible teaching (57% vs 40%) were available in secondary compared to primary schools. Flexibility included alternatives to physical education lessons (26%), support lessons and options to drop a GCSE in mainstream schools and totally individual lesson plans in SEN schools. More support was reported in primary school with: help to understand tasks (62% vs 21%), staying on task/focused (60% vs 36%), one to one support (47% vs 47%), writing/scribing (42% vs 26%), personal care (47% vs 26%), behavior (40% vs 21%), moving around school (30% vs 26%), physical education (25% vs 10%). The most commonly used specialist equipment in primary schools were chairs, pencil grips and laptops/ipads (all15%). In secondary school, hearing and radio aids (26%), specialist chairs (21%) and laptops/ipads (15%) were commonly used. Individuals received input from a range of professionals at primary and secondary school; speech and language (65% and 42%), physiotherapists (50% and 42%), SEN coordinators (45% and 68%), educational psychologists (42% and 31%) and occupational therapists (40% and 31%). Sixty three percent of individuals felt that their support needs had changed from primary to secondary education; decline in mobility/more help to move around larger schools (31%), difficulty understanding work (57%), deteriorating health/surgery (15%). Of the13 individuals aged 16 or over, 69% had obtained GCSE or equivalent qualifications. Ten individuals were attending or had attended further education including 6th form, college and university. All received support; one to one (30%), support unit/group (30%), physical/medical (50%), scribe (30%), extra time (10%). Of the 8 individuals who had completed their education 50% were working in voluntary or paid employment. Conclusions: There is a wide range of support available for individuals with MPS II in both SEN and mainstream education. Educational needs for those with CNS involvement cannot always be met in mainstream primary schools, but is usually recognized, and more suitable SEN schooling found by age seven. Support requirements and the need for a flexible approach appear to change from primary to secondary school due to disease progression, the demands of moving around a larger school and the difficulty of the school work. Half of the MPS II sufferers surveyed who had completed their education had gained further education qualifications and found employment in the voluntary or paid sectors.
The Educational Journey of Individuals With MPS IV Morquio Disease
S. Thomas1, A. Morrison1
1The Society for Mucopolysaccharide Disease (MPS Society), Amersham
Objectives: To determine the educational and employment history of individuals with MPS IV. Methods: Individuals with MPS IV identified by the MPS Society were invited to take part in the survey via postal questionnaire in April and May 2014. A specifically designed questionnaire was used to assess the individual’s educational attainment and need for support from primary through to further education as well as their employment history. Results: Forty six individuals responded to the survey. The majority of individuals attended mainstream schools; primary 89% (41/46), secondary 83% (25/30). Most followed the National Curriculum (78% primary, 73% secondary). Sixty one percent had a statement of educational need in primary school that was issued at an average age of 5.3 years (range 3 to 12 years). This rose to 66% in secondary school. Seventy percent of individuals needed additional help at primary school compared to 63% at secondary. The most common requirements at primary school were help moving around the school (37%), getting ready for physical education (PE) (35%) and with writing (26%). Similar needs were reported at secondary school: moving around school/carrying things (43%), personal care/dressing (40%), writing (27%), PE (20%) and one to one support (17%). Special equipment used in primary schools included typewriters/laptops/word processors (35%), special chairs (35%) and pencil grips (20%). Typewriters/laptops were commonly used in secondary schools as well (30%). In primary school 13% of individuals used wheelchairs, 7% used scooters/buggies and 4% had walking frames. Wheelchair use increased to 40% in secondary school and 10% used scooters. There was considerably less input from professionals in primary school. The most frequently seen professionals in secondary school were: occupational therapist (40%), physiotherapist (37%), special educational needs coordinator (37%) and educational psychologist (17%). No one reported input from these professionals in primary school, where the most frequently seen specialists were advisory teachers for hearing and physical disabilities (4%). Fifty four percent felt that the number of medical appointments impacted on their primary education, versus 46% on secondary education. Overall 91% reported their experience of primary school as positive, compared to 86% positive for secondary school. Eighty five percent (23/27) of individuals were studying for, or had gained, GCSE qualifications. A further 11% had gained other secondary school qualifications and only 1 individual had not taken exams at school. Twenty one individuals were in or had completed further education, of which 47% were studying for or had gained honors or higher degrees. Only one individual had gone straight from secondary school to employment. Of the 16 individuals who had completed their education, 81% were or had been employed. Conclusions: The educational needs for most individuals with MPS IV were met by mainstream schools. Individuals received very little specialist input in primary school, but were well supported in secondary school. Most individuals had gone on to further education and a high proportion attended university.
Quality of Family Life in Patients With Morquio Type IV A Syndrome: The Perspective From the Colombian Social Context (South America)
D. Ortiz Quiroga1, Y. Ariza-Araujo1, H. Pachajoa1
1Universidad Icesi, Cali
Purpose: People living with “rare diseases” have to face many barriers in order to have access to the services offered by the Colombian Health System. Eighteen years ago, organizations like the Colombian Association of Patients with Lysosomal Storage Diseases (in Spanish Asociación Colombiana de Pacientes con Enfermedades de Deposito Lisosomal—ACOPEL) sought to facilitate this by providing legal assessment and emotional and economical support in order to improve the quality of life of patients and their families. However, previously an integral approach towards the patient’s disability and the quality of life of the family had not been considered. This paper seeks to characterize the quality of life of the family of patients with Morquio type IV A syndrome that are currently active members of ACOPEL. Methodology: A survey was undertaken on key members of the families in which at least one member has a diagnosis of Morquio type IV A syndrome and that are active members of ACOPEL. The following tools were used: Kansas’ University Quality of Family Life Survey and the Scale of Quality of Family Life (SQFL) adapted for Colombia. The tool evaluates five variables: family interaction, parental role, health and security, general resources and support for patient with disability (PWD). Each variable was evaluated according to the importance and degree of satisfaction according to the caretaker. The Map of Quality of Family Life (MQFL) that points to two areas (critical and strong) was also used. The information was stored and processed using Excel. Results: From 121 patients enrolled to ACOPEL in Colombia until 2015, 102 (84%) were surveyed, which corresponds to 81 families. The results of the MQFL demonstrated that for all of the questioned variables, the most frequent answer was “satisfied”. However, the proportion of satisfied families varied from one factor to another. The reported frequencies of “satisfied” were: family interaction (91%), parental role (90%), health and security (77%), family resources (56%), and support to the PWD (59%). The families reported “less satisfied” in the factors: family resources (29%) and support to the PWD (35%). These areas were considered as “critical” in the MQFL, and recommendations were given on each case. Conclusions: Families related the dissatisfaction of their quality of life, as an immediate result of the health condition of the family member affected with Morquio type IV A syndrome. The participants cannot distinguish the terms: deficit and disability, which confirms the dominance of the biomedical model which is reinforced by the assistance practice provided by the programs of social action offered by the government and private organizations. This perspective limits the recognition of the capacity of the family to transform their reality. It is expected that this experience will be useful for the participants and for ACOPEL which will reconsider the traditional assistance practice that is currently being provided in order to give way to the self-determination of the families.
Social Vulnerability: The Reality of Patients With Morquio Type IV A Syndrome in Colombia
D. Ortiz Quiroga1, Y. Ariza-Araujo1, H. Pachajoa1, J. Gonzalez2
1Universidad Icesi, Santiago de Cali
2Acopel, Bogotá
Purpose: People with Morquio type IV A syndrome present a series of limitations in order to perform basic, instrumental and advanced activities of daily living in a functional and independent way. This limitation is a result of a series of conditions: on one side, the clinical severity of the disease, and on the other side, the barriers of the socio-cultural context of the patient. The family, as the main socializing context of people with a disability, becomes the most valuable source of information in order to identify the necessary services and types of supports in order to improve the patient’s quality of life. Therefore, this paper sought to identify which were the most important assistances, the patients and their families need and receive from the Colombian National Health System. Methodology: A survey was undertaken on key members of the families, in which at least one member had a diagnosis of Morquio type IV A syndrome and that are active members of the Colombian Association of Patients with Lysosome Storage Diseases (ACOPEL). The section “Support and Services” from the Scale of Quality of Family Life (SQFL) was used. The tool evaluated the services a person with Morquio type IV A syndrome and his/her family needs and receives. Additionally, a semi-structured interview was built and used by the team in order to deepen the productive situation of the population in all of the stages of the vital cycle. Results: From 121 patients enrolled to ACOPEL in Colombia until 2015, 102 (84%) were surveyed, which corresponds to 81 families. The results from the “Support and Services” section revealed that the most important services for patients and their families were: medical check-ups and receiving technique and transportation aids. In the same way, families consider that “transportation service” is their most urgent necessity, followed by the need of money and in third place to participate in support groups. On the other hand, the services that the families considered to have less importance included: technical formation for a job and vocational orientation, and language and behavioral support. Furthermore, the semi-structured interview revealed that 50 patients attended school in the previous year, out of which 19 manifested being bullied, especially those attending public institutions. Likewise, it was identified that 72% of the patients, which are 18 years or older, are currently unemployed. Conclusions: All services considered within the instrument were referred to as necessary for people with Morquio IV-A syndrome and their families, but health services were those ranked as priority. Thus, the results of this research are presented as a baseline, which sets the needs of people with Morquio syndrome and their families for the joint construction of a public policy that generates a change from care and access to health and social services.
Quality of Life and Activities of Daily Living in Alpha-Mannosidosis: Long-Term Data of Enzyme Replacement Therapy With Velmanase Alfa (Human Recombinant Alpha Mannosidase)
F. Cattaneo1, L. Borgwardt2, C. Dali2, A. Tylki-Szymanska3, F. Wijburg4, J. Van den Hout5, J. Fogh6, D. Ardigò1, A. Lund2, D. Phillips7
1Chiesi Farmaceutici S.p.A., Parma
2Rigshospitalet, Copenhagen
3The Children’s Memorial Health Institute, Warszawa
4Amsterdam Medical Centre, Amsterdam
5Erasmus MC-Sophia, Rotterdam
6Zymenex A/S, Hillerød
7University of North Carolina, Chapel Hill
Purpose: To investigate the Activities of Daily Living (ADL), physical function, pain, and overall health of patients affected by Alpha-Mannosidosis during treatment with velmanase alfa (human recombinant alpha mannosidase). Alpha-Mannosidosis is a rare, autosomal recessive lysosomal storage disorder with no approved therapeutic option. Velmanase-alfa is a recombinant version of human alpha mannosidase developed as intravenous enzyme replacement therapy for the treatment of Alpha-Mannosidosis. Methodology: A total of 33 patients, adults (N = 14, age range 18-35 years) and pediatrics (N = 19, age range 6-17), previously enrolled in phase I/II (N = 9; rhLAMAN-02, -03, -04) and phase III (N = 24; rhLAMAN-05) studies with velmanase alfa were assessed for efficacy and safety in a comprehensive, open-label, uncontrolled, pivotal clinical trial (rhLAMAN-10). Mean (SD) exposure was 890.5 (461.5) days (ranging from 357 to 1625 days). All patients included in the study received the intended clinical dose of 1 mg/kg/week by intravenous infusion for at least 12 months, whereas more than half (N = 19, 57.6%) for at least 24 months. The Childhood Health Assessment Questionnaire (CHAQ) and the Euro QOL 5D 5 L (EQ5D5 L) were administered at baseline and twice yearly. Two scales were derived from CHAQ: a Disability Index with 30 age-appropriate items in 8 subscales of ADL and a Discomfort (pain) Index based on a 100 mm visual analog scale (VAS). Both scales are scored from 0 to 3 (0 = functional independence; 3 = complete dependence on caregiver). A decrease of ≥ 0.13 was considered as the minimal clinically important difference (MCID) under treatment, similarly to that used in juvenile arthritis. The EQ5D5 L is a standardized measure of health designed to provide a simple, generic measurement through 5 dimensions (mobility, self-care, usual activities, pain/discomfort, and anxiety/depression). Each dimension is rated on a 5-point scale, from “no problems” (coded as 1) to “extreme problems” or “unable to complete” (coded as 5). The dimension scores can be used to create a single summary Health Index, ranging from 0 (worst) to 1 (best). An increase of 0.05 was considered as the MCID under treatment, similarly to that used in Multiple Sclerosis. EQD5D5L’s VAS ranged from 0 (“best health you can imagine”) to 100 (“worst health you can imagine”). Results: Mild to moderate disability was documented in the overall Alpha-Mannosidosis patients by the mean Baseline CHAQ Disability Index values. A greater level of disability and pain at Baseline on the CHAQ and poorer overall health on the EQ5D5 L was observed in pediatric patients, indicating a disease progression in the initial phase of the disease. The mean (SD) CHAQ Disability Index decreased from baseline to last observation from 1.22 (0.89) to 1.07 (0.68) for the pediatric group and from 1.55 (0.55) to 1.52 (0.65) for adults. A decrease (66.6%) in the number of seriously impaired patients according to CHAQ Discomfort Index was observed from baseline (N = 3, 9.4% of the overall population) to the last observation (N = 1, 3.0%). A corresponding increase in subjects who were not or poorly impaired was also observed (N = 24, 75% to 28, 84.8%, respectively). Although these changes in CHAQ scores were not statistically significant, the mean change in the Disability and Discomfort Indexes in pediatric patients was greater than the established MCID. EQ5D5 L Health Index values increased from baseline to the last observation from 0.697 (0.184) to 0.757(0.157) in subjects <18 years and from 0.568 (0.142) to 0.618 (0.183) in adults. A clear statistical trend for mean increase was observed in EQ5D5 L Health Index and VAS (p=0.036 for change from baseline to last evaluation for Health Index). Notably, all EQ5D5 L Health Index mean changes from Baseline to the last evaluation met the lower range for MCID ranging from 0.05 to 0.084, being the result applicable to the entire group and by age sub-analysis. Conclusions: Improvements in ADL and physical function following velmanase alfa treatment were detected in the pediatric group with a mean difference exceeding MCID for CHAQ Disability Index. Overall, a beneficial health improvement in the whole treated population was measured by EQ5D5 L Health Index, for which mean changes exceeding the established MCID were reached from baseline to the last evaluation. Pain was reduced in all groups and exceeded the MCID. Data support a beneficial impact of chronic treatment with velamanase alfa on quality of life and daily function in patients with Alpha-Mannosidosis.
Lysosomal Dysfunction Drives Neurodegeneration by Disrupting Presynaptic Maintenance
A. Fraldi1, I. Sambri1, R. D’Alessio1, Y. Ezhova1, T. Giuliano1, N. Sorrentino1, V. Cacace1, M. De Risi1, M. Cataldi2, E. De Leonibus1
1TIGEM, Naples
2University Federico II, Naples
Several neurodegenerative conditions are associated to the failure of lysosomal system. Seeking to understand the mechanisms underlying this functional link we discovered that lysosomal dysfunction disrupts presynaptic integrity by a α-synuclein-/CSPα-dependent pathway. In mouse models of neurodegenerative lysosomal storage disorders (LSDs), and in cultured neurons treated with lysosomal inhibitors, lysosomal pathology develops progressively with alterations in presynaptic structure and function. In these models, impaired lysosomal activity causes massive perikaryal accumulation of insoluble α-synuclein and increased proteasomal degradation of CSPα. As a result, the availability of both α-synuclein and CSPα at nerve terminals strongly decreases, thus deregulating the proteostasis of soluble NSF attachment receptor (SNARE) and inhibiting their assembly in functional complexes. Aberrant presynaptic SNARE phenotype is partially recapitulated in mice with genetic ablation of one allele of both CSPα and α-synuclein. Overexpression of CSPα in the brain of LSD mice efficiently re-established SNARE-complex levels, thereby ameliorating presynaptic function and attenuating neurodegenerative signs. Our data demonstrate that the loss of lysosomal function disrupts the proteostasis of SNAREs at nerve terminals. This pathway is mediated by the concurrent presynaptic depletion of α-synuclein and CSPα and is a key determinant of neurodegenerative processes in lysosomal diseases, thus suggesting alternative therapeutic interventions for these severe neurodegenerative conditions.
Synaptic Dysfunction in Sanfilippo Type C Syndrome
C. de Britto Para de Aragao1, L. Bruno1, C. Han2, G. Di Cristo1, P. McPherson2, C. Morales3, A. Pshezhetsky1
1CHU-Sainte Justine Research Center, Montreal
2Montreal Neurological Institute, Montreal
3McGill University, Montreal
Sanfilippo type C syndrome, also known as mucopolysaccharidosis IIIC (MPSIIIC) is the lysosomal storage disorder triggered by mutations in the HGSNAT (acetyl-CoA:α-glucosaminide N-acetyltransferase) gene causing lysosomal storage of glycosaminoglycan, heparan sulfate. In humans, MPSIIIC causes rapid neurological decline but mechanisms underlying the neuropathophysiology are still not well understood. In mice, inactivation of the Hgsnat gene triggered hyperactivity at the age of 6-8 months followed by cognitive and memory decline and significant neuronal loss at 10 months. To test the hypothesis that cognitive dysfunction occurring in the MPSIIIC mice prior to the age when significant neuronal loss is observed is associated with a decrease in neurotransmission, we analyzed the synaptic transmission in neuronal cultures established from embryonic day 16 of HGSNAT KO and C57BL/6 WT mice. Cultured neurons at 21 days were and analyzed by immunocytochemistry for several pre- and post-synaptic markers and also processed for transmission electron microscopy. Density and morphology of synaptic spines were studied using hippocampal cultures and brain sections from the MPSIIIC mice expressing GFP in hippocampal neurons. Cultured hippocampal neurons from the MPSIIIC mouse model have shown a decrease in expression of presynaptic proteins such as synaptophysin and synapsin. Synaptophysin, vGLUT1 and neuroligin-1 were not widely distributed along the axons as in the WT neurons but showed perinuclear localization in the soma. In post-synaptic MPSIIIC neurons, the expression of PSD95 and neuroligin-1 was drastically reduced. The spine density and maturity on hippocampal neurons from MPSIIIC mice was decreased already at postnatal day 20 and further declined with age. Besides, spines also showed irregular morphology and distribution along the dendrites; in culture it was observed that MPSIIIC neurons presented immature spines (filopodia shape). Cultured neurons analyzed by electron microscopy revealed massive lysosomal storage and compromised microtubules network. Synaptic terminals presented sparse and disorganized synaptic vesicles. Altogether our data demonstrate deficiency in synaptic vesicle trafficking in hippocampal neurons of MPSIIIC mice directly affecting pre- and post-synaptic neurons and presumably compromising the whole process of synaptic transmission.
Microglia Plays a Central Role in the Alteration of Brain Iron Metabolism in Sanfilippo Syndrome
V. Puy1, C. Gomila2, P. Bodet3, Z. Karim4, S. Vitry5, A. Hodroge1, F. Gonnet3, R. Daniel3, J. Ausseil2
1CHU AMIENS PICARDIE, Amiens
2Unité INSERM U1088, Amiens
3Université d’Evry Val d’Essonnes
4INSERM U1149, Paris
5Institut Pasteur, Paris
Purpose: In Sanfilippo syndrome, the deficiency of an enzyme involved in heparan sulfate catabolism leads to the accumulation of Heparane Sulfate Oligosaccharides (HSO) with different sizes and different degrees of sulfatation. This progressive accumulation induces series of pathological consequences including neurodegeneration, neuroinflammation and oxidative stress development. We have already shown that diffuse microgliosis and astrocytosis are present throughout the brains of MPSIIIB mice. Although neuroinflammation is a major component of the disease, it is not the main cause of neurodegeneration and oxidative stress. As subsequent cranial MRI imaging revealed progressive brain iron accumulation in deep brain nuclei of 2 MPSIIIB patients, we hypothesized that an alteration of local iron metabolism leads to its accumulation in the brain and finally generates oxidative stress by production of reactive oxygen and nitrogen species (ROS, RNS). ROS and RNS could participate to the neurodegeneration development. Methodology: The main objectives of our present work were 1/to investigate whether iron metabolism is dysregulated in this syndrome and 2/to determine by which mechanism this alteration could occur. Iron homeostasis is partly regulated by hepcidin which expression at transcriptional level is partly modulated by inflammation. Results: We showed that mRNA expression of hepcidin is already higher in MPSIIIB brain than in normal mice at 3 months of age, and this expression increased with aging. This overexpression inhibits ferroportin expression leading, at the end, to iron retention in brain cells. To better understand these dysfunction, we first aimed to determine whether HSO were directly involved in iron accumulation. We isolated HSO from MPSIIIB patient urines and we also produced specific HSO fragments by heparinase (I, II, III) digestion of commercial HS. These fragments were then purified according to their size. We showed that HSO isolated from MPSIIIB urines and most predominantly hexasaccharides produced by heparinase HS digestion, activate the production of hepcidin when added to microglia cultures. This overexpression is mostly the consequence of STAT3/Phospho STAT3 signaling pathway activation. Interestingly, none of these fractions have an effect on neuron culture indicating that increased expression of brain hepcidin is directly caused by HSO accumulation and microglia activation. Conclusion: In this study, we show a central role of microglia in hepcidin overexpression leading to brain iron accumulation, probably partly responsible of the neuropathology.
Processing and Trafficking of N-Acetyl-α-Glucosaminidase in Fibroblasts of Patients With Mucopolysaccharidosis Type IIIB
O. Meijer1, R. Ofman1, H. te Brinke1, L. IJlst1, N. van Vlies1, F. Wijburg1
1Academic Medical Center, Amsterdam
Background: Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal recessive disorder caused by a mutation in the gene encoding for the lysosomal enzyme N-acetyl-α-glucosaminidase (NAGLU), resulting in the accumulation of heparan sulfate (HS). Patients suffer from progressive neurocognitive deterioration. However, a wide spectrum of disease severity is seen, with patients with a classical, severe and rapidly progressing (RP) phenotype on one side of the spectrum and patients with a more attenuated, slowly progressing (SP) course, on the other side. Earlier studies showed that NAGLU activity in skin fibroblasts from patients with an SP phenotype when cultured at 30°C, have a significantly higher NAGLU activity (up to 10% of control) and lower HS levels than observed in fibroblasts from RP patients. This indicates that the enzyme exerts its catalytic function in the lysosome. We now studied the underlying processes that result in the increase of residual NAGLU activity after culturing at 30°C, in order to design targeted therapies. Methods: Skin fibroblasts from controls, RP MPS IIIB patients (mutations: p.A72_G79dup8 and p.A72_G79dup8; p.R297* and p.R297*) and SP patients (mutations: p.R565 W and p.E634 K; p.S612G and p.S612G; p.G170D and p.H248 R; p.R643C and p.R643C) were cultured at 37°C or 30°C and harvested at different time points to determine NAGLU activity and to assess NAGLU protein by Western blot analysis. Immunofluorescence was used to analyze intracellular trafficking of normal and mutant NAGLU at 37°C and 30°C. Results: NAGLU protein in control cell lines was present in two forms: a 85 kDa precursor and a 82 kDa mature form. In fibroblasts from SP MPS IIIB patients, only the 85 kDa band was detected at 37°C. However, culturing at 30°C revealed the presence of the 82 kDa NAGLU protein, which corresponded with an increase of enzyme activity. These effects were absent in fibroblasts from RP MPS IIIB patients. Conclusion: We show that the NAGLU protein contains two forms: a 85 kDa precursor and a mature form. Culturing fibroblasts of SP MPS IIIB patients at 30°C resulted in similar levels of NAGLU protein when compared to controls, and a significant increase in residual NAGLU activity. These data suggest that both processing and intracellular trafficking of mutant NAGLU strongly improves upon culturing at a lower temperature. A better understanding of these processes could provide new clues to therapeutically enhance residual enzymatic activity in MPS IIIB patients with an SP phenotype.
Virtual Screening and In-Vitro Evaluation of Two Potential Pharmacological Chaperones for N-Acetylgalactosamine-6-Sulfate Sulfatase
C. Alméciga-Diaz1, E. Guzmán1, S. Olerte-Avellaneda2, L. Pimentel1, A. Rodriguez-Lopez1
1Pontificia Universidad Javeriana, Bogota
2Universidad Colegio Mayor de Cundinamarca, Bogota
Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease produced by mutations in N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Currently, the approved treatment for Morquio A is based on enzyme replacement therapy (ERT) by using elosulfase alfa. However, elosulfase alfa has some limitations that include: i) a reduced effect on skeletal, corneal, and heart valvular issues, ii) a short half-life of the enzyme and rapid clearance from the circulation, iii) immunological issues, and iv) a high cost. In this sense, it is necessary to explore new treatment alternatives such as gene therapy, hematopoietic stem cell transplantation, or pharmacological chaperones. In this study, we report the in-silico and in-vitro evaluation of two potential pharmacological chaperones for GALNS enzyme. Previously, we reported the modelling of GALNS tertiary structure and its interactions with natural and artificial ligands. We used this model to identify potential GALNS chaperones through a virtual screening against a drug library. Two ligands (MoIEM-1 and MoIEIM-2) showed higher affinity energy than that of chondroitin-6-sulfate, and were selected for in-vitro evaluation. The selected compounds were tested against purified recombinant GALNS produced in Pichia pastoris, as well as during the production of recombinant GALNS in E. coli and P. pastoris. The results showed that both ligands compete with the artificial substrate for their binding to the active cavity of the enzyme. Addition of these compounds to cultures of E. coli and P. pastoris expressing human recombinant GALNS led to an activity increased of up to 5-fold. It is noteworthy that incubation of MoIEM-1 and MoIEIM-2 with recombinant iduronate-2-sulfatase did not produce an increase in enzyme activity, showing the specificity of these compounds. These results show that MoIEM-1 and MoIEIM-2 can be consider as potential pharmacological chaperones for GALNS towards the development of a treatment for Morquio A.
Changes in the Cell Cycle of MPS Fibroblasts and Genistein-Mediated Modulation of this Process
G. Wegrzyn1, M. Moskot2, M. Gabig-Ciminska2, J. Jakobkiewicz-Banecka1, E. Piotrowska1, E. Smolinska1, K. Bochenska1, M. Wesierska1
1University of Gdansk
2Institute of Biochemistry and Biophysics, Gdansk
Purpose: One of possible therapeutical options for mucopolysaccharidoses (MPS) is the use of small molecules, able to cross the blood–brain-barrier, which could impair synthesis of glycosaminoglycans (GAGs). This strategy is called substrate reduction therapy [1]. It was demonstrated that genistein (5, 7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) inhibited GAG synthesis in MPS fibroblasts in vitro [2]. This inhibition appears to be due to impairment of the epidermal growth factor receptor activity, and further down-regulation of the signal transduction, which normally leads to stimulation of expression of genes coding for enzymes involved in GAG synthesis [3]. Despite different laboratories reported various effects of genistein on GAG production and accumulation in different cell lines [2-7], recent studies, employing microarray analyses followed by real-time quantitative RT-PCR identified particular genes coding for enzymes necessary for GAG synthesis which were inhibited by genistein, while most of genes for GAG lysosomal hydrolases were stimulated by this isoflavone [8, 9]. This stimulation was apparently due to positive regulation of the transcription factor EB (TFEB), a master regulator for lysosomal biogenesis and function [8, 9]. Nevertheless, transcriptomic studies indicated also that expression of many other genes can be regulated by genistein. Therefore, we aimed to investigate effects of this isoflavone on the cell cycle in normal and MPS fibroblasts. Methodology: Human fibroblasts, derived from either a healthy person or MPS patients, were used. High throughput screening with microarrays, coupled to real-time quantitative Reverse Transcription PCR analyses, was employed to study the changes in expression of key genes associated with cell cycle regulation and/or DNA replication in response to genistein. Progression in the cell cycle was monitored by using the MUSE Cell Analyzer (Merck Millipore, Germany). Results: Genistein significantly modulated, in a time-and dose-dependent manner, activity of genes which products are involved in different phases of the cell cycle and in regulatory processes important for DNA replication and cell growth. It considerably reduced the efficiency of expression of genes coding for MCM2-7 and MCM10 helicases, as well as some other proteins involved in the S phase control. Moreover, genistein caused cell cycle arrest in the G2/M phase, which was accompanied by activation of CDKN1A, CDKN1C, CDKN2A, CDKN2B, CDKN2C, and GADD45A genes, as well as down-regulation of several mRNAs specific for this stage, demonstrated by transcriptomic assessments. Effects of genistein on cell proliferation was similar in both normal and MPS fibroblasts. However, in non-treated fibroblasts, fractions of cells in the G0/G1 phase were higher, and number of cells entering the S and G2/M phases was considerably lower in MPS II fibroblasts relative to control cells. Somewhat similar tendency, though significantly less pronounced, could be noted in MPS I, but only at longer times of incubation. On the other hand, this was not observed in MPS IIIA and MPS IIIB fibroblasts. Genistein was found to be able to partially correct the disturbances in the MPS II cell cycle, and to some extent in MPS I, at higher concentrations of this compound. The tendency to increase the fractions of cells entering the S and G2/M phases was also observed in MPS IIIA and IIIB fibroblasts treated with genistein. Conclusions: Cell cycle is disturbed in MPS I and MPS II fibroblasts. Genistein modulates cell cycle in wild-type and MPS cells through modulation of expression of genes which products are involved in the regulation of this process, as well as in DNA replication and cell growth. Genistein can also partially correct the cell cycle progression in MPS fibroblasts. Literature: [1] Cox T.M. (2015) Best Pract. Res. Clin. Endocrinol. Metab. 29:275-311; [2] Piotrowska E. et al. (2006) Eur. J. Hum. Genet. 14:846-852; [3] Jakobkiewicz-Banecka J. et al. (2009) J. Biomed. Sci. 16:26; [4] Arfi A. et al. (2010) J. Inherit. Metab. Dis. 33:61-67; [5] Kloska A. et al. (2011) Metab. Brain. Dis. 26:1-8; [6] Otomo T. et al. (2012) Mol. Genet. Metab. 105:266-269; [7] Kingma S.D. et al. (2014) J. Inherit. Metab. Dis. 37:813-821; [8] Moskot M. et al. (2014) J. Biol. Chem. 289:17054-17069; [9] Moskot M. et al. (2015) Sci. Rep. 5:9378.
IVA336 A Potential Substrate Reduction Therapy for Mucopolysaccharidose Type-VI, -I, and -II Diseases
M. Tallandier1, P. Masson1, E. Entchev1, S. Jacquet1, I. Jantzen1, O. lacombe1, V. Douet1, P. Broqua1, J. Junien1, J. Abitbol1
1Inventiva, Daix
Purpose: IVA336 is an orally-active competitor of the galactosyl transferase I substrate. It allows the synthesis of soluble Glycosaminoglycans (GAGs), mainly chondroitin sulfate (CS) and dermatan sulfate (DS). The elimination of neosynthesized soluble GAGs is mainly via urine excretion and they do not enter the classical lysosomal degradation pathway. IVA336 was originally, developed as an antithrombotic agent for the prevention of venous and arterial thromboembolism due to its action on heparin cofactor 2. Antithrombotic activity of IVA336 is directly proportional to IVA336 dose and subsequent soluble DS synthesized. IVA336 was administered to more than 1800 subjects in phase I and Phase II trials. It was safe and well tolerated. Medical need for the treatment of MPS is still very high as ERTs have not been able to act in certain regions of the CNS, ophthalmological system and joints due to the blood-brain barrier and comparatively poor vascularization of the joints preventing penetration of the enzymes to these regions. We evaluated the effect of IVA336, in vitro and in vivo, on GAG storage. Methodology: In vitro, the effect of IVA336 was tested on intracellular and extracellular GAG contents in fibroblasts isolated from donors affected with MPS I, VI, or II or from non-affected donors (cells originate from the Coriell Institute repository). In vivo, effects of IVA336 on GAG storage was studied in mice disease relevant model. Tissue distribution of IVA336 was studied in vivo in rats after oral administration. Results: In vitro, treatment with IVA336 led to a complete dose-dependent inhibition of intracellular CS storage in fibroblasts. Though the basal CS load was much higher in the fibroblasts from MPS donors compared to non-affected donors, the effect was comparable in both cell types (IC50 in the μM range). Interestingly, IVA336 inhibitory effect is observed when treatment is applied early or late after the cell plating (i.e. when CS accumulation is lower or very high, respectively). In vivo, on relevant MPS mice model, IVA336 reduced GAG accumulation in different organs (including liver). In vivo activity is currently being further characterized. Analysis of the tissue distribution of IVA336 after oral administration in rats (10 mg/kg for 5 days), demonstrates that organs that are poorly targeted by ERTs such as bones, cartilages, retina, and heart are exposed at pharmcological active concentrations. Conclusion: IVA336 is a small molecule orally active that has demonstrated safety and tolerability in adult subjects in clinical trials. IVA336 is reducing GAG storage in vitro and in vivo. After oral administration, IVA336 is distributed throughout the body and is found at pharmacological active concentrations in bones, cartilages, retina, and heart. IVA336 is proposed a novel substrate reduction therapy for MPS in which CS and/or DS accumulate. Together, the data support the clinical investigation of IVA336 for the treatment of MPS I, II, and VI. A clinical trials in MPS VI patients is planned to start within a year.
Subunit Interactions of Mucolipidosis II and III-Related Hexameric GlcNAc-1-Phosphotransferase Complex
R. Voltolini Velho1, R. De Pace1, T. Braulke1, S. Pohl1
1University Medical Center Hamburg-Eppendorf, Hamburg
Defects in the Golgi-resident GlcNAc-1-phosphotransferase cause the inherited lysosomal storage disorders mucolipidoses type II and III. The GlcNAc-1-phosphotransferase is a hexameric complex consisting of two α-, two β- and two γ-subunits that catalyzes the formation of mannose 6-phosphate (M6P) recognition marker on lysosomal enzymes, required for their efficient receptor-mediated transport to lysosomes. GNPTAB and GNPTG encode the membrane-bound α/β-subunit precursor membrane protein and the soluble γ-subunit, respectively. The cleavage of the α/β-subunit precursor to mature α- and β -subunits by the site-1 protease in the Golgi lumen is essential for GlcNAc-1-phosphotransferase activity. Performing extensive mutational analysis followed by pull-down experiments, we identified the regions in GlcNAc-1-phosphotransferase for the interaction between the subunits. The γ-subunit binding in a previously uncharacterized domain of the glycosylated α-subunit, named GNPTG binding (GB) domain. Both deletion of GB preventing γ-subunit binding and CRISPR/Cas9-targeted deletion of GNPTG led to significant reduction of GlcNAc-1-phosphotransferase activity. We also identified the cysteine residue responsible for covalent homodimerization of α-subunits. Homodimer formation of α-subunits is, however, neither required for interaction with γ-subunits nor for catalytic activity of the enzyme complex. Finally, binding assays using various γ-subunit mutants revealed that both N-terminal (aa 26-69) as well mid regions (aa 130-238) of the γ-subunit contribute to the interaction with α-subunit most likely by forming a conformation-dependent protein domain. These studies provide new insight into the assembly of the GlcNAc-1-phosphotransferase complex, and the functions of distinct domains of the α- and γ-subunits.
Mucopolysaccharidosis IIIA Storage Substrate Drives an Innate Immune Neuroinflammatory Response
H. Parker1, H. Boutin1, F. Wilkinson2, R. Holley1, D. Brough3, E. Pinteaux3, B. Bigger1
1Faculty of Medical & Human Sciences, University of Manchester
2Faculty of Life Sciences, University of Manchester
3School of Healthcare Science, Manchester Metropolitan Univ., Manchester
Introduction & Purpose: Mucopolysaccharidosis type IIIA (MPSIIIA) is a pediatric lysosomal storage disease characterized by mutations in the sulfo-glucosamine sulfo-hydrolase (SGSH) gene, resulting in reduced lysosomal SGSH enzyme activity. Subsequently, an accumulation of highly sulfated, partially degraded heparan sulfate oligosaccharides occurs in lysosomes and the extracellular matrix with disease progression, alongside secondary accumulation of GM gangliosides, cholesterol and amyloid beta. MPSIIIA patients develop behavioral disturbances and progressively worsening cognitive deficits, ultimately leading to dementia and premature death, a possible consequence of neuro-inflammation. Brains from MPSIIIA mice demonstrate markedly increased astrocytosis and microgliosis, particularly in the cortex.1 This, coupled with up-regulation of IL-1α, IL-1β and TNFα, suggests the development of a pro-inflammatory environment in MPSIIIA. However, the molecular mechanisms responsible for neuro-inflammation in MPSIIIA remain unclear. This project aims to understand how HS and secondary storage substrates affect the CNS and neuro-inflammatory pathways. Methodology & Results: Here we show that glycosaminoglycans (GAGs) isolated from MPSIIIA mice induce pro-inflammatory TNFα, IL-6, IL-1α and IL-1β responses when applied to a primary mixed glial culture, where normalized levels of WT GAGs did not elicit a response. The data also shows that qualitative alterations, rather than amount, in MPSIIIA GAGs are responsible for inflammatory responses. MPSIIIA GAGs act as an inflammatory priming stimulus via toll-like receptor 4 (TLR4) as inhibition of the intracellular domain of TLR4 completely abrogated the inflammatory response (p≤0.001). MPSIIIA GAGs did not initiate IL-1 dependent signaling alone in this in vitro model; indeed recombinant IL-1 receptor antagonist (IL-1Ra) did not reduce the inflammatory response. Secondary stimulation with NLRP3 inflammasome activators such as ATP or secondary storage substrates accumulated in MPSIIIA (cholesterol or amyloid beta oligomers), induced the release of intracellular IL-1β associated with MPSIIIA GAG priming (p≤0.001). Conclusion: In vitro data suggests that MPSIIIA neuro-inflammation may be dependent on IL-1, and driven by primary and secondary storage substrates. We are currently performing in vivo studies to confirm whether the MPSIIIA neuro-inflammatory response acts through IL-1 dependent mechanisms, and whether modulation of the innate immune response will slow disease progression.
Reference
WilkinsonFLHolleyRJLangford-SmithKJ. Neuropathology in Mouse Models of Mucopolysaccharidosis Type I, IIIA and IIIB. PLoS One. 2012;7(4):e35787.doi:10.1371/journal.pone.0035787 (2012).
A Longitudinal Kinematic Assessment Using a Head Drop Test in the Canine Model of Mucopolysaccharidosis Type IIIB Reveals Degenerative Cerebellar Function in Affected Animals
A. Hess1, S. Safayi1, B. Johnson1, K. Kline1, N. Jeffery1, M. Ellinwood1
1Iowa State University, Ames, IA
Purpose: Sanfillipo syndrome type IIIB, also known as Mucopolysaccharidosis type IIIB (MPS IIIB), is a recessive, fatal pediatric neuropathic lysosomal storage disease. Recessively-inherited loss of activity of the enzyme alpha-N-acetyl-D-glucosaminidase results in failed catabolism of the primary substrate heparan sulfate, a species of glycosaminoglycan. The prevalence of this rare lysosomal storage disease, which has no effective therapy, is approximately 1 in 211,000 live births. A canine model of MPS IIIB was established with primary clinical signs observed at ∼24 months of age. The clinical manifestation of this disease includes dysmetria, hind limb ataxia, and a wide-based stance with trunk swaying, which are consistent with cerebellar dysfunction. Development of a quantifiable assessment of cerebellar dysfunction is desirable in order to have a clinically-relevant endpoint in this model that can reliably assess clinical dysfunction as well as potential response to therapy. Herein we describe a kinematic evaluation of the head drop test, which is a clinically useful neurologic exam employed in veterinary medicine to assess animals with suspected cerebellar dysfunction. Methodology: The head drop test was used to assess cerebellar dysfunction by gently restraining an animal’s head in a vertical position followed by release of that restraint. Sudden elimination of the head support elicited a rotatory motion of the head in the sagittal plane, subsequent deceleration and then a restorative correction to normal posture. We hypothesized that measures of angular velocity would be altered between normal and affected MPS IIIB dogs when assessed kinematically. To this end, a Vicon Nexus system (Vicon®; Oxford Metrics, Oxford, UK) including six infrared cameras was used to capture head drop. The motion of the head was defined using three 15 mm optical markers attached to the nose (overlying the nasal bone), forehead (overlaying frontal bone) and caudal aspect of the head (overlying the occipital bone). Head movement was recorded with a frequency of 100 Hz tracking and captured at 100 frames per second, and x, y, and z coordinates were captured for each optical marker at each time point. From the raw data, measures of angular velocity relative to duration of the head drop for each individual dog were calculated using R Statistical Package software for each of the four sessions. The time period for each recording was defined as maximum height of the nasal marker position until the dog’s head moved upward. A smoothed curve for each coordinate for each marker was fitted for each recording, and the angle of head rotation in sagittal plane at each time point in the recording was calculated. Angular velocity was estimated using linear regression spanning 5 consecutive time points, from which the maximum angular velocity was retained for each recording. Results: The data included 4 unaffected and 4 MPS IIIB affected intact male and female canines evaluated over 4 sessions: ∼7-9 months, ∼10-11 months, ∼13-15 months, and ∼17-17.5 months of age. The test was repeated and recorded 10 times for each individual dog during a session. Maximum angular velocity was log transformed and used to compare MPS IIIB affected and unaffected dogs using statistical linear mixed animal model in ASReml v3.0, which included an Age*Status interaction to estimate the slope of maximum angular velocity in the affected MPS IIIB group over time vs the unaffected group. Statistical analysis revealed that the maximum angular velocity of unaffected animals did not change over time (1.19E-04 ± 2.31E-04 [log10(degrees/second)]/day; P=0.61), while the maximum angular velocity for affected animals declined over time (-5.22E-04 ± 1.76E-04 [log10(degrees/second)]/day; P=0.01). These slopes were significantly different from each other (P=0.028). Conclusion: These results demonstrate that maximum angular velocity decays over time in MPS IIIB affected animals, but not in unaffected animals, suggestive of cerebellar dysfunction. Given the strong trend in differences in head drop over time between unaffected and MPS IIIB affected dogs, we expect that this model will be useful to quantitatively assess responses to therapy. Therefore, this technique is expected to be of use in the canine MPS IIIB model as a method to evaluate preclinical treatments developed for human use.
Progressive Neuronal and Somatic Pathology in a New Mouse Model of MPSIIID
C. Roca1, S. Motas1, S. Marco1, A. Ribera1, V. Sánchez1, X. Sánchez1, J. Bertolín1, J. Ruberte1, V. Haurigot1, F. Bosch1
1CBATEG-UAB, Bellaterra
Purpose: Mucopolysaccharidosis type IIID (MPSIIID) is a rare inherited metabolic disorder caused by deficiency in the activity of the enzyme N-acetylglucosamine-6-sulfatase (Gns) which is required for the breakdown of the glycosaminoglycan (GAG) heparan sulfate (HS). Due to pathologic build-up of undegraded HS in the lysosomes, cellular damage occurs, affecting the central nervous system (CNS) and peripheral tissues. Clinically, the first symptoms of MPSIIID usually manifest between 2 and 6 years of age as delayed psychomotor development, progressing to neurologic degeneration and severe dementia by late infancy. Death typically occurs during the second or third decade of life. Currently there is no specific treatment available for MPSIIID and the lack of animal models of MPSIIID precludes the development of therapeutic approaches that have proven successful for related disorders. Therefore, the purpose of the present work was to generate and phenotype a new mouse model of MPSIIID. Methodology: C57BL/6N-A/a Embryonic stem cells carrying a reporter (LacZ) gene tagged insertion in the Gns gene were microinjected in blastocysts following standard methods. Under this design, successful targeting allows for the identification of sites of endogenous expression of the knocked-out gene through detection of β-Galactosidase activity. The resulting male chimeras were bred with C57Bl/6 N females to generate Gns knock-out offspring. A full phenotyping of GNS-deficient mice was undertaken through evaluation of GNS expression and activity, GAG content, lysosomal distention and function, neuroinflammation, behavior and survival rate. Results:Gns disruption was evidenced by lack of expression and activity of the enzyme in brain, liver and kidney. Through detection of β-Galactosidase, Gns endogenous expression was mapped, and found to be widely distributed in brain, liver, and blood vessels of the heart and spleen, in agreement with the development of CNS and somatic pathology in MPSIIID patients. At biochemical level, 2-month-old MPSIIID mice showed GAG accumulation and enlargement of the lysosomal compartment in different regions of the brain and peripheral organs such as the liver, heart or lung. Disturbance of normal lysosomal function was confirmed by deregulation of the activity of several lysosomal hydrolases in the brain and liver of MPSIIID mice. Neuroinflammation was detected in different areas of the brain by immunohistochemical analysis with specific stainings for activated astrocytes and microglia. Furthermore, many of these pathological findings were exacerbated in 6 month-old animals, suggesting worsening of the pathology as animals aged. Accordingly, Gns−/− mice behaved normally at 2 months of age but showed hypoactive behavior at 6 months. Finally, MPSIIID mice had shortened lifespan. Conclusions: The GNS-deficient mouse generated here recapitulates human MPSIIID at biochemical, histological and functional level and should proof a valuable tool for the study of the pathophysiology of the disease as well as for the development of novel therapeutic approaches.
Initial Characterization of a Murine Model of Mucopolysaccharidosis Type IIID: Similar Pathology to MPS IIIB and IIIA Murine Models
M. Jamil1, E. Snella1, S. Le2, S. Kan2, B. Birtcil3, P. Dickson2, N. Ellinwood1, J. Smith3
1Iowa State University College of Agriculture & Life Sciences, Ames
2Los Angeles Biomedical Research Institute at Harbor UCLA, Torrance
3Iowa State University College of Veterinary Medicine, Ames
The Mucopolysaccharidoses (MPSs) are a class of hereditary disorders caused by the primary lysosomal storage of single or multiple classes of glycosaminoglycan (GAG). All the MPSs are autosomal recessive disorders with the exception of MPS II which is an X-linked disorder. Mucoploysaccharidosis type III, or Sanfilippo syndrome, is characterized by the primary lysosomal storage of heparan sulfate (HS), and the secondary lysosomal accumulation of other compounds e.g. gangliosides. The MPS IIIs are pediatric neuropathological disorders usually resulting in an early death. In humans there are four types of MPS III (A, B, C, and D), all of which have murine models except type IIID. Thus far, there are no approved therapies for any of the MPS IIIs which greatly increase the value of animal models of these disorders and the need specifically for a murine MPS IIID model. Herein, we have characterized a first murine model of MPS IIID. The MPS IIID mouse is a knockout model produced by Taconic Artemis GmbH by removing exons 2 to 13 of the 14 exons from N-acetyleglucoseamine-6-sulfatase (GNS) gene. This gene encodes for an enzyme which takes part in the HS degradation pathway. Both the embryonic stem cell donor and the chimera mates were from the C57BL/6 line, eliminating the need for several rounds of backcross. The production of GNS−/− mice followed simple Mendelian inheritance and were diagnosed by PCR. The GNS−/− animals were compared with GNS+/+ animals by phenotype, biochemistry, histopathology, and behavior at four, eight and thirteen months of age. At four months GNS−/− mouse was also compared with already existing MPS IIIA and IIIB mice. At thirteen months clinical signs consisted of enlarged bladder (∼1.1 ml urine collected), hepatomegaly, and rough hair coat. For biochemistry, at four months no GNS activity was detected by a 4MU based enzyme assay and the GAG assay for total soluble sulfated GAGs showed an exponential increase in GAG in all affected animals including MPS IIIA and IIIB. A progressive increase in the accumulated GAGs was seen in GNS−/− animals at eight and twelve months. For histology all tissues were stained with hematoxylin and eosin (H&E) and the brain sections from GNS−/− animals were further stained with luxol fast blue (LFB). At four months MPS IIID and IIIA and IIIB affected animals had mild vacuolation in the CNS whereas preliminary assessment of the tissues of liver and kidney did not show any clear difference compared to the wild type. The CNS had infrequently observed LFB positively stained neurons. At eight months in GNS−/− animals, the CNS histopathology findings was not fully intermediate between the four and twelve month animals as the findings were more similar qualitatively to storage seen at the four month time point. In thirteen month old GNS−/− animal extensive vacuolation in CNS, hepatocytes, and renal tubular cells, and severe Purkinje cell loss was seen. Numerous neurons in the LFB stained CNS sections had cytoplasmic granular storage. Animals (affected and wild type) are currently being tested every month, beginning at 2 months of age for motor function using the rocking rotarod protocol. To date, the latest age of affected animal evaluated is nine months, a time point at which the MPS IIIB murine model first begins to manifest deficits using this protocol. Hence the early stage of this evaluation and preliminary nature of the data precludes any statistical assessment. The MPS IIID murine model showed similar gross and histological findings to other MPS III murine models, including urine retention, and marked liver, renal, and CNS storage. The MPS IIID mouse had no GNS activity. The measurement of increased tissue levels of sulfated GAGs over time reflects the expected progressive accumulation of heparan sulfate. Neuronal vacuolation and severe Purkinje cell loss is comparable to findings in other MPS III models. Extensive hepatocellular vacuolation is consistent with the predicted heparan sulfate storage, and the LFB positive staining of neurons is consistent with the known secondary CNS storage of glycosphingolipids in MPS III. Hence this model of murine MPS IIID appears to manifest the expected pathology of MPS III based on previously studied MPS III models. This new model will prove critical to further study of the pathogenesis of and therapy for human MPS IIID.
Impaired Wnt Signaling Contributes to Delayed Epiphyseal Chondrocyte Differentiation in Mucopolysaccharidosis VII Dogs
S. Peck1, E. Shore1, N. Malhotra1, J. Tobias1, M. Pacifici2, M. Haskins1, L. Smith1
1University of Pennsylvania, Philadelphia
2The Children’s Hospital of Philadelphia
Introduction: Mucopolysaccharidosis (MPS) VII patients present with severe skeletal disease, particularly in the spine [1]. Vertebral dysplasia due to failed cartilage-to-bone conversion during growth leads to kyphoscoliosis and spinal cord compression. Using the naturally-occurring canine model, we previously identified the developmental window when failed vertebral bone formation first manifests in MPS VII and that epiphyseal chondrocytes fail to undergo hypertrophic differentiation (Figure 1) [2]. Our objectives in this study were to identify pathways that fail to activate in MPS VII epiphyseal cartilage using whole-transcriptome sequencing (RNA-Seq) and examine cellular responses to related secreted growth factors using a cartilage explant model. Methods: Vertebral epiphyseal cartilage from unaffected control and MPS VII dogs was collected postmortem at 9 and 14 days (n = 5, schematic, Figure 1B). Total RNA was extracted, RNA-Seq performed, and differential mRNA expression confirmed with qPCR. Nuclear β-catenin protein levels were measured via Western blot. Vertebral epiphyseal cartilage explants from 9 day control (n = 4) and MPS VII (n = 2) animals were cultured for 1, 3, or 7 days in minimal medium in the presence or absence of 500 ng/mL Wnt3a. RNA was isolated from explants, and SOX9 mRNA expression was determined with qPCR. Results: Principal component analysis (PCA) of RNA-Seq results showed clustering of global gene expression for each sample group (Figure 2A). A total of 411 and 1104 genes were significantly differentially expressed (fold-change>2) between control and MPS VII at 9 and 14 days, respectively. At both ages, the Wnt/β-catenin pathway was the most dysregulated bone formation pathway with 14 and 54 pathway-associated genes differentially expressed at 9 and 14 days, respectively, verified by qPCR (Figure 2B). Immunoblots showed significantly higher levels of nuclear β-catenin protein at 14 compared to 9 days in controls but no change in MPS VII (Figure 2C). Control explants treated with Wnt3a exhibited a significant decrease in SOX9 mRNA after 1 and 3 days (Figure 3). In contrast, MPS VII explants exhibited no significant response to Wnt3a at day 1, but did exhibit significant decreases in SOX9 mRNA after 3 and 7 days of treatment. Unlike untreated controls, untreated MPS VII explants did not show decreased SOX9 expression after 3 or 7 days. Discussion: These results suggest that Wnt/β-catenin signaling does not activate at the appropriate developmental stage to initiate chondrocyte differentiation in MPS VII and that MPS VII chondrocytes have impaired capacity to respond to Wnt signaling. This study provides the basis for further mechanistic investigations of skeletal disease in MPS VII and identifies the Wnt/β-catenin pathway as a promising therapeutic target.
Representative mid-coronal μCT images of vertebrae. Red boxes: bone formation in secondary ossification centers in 14-day control animals. B. Schematic of vertebral epiphyseal cartilage excision (mid-coronal ABPR-stained histological section). C. mRNA levels of COL10A1 (hypertrophic marker) and BGLAP (bone marker) in vertebral epiphyseal cartilage at 9 and 14 days-of-age. N=5; scale=1mm; *p<0.05 vs all. S: Secondary and P: Primary ossification center.
A. RNA-Seq PCA plot. B. mRNA levels of Wnt/β-catenin signaling pathway genes measured by qPCR. C. Nuclear β-catenin protein expression in control and MPS VII vertebral epiphyseal chondrocytes. N=5; *p<0.05; #p<0.05 vs all.
SOX9 mRNA for control (n=4) and MPS VII (n=2) explants with Wnt3A treatment after 1, 3 and 7 days. *p<0.05, #p<0.05 vs day 1.
Acknowledgments
NIH; Penn Center for Musculoskeletal Disorders; National MPS Society.
References
de KremerRD. Mucopolysaccharidosis type VII (beta-glucuronidase deficiency): a chronic variant with an oligosymptomatic severe skeletal dysplasia. Am J Med Genet. 1992;44:145–152.PeckS. Delayed hypertrophic differentiation of epiphyseal chondrocytes contributes to failed secondary ossification in mucopolysaccharidosis VII dogs. Mol Genet Metab. 2015;116:195–203.
Progression of Vertebral Bone Disease in Mucopolysaccharidosis VII Dogs From Birth to Skeletal Maturity
S. Peck1, J. Kang1, N. Malhotra1, P. O’Donnell1, M. Haskins1, M. Casal1, L. Smith1
1University of Pennsylvania, Philadelphia
Introduction: Mucopolysaccharidosis VII patients exhibit severe skeletal abnormalities that are prevalent in the spine. Previously, we showed the presence of cartilaginous lesions in MPS VII vertebrae that represent failed secondary ossification during postnatal development. For optimal therapeutic intervention it is critical to first elucidate temporal patterns of disease manifestation during postnatal development. Therefore, the objective of this study was to establish the nature, timing, and progression of vertebral bone disease in MPS VII from birth to skeletal maturity, using the naturally-occurring canine model. Methods: We used the naturally-occurring MPS VII canine model that mimics both the progression and pathological phenotype of the skeletal abnormalities found in human patients. Control and MPS VII dogs (n = 1-5) were euthanized at 9, 14, 30, 42, 90, 180, and 365 days. Progression of vertebral bone formation in primary and secondary ossification centers was analyzed using micro-computed tomography (µCT). Serum was collected at 90, 180, and 365 days-of-age and bone-specific alkaline phosphatase (BAP) activity was measured. Results: Secondary ossification was not evident in MPS VII until 30 days, compared to 14 days in controls, was highly non-uniform, and ceased progressing between 42 and 90 days (Figure 1). At 365 days, there was abnormal persistence of growth plate cartilage. In primary ossification centers, bone content was normal at early ages, but beyond 30 days, was significantly lower in MPS VII (Figure 2A, B). Serum BAP levels were lower in MPS VII animals at 90 and 180 days, but not at 365 days (Figure 2C). Discussion: This work establishes that vertebral bone disease in MPS VII manifests differently in primary and secondary ossification centers in a temporally-dependent manner, informing optimal targeting and timing for potential therapeutic interventions. Vertebral secondary ossification in MPS VII was found to be not only markedly delayed, but also highly non-uniform, suggesting that early developmental signals for bone formation are both impaired and spatially dysregulated. Results also suggest that serum BAP may be a robust, non-invasive diagnostic tool for assessing bone disease progression in MPS patients.
Representative μCT images of control and MPS VII vertebrae. A, Axial view. B, Midsagittal view. At 365 days-of-age, the growth plate has closed in control animals, and thus, a distinct secondary ossification center no longer exists. Numbers indicate postnatal days-of-age. Gray: primary ossification centers; Purple: secondary ossification centers. Scale = 1 mm.
A, Bone volume fraction (BV/TV) and B, Bone mineral density (BMD) for control and MPS VII vertebral primary ossification center trabecular bone from birth to skeletal maturity. C, Serum bone alkaline phosphatase (BAP) activity for control and MPS VII animals at older ages. Nine days (n = 5), 14 days (n = 5), 30 days (n = 2), 42 days (n = 2), 90 days (n = 1), 180 days (n = 1), and 365 days (n = 1).
Acknowledgments
NIH; Penn Center for Musculoskeletal Disorders; National MPS Society.
In Vivo Expression of the γ-Subunit of MLIII-Related GlcNAc-1-Phosphotransferase
G. Di Lorenzo1, R. De Pace1, M. Schweizer1, T. Braulke1, S. Pohl1
1University Medical Center Hamburg-Eppendorf, Hamburg
Mucolipidosis III gamma (MLIII) is clinically characterized by onset of signs at ∼ 5 years such as stiffness of hands and shoulders, claw hand deformities, scoliosis and progressive destruction of hip joints. The disease is caused by mutations in GNPTG encoding the γ-subunit of a phosphotransferase complex responsible for the modification of about 50 lysosomal enzymes with mannose 6-phosphate (M6P) residues. M6P residues are required for efficient transport of lysosomal enzymes to lysosomes. Defective γ-subunits resulted in reduction of phosphotransferase activity and subsequently M6P-dependent sorting of multiple lysosomal enzymes associated with the accumulation of non-degraded material in lysosomes. Real-time PCR and western blot analysis revealed organ-specific Gnptg mRNA and protein level in 12 weeks old mice. To examine cell-specific expression of γ-subunit during postnatal development in vivo, we generated a GnptgLacZ reporter mouse encoding bacterial β-galactosidase under the Gnptg promoter. LacZ and haematoxylin staining of cryosection of various tissues prepared on postnatal day 3, and 12 and 40 weeks after birth showed that Gnptg is strongly expressed in the hippocampus, Purkinje cells of cerebellum, in the retina, salivary gland, vertebral bodies, in heart muscle cells, hair roots and distinct cells of the kidney. Furthermore, mass spectrometric analysis of the lysosomal proteome of γ-subunit-lacking mouse cells revealed that only few lysosomal enzymes are completely missorted. The role of these enzymes in bone and cartilage homeostasis has to be studied in more detail and might represent targets of therapeutic strategies in MLIII.
Neurodegeneration in the Brain and the Retina in a Mouse Model Deficient for the Lysosomal Membrane Protein Cln7
L. Brandenstein1, W. Jankowiak1, C. Hagel1, M. Schweizer1, J. Sedlacik1, J. Fiehler1, U. Bartsch1, S. Storch1
1University Medical Center Hamburg-Eppendorf, Hamburg
Purpose: Generation of a mouse model for CLN7 disease. Methodology: Phenotypic analysis of a Cln7 KO mouse model using immunoblotting, pulse-chase analyses, immunohistochemistry, electron microscopy analyses, and magnetic resonance imaging (MRI). Results: CLN7 disease is an autosomal recessive, neurodegenerative lysosomal storage disorder of childhood caused by the defective lysosomal membrane protein CLN7. We have disrupted the Cln7/Mfsd8 gene in mice by targeted deletion of exon 2 generating a novel knockout (KO) mouse model for CLN7 disease, which recapitulates key features of human CLN7 disease pathology. In Cln7 KO mice a neurological phenotype including hind limb clasping and myoclonus mice and increased mortality were observed. Lysosomal dysfunction in the brain of mutant mice was shown by the accumulation of autofluorescent lipofuscin-like lipopigments, subunit c of mitochondrial ATP synthase (SCMAS) and saposin D and increased expression of lysosomal cathepsins B, D and Z. By immunohistochemical co-stainings, elevated cathepsin Z expression restricted to Cln7-deficient microglia and neurons was detected. Ultrastructural analyses revealed large storage bodies in Purkinje cells of Cln7 KO mice containing inclusions composed of irregular, curvilinear and rectilinear profiles as well as fingerprint profiles. Generalized astrogliosis and microgliosis in the brain preceded neurodegeneration in the olfactory bulb, cerebral cortex and cerebellum in Cln7 KO mice. MRI analyses revealed neurodegeneration in the olfactory bulb, cerebral cortex and cerebellum of Cln7 KO mice late in disease. Increased levels of microtubule-associated protein 1 light chain 3-II (LC3-II) and the presence of neuronal p62- and ubiquitin-positive protein aggregates suggested that impaired autophagy represents a major pathomechanism in the brain of Cln7 KO mice. Morphological analyses of the retina of Cln7 KO mice revealed an early onset and rapidly progressing degeneration of photoreceptor cells in Cln7 KO mice, resulting in the loss of about 70% photoreceptors by 4 months of age. The absence of Cln7 in the retina led to increased expression of multiple lysosomal proteins, accumulation of SCMAS and saposin D and reactive astrogliosis and microgliosis. Conclusion: The data suggest that loss of the putative lysosomal transporter Cln7 leads to lysosomal dysfunction, impaired constitutive autophagy and neurodegeneration in the brain late in disease and early, rapidly progressing neurodegeneration in the retina.
Progressive Eye Pathology in MPS I Mice and Effects of Intravenous Enzyme Replacement Therapy
G. Baldo1, E. Gonzalez2, G. Pasqualim2, T. de Carvalho2, F. Hehn de Oliveira3, R. Giugliani4, U. Matte1
1Gene Therapy Center, Porto Alegre
2Postgraduate program in Genetics and Molecular Biology, Porto Alegre
3Experimental Pathology Service, Porto Alegre
4Medical Genetics Service, Porto Alegre
Purpose: Mucopolysaccharidosis type I (MPS I) is an inherited disorder caused by deficiency of alpha-L-iduronidase (IDUA) which results in accumulation of glycosaminoglycans in multiple tissues. Ocular manifestations are common in MPS I patients and often lead to visual impairment. Accumulation of GAG in corneal stromal cells causes corneal opacity and reduced vision. Other typical ocular and neurosensory complications include retinopathy and degeneration of the retina. Therefore, the purpose of this study was to determine the progression of eye disease as well as the effectiveness of intravenous enzyme replacement (ERT) on the number of photoreceptors in the outer nuclear layer (ONL) and ocular GAG accumulation in MPS I mice. Methodology: Ocular tissues were obtained from 2, 6 and 8 months old of normal and MPS I mice. A group of MPS I mice received 1.2 mg laronidase/kg every 2 weeks from 6 to 8 months of age (ERT 6-8mo) and was sacrificed 2 weeks after. Histological analyses were performed by Alcian Blue/Hematoxylin eosin staining. We analyzed the presence of ocular accumulation of GAG and measured the thickness of the ONL layer over time. ONL thickness was compared between normal and MPS I mice at 2, 6 and 8 months. Measurements in ERT 6-8mo group was compared with animals of the same age. ONL thickness served as a measure of number of photoreceptor cell bodies to study retinal degeneration. Results: Large vacuoles with positive staining for GAGs were found in corneal stromal cells from MPS I (at 2, 6 and 8 mo) and ERT 6-8mo mice. ONL layer was similar in Normal and MPS I mice at 2 months. At 6 months, the ONL layer was significantly reduced in MPS I mice (24% reduction, p< 0.01) compared to normal mice. At 8 months-old, photoreceptor loss was accentuated, with a decrease of 51% (p< 0.01) in the ONL thickness. ERT 6-8mo group showed a decrease in the ONL thickness of 46% (p< 0.01) at 8 months, similar to untreated MPS I mice. Conclusion: Ocular pathologies occur in MPS I mice during childhood and adulthood and involve corneal and retinal tissues. Here we demonstrated that there is a progressive decrease in the ONL layer in MPS l mice. ERT therapy started late does not revert the photoreceptor loss in ONL nor does the GAGs accumulation in the cornea. Finally, we believe that decreased ONL thickness in MPS I mice is likely to cause some change in the visual properties of the eye in this model.
Comparative Study of Idursulfase Beta and Idursulfase in IDS KO Mice
C. Kim1, J. Seo2, J. Lee2, H. Ji2
1MOGAM Research Institute, Yongin
2Green Cross Corporation, Yongin
Objective: Mucopolysaccharidosis II (MPS II, Hunter syndrome; OMIM 309900) is an X-linked lysosomal storage disease caused by a deficiency in the enzyme iduronate-2-sulfatase (IDS), leading to accumulation of GAGs. For enzyme replacement therapy of Hunter syndrome, two recombinant enzymes, idursulfase (Shire Human Genetic Therapies, Lexington, MA) and idursulfase beta (Green Cross Corp., Yongin, Korea), are currently available in Korea and several countries. These two therapeutic drugs showed significant differences in various clinical parameters in a recent clinical trial. To investigate similarities and differences of these, we compared two drugs in vitro, in vivo efficacy, and various factors of biochemical property. Methods: To compare in vivo efficacy of these, we used Hunter syndrome disease mouse model, IDS KO. 1mg/kg of the two drugs was administered for six months and analyzed urine and tissue GAGs, evaluation of bone improvement, and anti-drug antibody formation. Biochemical characteristics were compared by glycosylation, glycan structure, and purity. Results: Idursulfase beta was uptake into the cell by receptor-mediated manner and degraded GAGs accumulated in patient fibroblasts. In vivo studies of two drugs in mouse models of MPS II showed a similar organ distribution and decreasing urinary GAGs excretion. However idursulfase beta showed more effective in vitro efficacy in the lower concentration treatment (less than 312 pM, p<0.001) and in vivo efficacy in degradation of tissue GAGs and improvement of bones. Especially, Antibody formation shows the difference between them following 1mg/kg administered for long-term. Idursulfase contained high-mannose type and complex type in N-glycans and showed higher aggregation percentage compared to idursulfase beta. Conclusions: Two drugs have same amino acid sequences but show a little different biochemical characteristic which come from different producing cell line and production process. In vitro and in vivo efficacy may affect complexly by these differences. Our comparison data indicate two drugs are very useful and helpful for therapy of Hunter syndrome patient.
Neurological Correction of Mucopolysaccharidosis IIIB Mice by Hematopoietic Stem Cell Gene Therapy
R. Holley1, S. Ellison1, D. Fil1, J. McDermott1, N. Senthivel1, A. Langford-Smith1, F. Wilkinson1, S. Kan2, P. Dickson2, B. Bigger1
1Faculty of Medical & Human Sciences, University of Manchester
2Los Angeles Biomedical Research Institute at Harbor-UCLA, Torrance
Purpose: Mucopolysaccharidosis Type IIIB (MPSIIIB) is a pediatric, autosomal recessive lysosomal storage disease (LSD) caused by deficiency of α-N-acetylglucosaminidase (NAGLU), an enzyme involved in the degradation of heparan sulfate (HS). Thus absence of NAGLU leads to the accumulation of partially degraded HS glycosaminoglycan in lysosomes, giving rise to cellular dysfunction with devastating clinical consequences. Affected Individuals exhibit severe central nervous system degeneration with progressive cognitive impairment and behavioral problems, alongside more attenuated somatic symptoms. There are currently no effective treatments available. Enzyme replacement therapy with recombinant NAGLU enzyme is ineffective as the NAGLU enzyme cannot cross the blood brain barrier (BBB) to where it is needed. Here we have developed a hematopoietic stem cell gene therapy approach (HSCGT) in a mouse model of MPSIIIB to effectively deliver NAGLU enzyme to the brain via monocyte trafficking and engraftment, to enable neurological disease correction. Methodology: NAGLU was codon-optimized and inserted into our high-titer lentiviral vector under control of the myeloid-specific CD11b promotor. Hematopoietic stem cells (HSCs) from autologous MPSIIIB mice were transduced with LV.CD11b.NAGLU and transplanted into myeloid ablated 6-8 week-old MPSIIIB mice. Mice were then compared to wild-type, untreated MPSIIIB and MPSIIIB receiving a wild-type bone marrow transplant at 8 months of ages for enzyme activity, HS storage, neuroinflammation and neurocognitive correction. Results: We observed correction of the MPSIIIB behavioral phenotype in treated mice to wild-type levels with normalization of path length, average speed, frequency entering the center and duration of speed >100mm/s in open field tests. In addition, we observed a significant correction of astrogliosis and lysosomal compartment size in the brains of CD11b.NAGLU LV treated mice with an accompanied normalization of inflammatory cytokines TNFα, IL1B and IL1RN. Furthermore, NAGLU enzyme activity was substantially increased in the brain. Interestingly, wild-type transplant alone was able to mediate a partial brain correction, although levels of inflammation and lysosomal storage remain high. Conclusion: Here we present for the first time neurological correction of MPSIIIB mice by HSCGT. Significantly correction was superior to wild-type transplant alone, demonstrating proof of principle for the development of a clinical trial to improve neurological function in patients.
Twenty-Six Week or Longer Intracerebroventricular Infusion Study of BMN 250 Administered Once Every 2 Weeks in a Canine Model of Mucopolysaccharidosis Type IIIB
N. Ellinwood1, B. Valentine1, W. Ware1, S. Hostetter1, G. Ben-Shlomo1, N. Jeffery1, S. Safayi1, S. Millman1, A. Hess1, M. Butt2, J. Cooper3, I. Nestrasil4, D. Wendt5, D. Kennedy5, A. Grover5, A. Melton5, A. Cherukuri5, J. Wait5, J. Pinkstaff5
1Iowa State University, Ames
2Tox Path Specialists, LLC, Frederick
3Kings College London
4University of Minnesota, Minneapolis
5BioMarin Pharmaceutical Inc, San Rafael
Mucopolysaccharidosis type IIIB (MPS IIIB) is a fatal neurodegenerative lysosomal disorder disease caused by genetic deficiency of the enzyme N-acetyl-alpha-D-glucosaminidase (Naglu). Deficiency of Naglu leads to accumulation of the glycosaminoglycan (GAG) heparan sulfate (HS) in the central nervous system (CNS) and other tissues. In humans, the resulting progressive neurodegeneration is typically fatal during the second decade of life. Naglu-null dogs are a relevant large animal model of MPS IIIB. These animals display HS accumulation in blood, liver, kidney, cerebrospinal fluid, urine, and CNS tissues. Gross clinical signs are present at approximately 18-24 months of age. Progressive neurological decline follows leading to mortality at 3-5 years of age. End-stage CNS histopathology includes severe cerebellar atrophy, Purkinje cell loss, and cytoplasmic vacuolation. BMN 250 is a fusion protein of recombinant human Naglu with an insulin-like growth factor 2 (IGF2), in development as a potential enzyme replacement therapy for MPS IIIB. The IGF2 moiety or GILT (glycosylation independent lysosomal targeting) tag allows for enhanced lysosomal uptake via the IGF-2 receptor (Maga et al., 2013). Direct delivery to the CNS via intracerebroventricular (ICV) infusion is the planned clinical route of administration for BMN 250. The objectives of this study are 1) to characterize the safety of BMN 250 in Naglu-null dogs and normal littermate controls; 2) to determine the pharmacodynamic effects of BMN 250 on lysosomal storage accumulation, brain morphology, and functional assessment of gait and cognition in this relevant dog disease model; and 3) to describe the pharmacokinetics, CNS distribution, and immunogenicity of ICV-administered BMN 250. This ongoing blinded study is fully enrolled with 24 dogs; 12 normal and 12 Naglu-null, MPS IIIB affected dogs, distributed to 3 groups of 4 dogs each designed to be treated with vehicle, 12 mg, or 48 mg of BMN 250 administered every two weeks via ICV catheters.
Reference
MagaJA. Glycosylation-independent lysosomal targeting of acid α-glucosidase enhances muscle glycogen clearance in pompe mice. J Biol Chem. 2013;288:1428–1438.
Normalization of Pathological Lysosomal Storage and Biochemistry in the Mucopolysaccharidosis IIIB (MPS IIIB, Sanfilippo B) Mouse Model by Intracerebroventricular Enzyme Replacement Therapy With BMN 250, a NAGLU-IGF2 Fusion Protein
M. Aoyagi-Scharber1, D. Crippen-Harmon1, J. Vincelette1, H. Prill1, G. Yogalingam1, B. Yip1, B. Baridon1, C. Vitelli1, A. Lee1, O. Gorostiza1, W. Minto1, B. Yates1, S. Rigney1, K. Webster1, T. Christianson1, P. Tiger1, M. Lo1, J. Holtzinger1, P. Fitzpatrick1, J. Brown1, S. Bullens1, R. Lawrence1, A. Bagri1, J. LeBowitz1, B. Crawford1, S. Bunting1
1BioMarin Pharmaceutical Inc., Novato
Introduction/Purpose: Mucopolysaccharidosis IIIB (MPS IIIB), caused by heritable deficiency of alpha-N-acetylglucosaminidase (NAGLU) required for lysosomal degradation of heparan sulfate (HS), is a devastating pediatric neurodegenerative disorder with no approved treatment. BMN 250 is an enzyme replacement therapy drug candidate, comprised of NAGLU fused with insulin-like growth factor 2 for receptor-mediated, glycosylation-independent lysosomal targeting. Cell-type specific targeting of BMN 250 was characterized by in vitro uptake in rat brain-derived primary cultures. Total, regional and cell-type specific distribution and efficacy of intracerebroventricularly (ICV) administered BMN 250 were evaluated in MPS IIIB mice. Methodology: In cell uptake studies, normal rat-derived neurons, astrocytes and microglia were incubated with ≤200nM BMN 250 and assayed for intracellular NAGLU activity. In MPS IIIB mouse studies, BMN 250 was infused four times over two weeks at 100-microgram doses. Brain and liver were harvested 1 or 28 days post-last-infusion and analyzed for NAGLU, disease-specific HS Non-Reducing Ends (NREs; Sensi-Pro) and other pathological markers. Results: BMN 250 exhibited highly efficient uptake in rat-derived neurons, astrocytes and microglia in vitro. ICV-administered BMN 250 resulted in NAGLU activity above normal levels, clearance of HS NREs (92.4±2.2% brain; 100.0±0.0% liver) and reduction of beta-hexosaminidase activity (30.0±3.2% brain; 70.1±2.5% liver) at 1 day post-last-dose, relative to vehicle-treated MPS IIIB mice. Immunohistochemistry confirmed broad biodistribution and uptake in all cell types. Compared to ICV delivery, intravenous (IV) administration of the equivalent 100-microgram doses did not lead to significant reduction of brain HS NREs (27.9±3.8%) or beta-hexosaminidase activity (4.0±2.6%) at 1 day post-last-dose, relative to the vehicle-treated MPS IIIB mice. Furthermore, IV-delivered BMN 250 was not readily detected in the brain by NAGLU enzyme activity or immunohistochemical staining. Efficacy of ICV-delivered BMN 250 persisted even after 28 days post-last-dose with complete clearance of HS NREs (100.0%) and further reduction of beta-hexosaminidase activity (55.5±1.3%), relative to the vehicle-treated MPS IIIB mice, to a level close to the wild-type control. ICV-delivered BMN 250 normalized additional brain pathologies: elevated LAMP2 levels, reactive astrocytosis and neuro-inflammation. Conclusion: In the MPS IIIB mouse brain, ICV-administered BMN 250 results in higher NAGLU levels than IV administration, widespread NAGLU distribution, uptake in all cell types, and unlike the IV doses, >90% clearance of disease-specific HS NREs and normalization of secondary pathologic markers, warranting clinical evaluation.
Enzyme Replacement Therapy for Sanfilippo Syndrome MPS IIID
P. Dickson1, T. Chou1, D. Moen1, S. Kan1, X. Zhang1, S. Le1, N. Ellinwood2, J. Wood3, S. Ekins3
1LABioMed at Harbor-UCLA Medical Center, Torrance
2Iowa State University, Ames
3Phoenix Nest, Brooklyn, New York
Sanfilippo disease (mucopolysaccharidosis type III; MPS III) is a devastating neurodegenerative lysosomal storage disorder of childhood. There is no cure or effective treatment available. The fundamental cause of MPS III is an inherited mutation in one of the 4 enzymes required to catabolize heparan sulfate (HS). Because each type of MPS III is individually rare, motivation for pharmaceutical and biotechnology companies to develop new therapies has been limited. We are currently developing an enzyme replacement treatment (ERT) for MPS IIID that aims to ameliorate or reverse the catastrophic and fatal neurologic decline caused by this disease. Our strategy proposes to deliver recombinant human alpha-N-acetylglucosamine-6-sulfatase (rhGNS) intrathecally (directly into the spinal fluid) to effectively treat the underlying causes of the neurologic symptoms that dominate MPS III pathology. Encouraging preliminary data showed robust expression of rhGNS in Chinese hamster ovary cells that suggested scale-up would be feasible. We are currently completing a phase I STTR in which we had 8 key milestones proposed. 1) Produce rhGNS (≥100 μg) in CHO cells; 2) purify it to a specific activity of ≥100,000 activity units/mg, 3) demonstrate enzymatic activity at lysosomal pH (with low activity at neutral pH), 4) demonstrate enzymatic activity at 37ºC; 5) develop a storage buffer that will enable at least 1 month of storage; 6) demonstrate intracellular enzymatic activity of rhGNS in MPS III fibroblasts when rhGNS is added to the media; 7) determine rhGNS colocalizes with lysosomal markers using confocal microscopy and 8) confirm radiolabeled HS diminished in MPS III fibroblasts treated with rhGNS. We now present the results of this project prior to performing in vivo proof of concept studies in MPS IIID mice and developing a scalable purification scheme needed for GMP manufacturing to enable needed prior to human clinical studies.
Intrathecal AAVrh10 Corrects Biochemical and Histological Hallmarks of Mucopolysaccharidosis VII Mice and Improves Bone Pathology, Behavior and Survival
A. Bosch1, G. Pagès1, L. Giménez-Llort1, B. Garía-Lareu1, L. Ariza1, A. Sánchez Osuna1, G. García-Eguren1, M. Navarro2, J. Ruberte2, C. Casas1, M. Chillón1
1Institute of Neurosciences, Cerdanyola del Vallès
2CBATEG, Cerdanyola del Vallès
Adenoassociated viruses have significant potential as gene therapy vectors for chronic inherited diseases. Among the different AAV serotypes, AAV9 and AAVrh10 show greater transduction capacity in the central nervous system (CNS). However, preexisting antibodies present in patients’ serum before therapy may impair efficient transduction. The use of a non-human serotype like AAVrh10 may avoid the neutralization by anti-AAV immune factors raised in human sera after natural AAV infections. Here, we propose a gene therapy strategy for Mucopolysaccharidosis (MPS) type VII, a lysosomal storage disease due to ß-glucuronidase deficiency, causing glycosaminoglycan accumulation into enlarged vesicles and resulting in peripheral and neuronal dysfunction. By lumbar puncture, a poorly invasive technique, we performed a single intrathecal injection of 8.75x1011 vg.kg-1 of an AAVrh10 coding for the β-glucuronidase gene to adult MPS VII mice. We show that vector delivery to the cerebrospinal fluid (CSF) allowed global transduction of CNS structures. In addition, the drainage of the vector from the CSF to the bloodstream resulted in transduction of somatic organs such as liver. This provided a systemic β-glucuronidase source that achieved serum enzymatic activity comparable to wild type mice, sufficient to attain correction of the biochemical and histopathological hallmarks of the disease in CNS and somatic organs at short and long term. Moreover, progression of bone pathology was also reduced. Importantly, the biochemical correction lead to a significant improvement of physical, cognitive and emotional characteristics of MPS VII mice and entailed the doubling of MPS VII mouse life span. This strategy may have implications for gene therapy in patients with lysosomal storage diseases.
Instituto de Salud Carlos III and European Regional Development Fund (PI15/01271), 2014 SGR 1354.
RTB Lectin as an Effective Platform to Carry Lysosomal Enzymes Across the Blood-Brain Barrier
W. Acosta1, L. Ou2, J. Ayala1, J. Condori1, V. Katta1, A. Flory1, R. Martin1, C. Whitley2, D. Radin1, C. Cramer1
1Biostrategies-LC, State University
2Gene Therapy Center, Minneapolis
In the past decades, enzyme replacement therapies (ERTs) have provided extraordinary advances in the treatment of inherited metabolic disorders. Even though ERTs are currently available for multiple lysosomal diseases resulting in improvement in many organs, not all tissues receive corrective doses of the enzyme (e.g., CNS, eye, bone, lung, etc). All approved ERTs exploit MMR or M6P receptors to direct cell uptake, which can be limited by receptor abundance in the cell type or developmental stage of the tissue. RTB lectin uses two galactose/galactosamine binding domains to trigger endocytosis by binding to any galactose-terminated glycoprotein on the cell surface. Corrective enzymes for MPSI and MPSIIIA were fused to RTB and recombinantly expressed in a plant transient expression system. Fusion proteins retained both lectin binding and enzymatic activity, directed uptake, and corrected disease phenotype in human fibroblast cells. Animals injected with RTB fused α-L-Iduronidase showed broad bio-distribution of the enzyme in visceral and central nervous system organ 24 hours after the infusion. Weekly infusions of the lectin-enzyme for 8 weeks, showed reduction of substrate to normal levels in visceral organs, brain and cerebellum, and significant neurological improvements in Barnes behavioral test. The RTB lectin technology platform effectively delivers therapeutic macromolecules across the blood-brain barrier treating pathologies of the central nervous system.
Intranasal Gene Delivery of AAV9 Iduronidase: A Non-invasive and Effective Gene Therapy Approach for Prevention of Neurologic Disease in a Murine Model of MPS Type I
L. Belur1, A. Temme1, K. Podetz-Pedersen1, M. Riedl1, L. Vulchanova1, L. Hanson2, C. Fairbanks1, K. Kozarsky3, W. Frey II2, W. Low1, R. McIvor1
1University of Minnesota, Minneapolis
2Regions Hospital, St. Paul
3REGENXBIO Inc., Rockville
Purpose: Mucopolysaccharidosis type I (MPS I) is an autosomal recessive storage disease caused by deficiency of α-L-iduronidase (IDUA), resulting in accumulation of heparin and dermatan sulfate glycosaminoglycans (GAGs). Current treatments for this disease include allogeneic hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT). However, ERT is ineffective in treating CNS disease due to the inability of lysosomal enzymes to traverse the blood-brain barrier, and while there is neurologic benefit to HSCT the procedure is associated with significant morbidity and mortality. Methodology: We have taken a novel approach to the treatment of neurologic disease associated with Hurler syndrome, using intranasal administration of an IDUA-encoding AAV9 vector. A CAGS (CMV enhancer/ß-actin promoter/globin intron) regulated AAV9 -IDUA vector was infused intranasally into adult mice (2-3 months of age) that had been immunotolerized at birth with human iduronidase (Aldurazyme) to prevent anti-IDUA immune response. Results and Conclusions: Mice sacrificed at 3 months post-infusion exhibited IDUA enzyme activity levels that were 100-fold that of wild type in the olfactory bulb, with wild type levels of enzyme restored in all other parts of the brain. Intranasal treatment with AAV9-IDUA also resulted in clearance of tissue GAG storage materials in all parts of the brain. There was strong IDUA immunofluorescence staining of tissue sections observed in the nasal epithelium and olfactory bulb but there was no evidence for the presence of transduced cells in other portions of the brain. This indicates that clearing of storage materials most likely occurred as a result of enzyme diffusion from the olfactory bulb and the nasal epithelium into deeper areas of the brain. At 6 months of age, intranasally treated animals along with age-matched heterozygote and IDUA-deficient control animals were subjected to neurocognitive testing using the Barnes maze. Unaffected heterozygote animals exhibited improved performance in this test while MPS I mice displayed a deficit in locating the escape. Remarkably, MPS I mice treated intranasally with AAV9-IDUA exhibited behavior similar to the heterozygote controls, demonstrating prevention of the neurocognitive deficit seen in the untreated MPS I animals. This novel, non-invasive strategy for intranasal AAV9-IDUA administration could potentially be used to treat CNS manifestations of MPS I.
Lentiviral-Driven Stem Cell Gene Therapy for Severe Mucopolysaccharidosis Type II
H. Gleitz1, C. O’Leary1, R. Holley1, B. Bigger1
1Faculty of Medical & Human Sciences, University of Manchester
Purpose: Mucopolysaccharidosis type II (MPS II) is an X-linked genetic disorder characterized by mutations in the iduronate-2-sulphatase (IDS) gene, which normally degrades complex sugars in lysosomes. These mutations lead to cellular accumulation of glycosaminoglycans in the brain and skeleton, and culminate by death in teenage years. Severe MPS II is non-responsive to enzyme replacement therapy or standard hematopoietic stem cell transplantation. We previously demonstrated improved neuropathology using ex vivo stem cell gene therapy in MPS IIIA using the hCD11b promoter in a third-generation lentiviral vector, and are applying a similar principle to treat the brain in MPS II. Methodology: We have successfully designed and generated a high-titer lentiviral vector expressing codon-optimized human IDS and tested its efficacy in vitro. Autologous MPSII hematopoietic stem cells (HSCs) were transduced with LV.CD11b.IDS vector expressing codon-optimized IDS and transplanted into busulfan-conditioned 6-week-old MPSII mice. An additional cohort of male MPS II mice was also transplanted with wild-type HSCs after receiving busulfan conditioning at 6 weeks of age. MPS II mice were evaluated for neurocognitive, skeletal and activity deficits using the rotarod, spontaneous alternation test, social novelty preference test and whole body x-ray. Results: In vitro data shows significant overexpression of IDS after transduction in microglial cells and high secretion of active IDS enzyme into surrounding media by up to 60-fold. LV.CD11b.IDS-transduced HSCs were analyzed for enzyme activity and hematopoietic proliferation and differentiation at transplant. Peripheral blood chimerism indicative of engraftment was measured at 4- and 8-weeks post-transplant and vector copy number in white blood cells (WBCs) was analyzed at 4-months post-transplant. Conclusion: We report that the myeloid-driven LV.CD11b.IDS lentiviral vector can produce supraphysiological levels of active IDS enzyme in both microglial cells and HSCs. High HSC engraftment is seen at 4-weeks post-transplant after busulfan conditioning and we obtained satisfactory vector copy number in WBCs 4-months post-transplant. We demonstrate compelling evidence that autologous lentiviral-driven stem cell gene therapy to treat the brain in MPS II is a highly promising and clinically relevant therapy.
Long-Term Efficacy and Safety Evaluation in Mice and Dogs of Intra-CSF Administration of AAV9-Sulfamidase for the Treatment of MPSIIIA
S. Marcó1, A. Ribera1, M. García1, S. Motas1, M. Jaén1, A. Andaluz1, S. Añor1, J. Ruberte1, V. Haurigot1, F. Bosch1
1Universitat Autònoma de Barcelona, Bellaterra
Purpose: Mucopolysaccharidosis Type IIIA (MPSIIIA) or Sanfilippo A syndrome is a rare autosomic recessive Lysosomal Storage Disease (LSD) caused by deficiency in sulfamidase, a sulfatase involved in lysosomal degradation of the glycosaminoglycan (GAG) heparan sulfate (HS). Accumulation of undegraded HS leads to lysosomal pathology, which in turn results in severe progressive neurological deterioration with relatively mild somatic pathology. Patients usually die within the first 2 decades of life. We previously demonstrated in MPSIIIA mice that 4 months after intra-cerebrospinal fluid (intra-CSF) administration of AAV9 vectors encoding sulfamidase there was whole-body disease correction through transgene expression throughout the CNS and liver. The aim of the present study was to evaluate the long-term therapeutic efficacy and safety of this gene therapy approach in mice and dogs. Methodology: Two-month-old MPSIIIA mice received an intracisternal administration of 5x1010 viral genomes (vg) of serotype 9 Adeno-Associated viral vector (AAV9) encoding for murine sulfamidase. Long-term safety and efficacy on CNS and somatic pathology was evaluated through biochemical, histopathological and functional measurements. In dogs receiving 1x1012 vg of AAV9 vectors encoding for canine sulfamidase, transgene expression was followed for more than 3 years through measurement of sulfamidase activity in CSF. Safety was evaluated through biochemical and hematological parameters as well as magnetic resonance of the brain and abdominal ultrasound. Results: Delivery of AAV9-Sulfamidase vectors to the CSF of adult MPSIIIA mice lead to safe and long-term correction (>10 months of follow-up) of CNS and somatic pathology. Moreover, a single intra-CSF administration of AAV9 vectors encoding canine sulfamidase to healthy Beagle dogs at the therapeutic dose resulted in long-term stable increase in sulfamidase activity in the CSF, in the absence of any safety concerns. Conclusion: These results demonstrate the long-term efficacy and safety of intra-CSF AAV9 sulfamidase gene transfer and support the clinical translation of this therapeutic approach for the treatment of MPSIIIA and other LSD with CNS involvement.
ZFN-Mediated Correction of Murine MPS I and MPS II Models by Expression of the Human Alpha-L-Iduronidase and Iduronate 2-Sulfatase cDNAs From the Albumin “Safe Harbor” Locus
T. Wechsler1, R. DeKelver1, K. Laoharawee2, L. Ou2, S. Tom1, R. Radeke1, M. Rohde1, S. Sproul1, M. Przybilla2, B. Koniar2, K. Podetz-Pedersen2, R. Cooksley2, Y. Santiago1, K. Meyer1, S. McIvor2, C. Whitley2, M. Holmes1
1Sangamo BioSciences, Richmond
2University of Minnesota, Minneapolis
Mucopolysaccharidosis type I (MPS I/Hurler Syndrome) and type II (MPS II/Hunter Syndrome) are caused by deficiencies of alpha-L-iduronidase (IDUA) and iduronate 2-sulfatase (IDS), respectively, and subsequent systemic accumulation of glycosaminoglycans (GAGs) leading to widespread complications and a shortened lifespan. Currently, these diseases are treated by stem cell transplant (with significant risk of morbidity and mortality) and enzyme replacement therapy (ERT), which requires costly, weekly therapeutic infusions. Due to the unmet need, a ZFN-mediated genome editing strategy is proposed to permanently modify patient cells by genetically complementing either the hIDUA or hIDS defect. We have previously demonstrated ZFN-driven in vivo targeting of the albumin locus in the liver as a “safe harbor” for expression of coagulation factors, correcting clotting defects in hemophilic mice. This approach ensures long-term expression of the AAV-delivered transgene by exploiting the high level transcriptional activity of the native albumin enhancer/promoter in stably modified hepatocytes and utilizing an endogenous promoter obviating this requirement in the AAV payload. For proof-of-concept studies both hIDUA-deficient MPS I and hIDS-deficient MPS II mice were treated with a single intravenous infusion of a mixture of ZFN-encoding AAV vectors and an AAV vector encoding either the hIDUA or hIDS cDNA flanked by albumin sequence homology arms (i.e. the transgene donor). Wild-type littermates, untreated MPS I/II mice, and MPS I/II animals infused only with the hIDUA/hIDS donor vector (without ZFN-encoding vectors) were included as controls. Successful ZFN-mediated insertion of the hIDUA/hIDS coding sequence at the albumin locus results in enzyme expression regulated by the endogenous albumin promoter. Following ZFN+Donor AAV administration robust hIDUA/hIDS protein expression was detected in the liver, leading to supraphysiological enzymatic activities (60-200 fold above wildtype levels). Similarly, in the plasma of treated animals, stable, supraphysiological activity levels of IDUA or IDS were detected throughout the study (4 months), while undetectable or very low levels of enzymatic activity were found in the control animals. Significant IDS/IDUA enzyme activity could also be found in respective peripheral tissues including spleen, kidney, lung, heart and muscle and ranged from 6% to 200% of wild-type levels. In addition, up to 2% of wild-type IDUA / IDS activity was detected in the brain of ZFN+Donor-treated animals, while little to no increase in IDUA/IDS activity was observed in the tissues of animals infused with the respective transgene donor vector alone. Urine GAGs were reduced in all of the ZFN+Donor treatment groups in both MPS disease models. Tissue GAGs in the treatment groups were also significantly decreased in liver and most peripheral tissues that had markedly increased IDUA/IDS activity. No tissue GAG reduction was observed in animals infused with hIDUA/hIDS donor only. Animals from both studies were tested 4 months after AAV injection in the Barnes Maze as neurobehavioral assessment to determine the neurological effect of targeted hIDUA/hIDS expression in the liver. Compared to untreated MPS I or MPS II mice the treated animals showed significant neurological benefits and behavior similar to wild-type mice. In summary, our data provide proof of concept for ZFN-mediated targeting of the albumin locus in hepatocytes as an in vivo protein replacement platform to express therapeutic amounts of hIDUA or hIDS, leading to GAG biomarker correction and neurological benefits in relevant disease mouse models.
A Cathepsin B Inhibitor Reduces Aortic Dilatation in MPS I Mice
G. Baldo1, E. Gonzalez2, G. Martins1, M. Viegas1, R. Giugliani3, U. Matte1
1Gene Therapy Center, Porto Alegre
2Postgraduate program in Genetics and Molecular Biology, Porto Alegre
3Medical Genetics Service, Porto Alegre
Purpose: Mucopolysaccharidosis type I (MPS I) is a hereditary disorder caused by mutations in the gene encoding for lysosomal enzyme alpha-L-iduronidase (IDUA), involved in the catabolism of glycosaminoglicans (GAG) heparan and dermatan sulfate. MPS I produce aortic dilatation that is possibly associated with the imbalance in the turnover of extracellular matrix proteins of aortic walls. The upregulation of cathepsin B has been described as a possible pathological mechanism. To test the role of this enzyme, a pharmacological inhibitor (Ca074-Me) was utilized. Methodology: Mouse aortas were collected to evaluate total elastase activity in the tissue. Also, cathepsin B localization in MPS I cells was analyzed by immunofluorescence. MPS I mice were treated by intraperitoneal injection with cathepsin B inhibitor (10 mg/kg/day) or equal volume of vehicle (DMSO 10%) for 12 weeks (from 2 to 6 months). Aortic root diameter was determined before euthanasia by digital caliper and elastin breaks were analyzed by Verhoef Van Gieson (VVG) staining. They were compared to control normal mice and MPS I untreated mice. All animals were sacrificed at 6 months. The study was approved by our local ethics committee. Results: MPS I mice presented increased total elastase activity in aortic tissue compared to normal mice (p = 0.02). In MPS I fibroblasts, Lamp-1-positive vesicles localized juxtanuclear and some Cathepsin B staining colocalized with Lamp-1. However, at the same time, Cathepsin B–positive vesicles were observed in the cytosol, indicating its upregulation and redistribution of these proteases from lysosomes to the cytosol. Aortic root diameter in MPS I mice was 67% higher compared to normal mice. Mice treated with Cathepsin B demonstrated decrease of 18% in aortic dilatation compared to MPS I mice (p<0.05), although were still not normalized. Elastin breaks in treated mice were also intermediate between normal and untreated mice. Assay for all cathepsins showed that, despite the use of the inhibitor in vivo, cathepsin B activity could still be detected at intermediate levels between normal and MPI mice in treated aortas. Conclusion: The results demonstrated that aortic dilatation in MPS I mice is associated with increased elastin breaks and elastase activity. Translocation of cathepsin B coupled with high activity and export outside of the cell, could be the main responsible for the elastase activity, elastin break and aortic dilatation observed in MPS I mice. Pharmacological inhibition partially inhibited cathepsin B activity in the aortic tissue and reduced aortic dilatation in MPS I mice.
Pentosan Polysulfate and Neuroinflammation in Mice With Mucopolysaccharidosis Type IIIA
C. Simonaro1, E. Schuchman1, N. Guo1, V. Deangelis1
1Icahn School of Medicine at Mount Sinai, New York
Purpose: Studies in murine models of Sanfilippo Syndrome (mucopolysaccharidosis type III; MPS III) have revealed that neuroinflammation and neurodegeneration are important components of the central nervous system (CNS) phenotype. We evaluated the anti-inflammatory properties of a FDA/EMA approved drug; pentosan polysulfate (PPS) on neuroinflammation, neurodegeneration and neurobehavioral defects in MPS IIIA mice. Methodology: PPS was administered subcutaneously (SQ) once weekly using two doses; 25 and 50 mg/kg. Animals were treated for 36 weeks beginning at 5-7 days of age or for 24 weeks beginning at 5 months of age. Analysis was focused on evaluating CNS endpoints, including neural cell death, expression of pathogenic neural proteins (e.g., P-tau), heparan sulfate and GM3 storage, and behavioral assessments. Immunohistochemistry and mass spectrometry was used. Systemic inflammation was measured in the plasma using Elisa methods for proinflammatory markers: IL-1 alpha, IL-1beta, MIP-1 alpha, MCP-1 and G-CSF. Results: Reduced neuroinflammation and P-tau expression was observed in the brains of the treated MPS IIIA mice. In addition, systemic inflammation as measured by inflammatory cytokines in the plasma was also reduced. Reduced hyperactivity was also seen in the treated mice by rotarod analysis. Conclusion: PPS has potent anti-inflammatory properties in the MPS disorders. We have previously demonstrated its efficacy in animal models of MPS VI and I, particularly in the skeletal and cardiac phenotypes (Simonaro et al., 2013; Frohbergh et al., 2014; Simonaro et al., 2016). In addition to reducing inflammation, PPS treatment substantially reduced glycosaminoglycan (GAG) storage in urine and tissues. It is not currently known whether systemically administered PPS crosses the blood brain barrier (BBB), although dogs with MPS I treated for 12 months with biweekly SQ PPS exhibited significant reduction of cytokines in their cerebrospinal fluid. This study has shown the anti-inflammatory effects of PPS both systemically and in the CNS of MPS IIIA mice. Other neurological endpoints were also improved. Therefore, we conclude that PPS may crossing the BBB and maybe beneficial in neurological forms of MPS.
AAV8-Mediated Expression of N-Acetylglucosamine-1-Phosphate Transferase Attenuates Bone Loss in a Mouse Model of Mucolipidosis II
Y. Sohn1, A. Ko2, S. Cho3, J. Kim3, D. Jin3
1Ajou University Hospital, Suwon
2Samsung Biomedical Research Center, Seoul
3Samsung Medical Center, Seoul
Mucolipidoses II and III (ML II and ML III) are lysosomal disorders in which the mannose 6-phosphate recognition marker is absent from lysosomal hydrolases and other glycoproteins due to mutations in GNPTAB, which encodes two of three subunits of the heterohexameric enzyme, N-acetylglucosamine-1-phosphotransferase. Both disorders are caused by the same gene, but ML II represents the more severe phenotype. Bone manifestations of ML II include hip dysplasia, scoliosis, rickets and osteogenesis imperfecta. In this study, we investigated to determine whether a recombinant adeno-associated viral vector (AAV2/8-GNPTAB) could confer high and prolonged gene expression of GNPTAB and thereby influence the pathology in the cartilage and bone tissue of a GNPTAB KO mouse model. The results demonstrated significant increases in bone mineral density and content in AAV2/8-GNPTAB-treated as compared to non-treated KO mice. We also showed that IL-6 expression in articular cartilage was reduced in AAV2/8-GNPTAB treated ML II mice. Together, these data suggest that AAV-mediated expression of GNPTAB in ML II mice can attenuate bone loss via inhibition of IL-6 production. This study emphasizes the value of the MLII KO mouse to recapitulate the clinical manifestations of the disease and highlights their amenability to therapy.
Improved Neurocognitive Performance and Hippocampal Synaptic Plasticity after Long-Term Enzyme Replacement Therapy in Immunotolerant Alpha-Mannosidosis Mice
S. Stroobants1, M. Damme2, A. Van der Jeugd1, B. Vermaercke1, C. Andersson3, J. Fogh3, P. Saftig2, J. Blanz2, R. D’Hooge1
1KU Leuven
2University of Kiel
3Zymenex, Hillerød
Alpha-mannosidosis is a glycoproteinosis caused by deficiency of lysosomal acid alpha-mannosidase (LAMAN), which markedly affects neurons of the central nervous system (CNS), and causes pathognomonic intellectual dysfunction in the clinical condition. Cognitive improvement consequently remains a major therapeutic objective in research on this disorder. Immune-tolerant LAMAN knockout mice were developed to evaluate the effects of prolonged enzyme replacement therapy (ERT). Biochemical evidence suggested that hippocampus may be one of the brain structures that benefits most from long-term ERT. In the present functional study, ERT was initiated in 2-month-old immune-tolerant alpha-mannosidosis mice and continued for 9 months. During the course of treatment, mice were trained in the Morris water maze task to assess spatial-cognitive performance, which was related to synaptic plasticity recordings and hippocampal histopathology. Long-term ERT reduced primary substrate storage and neuroinflammation in hippocampus, and improved spatial learning after mid-term (10 weeks+) and long-term (30 weeks+) treatment. Long-term treatment almost completely reversed the spatial-cognitive performance of mannosidosis mice, whereas effects of mid-term treatment were more modest. Detailed analyses of spatial memory indicated that even prolonged ERT did not completely restore higher cognitive abilities, but it did demonstrate marked therapeutic effects that coincided with improvements in hippocampal long-term potentiation (LTP). These experiments indicate that long-term ERT may hold promise, not only for the somatic defects of mannosidosis, but also to alleviate cognitive sequelae of the disorder.
Improvement in Biomarkers After Intrathecal Iduronidase for Children With MPS I-H
T. Lund1, W. Miller1, G. Raymond1, M. Pasquali2, P. Orchard1
1University of Minnesota, Minneapolis
2University of Utah School of Medicine, Salt Lake City, UT
Purpose: Mucopolysaccharidosis type I-Hurler (MPS I-H) is caused by a deficiency in alpha-L-iduronidase and defective metabolism of glycosaminoglycans (GAGs). Enzyme replacement therapy (ERT) in the form of recombinant iduronidase can be given to correct some of the visceral manifestations of disease, but does not cross the blood brain barrier or prevent fatal neurodegeneration that accompanies MPS-I-H. Neurologic correction is only by way of hematopoietic cell transplantation (HCT). To bypass the blood brain barrier, we tested the hypothesis that intrathecal (IT) iduronidase can be safely delivered to the brain and measured cerebral spinal fluid (CSF) biomarkers to assess biologic effect.
Methodology: Twenty MPS I-H patients received IT iduronidase at four time points: 8-12 weeks prior to HCT, 14 days prior to HCT, 100 days after HCT, and 180 days after HCT. We measured CSF white cells, protein, opening pressure (OP), HCIIT, and biomarkers of inflammation. Results: We found a significant reduction in the OP between the first and second doses of IT ERT (22.5 mm H2O versus 18 mm H2O, p = 0.03). We found significantly higher levels of CSF MCP1, SDF1, IL1-RA, IL-8, MIP-1b, and VEGF in MPS I-H patients when compared to non-MPS I-H patients, and intervention led to a decline in MCP1 and SDF1 levels over the four doses of IT ERT (mean of 976±126pg/mL to 169±53pg/mL for MCP1, mean of 396±31pg/mL to 302±48pg/mL for SDF1). Our analysis of CSF HCIIT levels showed a marked reduction in the amount of HCIIT between the first and second IT ERT doses (prior to BMT) followed by steady state levels after the third and fourth doses (4532±1365 pg/mL versus 2720±1180 pg/mL, p = 0.005). GAG levels measured by non-reducing ends drop a mean of 68% after the first dose of IT ERT (p < 0.0001) Conclusion: This is the first in-human experience with IT ERT in MPS-IH patients. MPS I-H patients tolerated IT ERT well; there were no adverse events observed and the delivery was safe without increased morbidity or mortality. We show significant declines in several biomarkers of MPS 1-H including: CSF opening pressure, HCIIT, GAG levels, and several CSF cytokines. The most significant results were found after the first dose of IT ERT prior to HCT. IT ERT may provide MPS I-H patients a “bridge” of CNS protection against the inflammatory component of MPS 1-H, prior to undergoing HCT.
Expert Opinion on the Management of Intracerebroventricular (ICV) Drug Delivery
I. Slavc1, J. Cohen-Pfeffer2, S. Gururangan3, E. Jurecki2, J. Krauser3, D. Lim4, M. Maldaun5, C. Schwering6, A. Shaywitz2, M. Westphal6
1Medical University of Vienna
2BioMarin Pharmaceutical Inc., Novato
3Duke University Medical Center, Durham
4University of California, San Francisco
5IEP-Hospital Sírio-Libanês, São Paulo
6University Medical Center Hamburg-Eppendorf, Hamburg
Background: The intracerebroventricular (ICV) route of administration has been used for many decades to treat pediatric and adult patients with a broad range of central nervous system (CNS) disorders. There is no consensus in management of ICV devices and associated rates of reported complications are highly variable. A systematic literature review revealed that noninfectious complication rates per patient range from 1-33%, while infectious complication rates range from 0-27%. Objectives: 7 healthcare professionals (neurosurgeons, neuro-oncologists, pediatricians, nurse practitioners) with expertise in ICV delivery met to discuss best practices in management of ICV devices and drug administration and to provide guidance on prevention of complications. Results: Experts share common practices in the management of ICV devices. Most are experienced in delivering drugs through a bolus injection, though one center has had clinical trial experience with an infusion. In either case, extreme care must be taken to follow strict aseptic/sterile techniques. Waiting a minimum of 5 days after device implantation before first use of device is recommended to allow proper wound healing and to reduce risk of backflow of the administered drug through the catheter tract. Experts differ in practice of hair removal over the device, on the type of skin prep solution used, and in use of gown and cap. All experts recommend use of sterile gloves and mask as well as skin disinfection with multiple separate swabs. Conclusions: ICV drug delivery is an effective way to deliver drugs into the CNS when stringent measures are taken to prevent complications.
Gene Editing Using the CRISPR-Cas9 System in Human MPS I Fibroblasts
G. Baldo1, T. Giacomet de Carvalho1, F. Matheus Pellenz1, R. Giugliani1, U. Matte1
1Gene Therapy Center, Porto Alegre
Purpose: Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder caused by mutations in the IDUA gene, which leads to accumulation of the glycosaminoglycans dermatan and heparan sulfate. Considering the limitations presented by existing therapies, the investigation of new treatments is needed. The recent progress in genome editing technology using the repeat-CRISPR-associated protein 9 (CRISPR-Cas9) system have enabled the possibility of precisely modifying target sites in the genome, and brings hope of a cure for many genetic diseases. Thus, the aim of this study was to use the CRISPR-Cas9 system to correct point mutations in fibroblasts from MPS I patients. Methodology: Fibroblasts of two MPS I patients with p.W402X mutation were grown and transfected with a CRISPR-Cas9 vector designed to cleave a region near the mutation plus an oligonucleotide with the corrected sequence for homologous recombination. The cells were kept in culture for up to 30 days. 48 hours and 30 days after transfection, IDUA activity in the cells and in the culture medium was quantified by fluorimetric assay, and the results were compared with normal and untreated fibroblasts. Results: In cells from both patients, we observed an increase of 8 times in IDUA activity 48 h after transfection, compared to untreated cells. This value was approximately 10% the IDUA activity measured in fibroblasts from healthy subjects. Enzyme activity in culture medium in which the cells were maintained for 48 hours after transfection showed no statistical difference among the cells transfected or not for both patients. Patient 1 cells were maintained in culture for 30 days after transfection, and the values of IDUA activity in these cells remained at approximately 10% of normal fibroblasts. Conclusion: The results showed that a percentage of the cells was corrected and went on to produce the missing enzyme in the patient’s cells even after 30 days, indicating a long term correction. This suggests a potential utility of Cas9-based therapeutic genome editing for MPS I.
CNS-Directed Gene Therapy for the Treatment of Neurologic and Somatic Mucopolysaccharidosis Type II (Hunter syndrome)
S. Motas1, M. Garcia1, S. Marcó1, A. Ribera1, C. Roca1, X. Sánchez1, V. Sánchez1, J. Ruberte1, V. Haurigot1, F. Bosch1
1CBATEG-UAB, Bellaterra
Purpose: Mucopolysaccharidosis type II (MPSII), or Hunter Syndrome, is a X-linked recessive Lysosomal Storage Disease (LSD) caused by deficiency inIduronate-2-sulfatase (IDS), an enzyme involved in the stepwise degradation of the glycosaminoglycans (GAGs) heparan sulfate (HS) and dermatan sulfate (DS). Undegraded HS and DS accumulate in lysosomes, leading to lysosomal pathology and cell dysfunction. The most severe form of MPSII, more prevalent than the milder form, is characterized by chronic and progressive neuropathy of the Central Nervous System (CNS) and multisystem dysfunction. Patients usually die during the second decade of life. Enzyme Replacement Therapy (ERT) based on recombinant human IDS (ELAPRASE®) periodic administrations is indicated for Hunter patients. However, weekly intravenous ERT-infusions are not sufficient to correct neurological impairment. The purpose of this work was to develop a gene therapy approach for MPSII based on CNS-directed gene transfer to simultaneously treat neurologic and somatic pathology. Methodology: Serotype 9 adeno-associated viral vectors (AAV9) encoding IDS were delivered by single intracisternal administration to the cerebrospinal fluid of MPSII males aged 2 months. The efficacy of the treatment was assessed 4 months after vector delivery through evaluation of IDS activity, GAG content, neuroinflammation, lysosomal distension and homeostasis, tissue ultrastructure, transcriptional signature, behavior and survival rate. Results: Treated mice showed a significant increase in IDS activity in both CNS and periphery, leading to reversion of GAG accumulation, neuroinflammation, normalization of brain transcriptional signature, restoration of lysosomal homeostasis, correction of behavioral deficits and extension of lifespan. In addition, results provide evidence of cross-correction of non-transduced organs by circulating enzyme. Conclusions: The delivery of IDS-encoding AAV9 vectors to the CSF of MPSII mice leads to the correction of both CNS and somatic pathology. The present work provides strong evidence supporting the clinical translation of this approach for the treatment of Hunter patients with cognitive impairment.
Treatment With Pentosan Polysulphate in Patients With MPS I: Results From an Open Label, Randomized, Monocentric Phase II Study
S. Gökce1, A. Solyom2, E. Mengel1, E. Schuchman3, C. Simonaro3, J. Hennermann1
1Villa Metabolica, Universitätsmedizin Mainz
2Plexcera Therapeutics, New York
3Icahn School of Medicine at Mount Sinai, New York
Purpose: Current treatment options for MPS I have limited effects on some organ systems, including the skeletal system. In MPS animal models pentosan polysulfate (PPS) reduces inflammatory markers and glycosaminoglycans (GAGs) in tissues and body fluids and improves cartilaginous and osseous pathologies. The goals of this study were to investigate primarily the safety and secondary the clinical effects of PPS treatment in MPS I patients. Methodology: Four MPS I-Hurler-Scheie/-Scheie patients aged 35.6±6.4 years with one male were included in the study. All patients were on enzyme replacement therapy since 9.45±3.75 years. PPS was applied subcutaneously with 1 mg/kg and 2 mg/kg, respectively, weekly for 12 weeks and then biweekly for 12 weeks. Results: The 24-week treatment with PPS was well tolerated by all patients. Laboratory results revealed no signs of hepatopathy or abnormal anticoagulation. Patients receiving 2 mg/kg PPS had higher values for aPTT and INR, as these patients were on additional treatment with phenprocoumon. Urinary GAG concentrations were reduced from 4.13±1.17 mg/mmol at baseline to 2.69±0.36 mg/mmol after 24-week treatment with 1 mg/kg PPS, and from 6.71±0.62 mg/mmol to 2.65±0.09 mg/mmol after 24-week treatment with 2 mg/kg PPS. The pain intensity score was reduced from 4.5±1.77 at baseline to 1.8±0.47 after 24-week treatment with 1 mg/kg PPS; patients with 2 mg/kg PPS had already minimal pain at start of the study (baseline: 2.0±0.35, after 24-week treatment: 1.9±1.59). An improvement in range of motion (ROM) of the lower limbs was noted in 3 out of 4 patients, and an improvement in foot ROM in 1 patient. Conclusion: PPS treatment in a small number of adult MPS I patients was well tolerated and resulted in clinical significant improvements in GAG excretion, pain intensity and ROM. Therefore, we conclude that PPS may be an effective treatment in MPS I and also other MPS forms.
Surgical Reconstruction for Severe Tracheal Obstruction in Morquio A Syndrome
S. Tomatsu1, C. Pizarro1, R. Davies1, L. Averill1, M. Theroux1
1Alfred I. duPont Hospital for Children, Wilmington
Purpose: Progressive tracheal obstruction is commonly seen in Morquio A syndrome, and can lead to life threatening complications including sleep apnea, airway obstruction, pulmonary hypertension and death. Two-thirds of this population die of respiratory failure. Although tracheostomy can address severe upper airway obstruction, additional features including a narrow thoracic inlet, the variable level of obstruction as well associated vascular compression can make tracheostomy a non-suitable intervention. Method: We describe three cases with Morquio A whose near fatal tracheal obstruction was relieved by timely surgical trachea vascular reconstruction with dramatic resolution of respiratory symptoms. Results: The ages at surgical intervention were 16, 23, and 27 years old. Respiratory status in all patients was declining. After surgical intervention, all patients recovered from fatal tracheal obstruction and activity of daily living was markedly improved. Two patients were under ERT. This is the first surgical intervention of tracheal correction to avoid tracheostomy which is difficult to manage in this population. Conclusion: We expect that this new surgical procedure rescues many lives of patients with Morquio A.
Mucopolysaccharidosis and Sports
T. Wittke1, H. Horstmann1, L. Grigull1
1Hannover Medical School, Hannover
Purpose: 15 children with Mucopolysaccharidosis (MPS) received stem cell transplantations (SCT) at Hannover Medical School. A follow-up program was developed for monitoring and supporting the patients and their families. This follow-up did not include sport intervention until 2014, although beneficial effects of physical activity have been reported for several chronic diseases in children 1. Therefor a sportive intervention program was initiated and the sport medicine was integrated into the yearly follow-up investigations of the patients after SCT. Methodology: In our opinion, we have three different, but interlocking strategies to increase physical activity. We created different approaches depending on intrinsically motivation of the MPS affected children and adolescents, involving the parents and involving the teachers: A) Increase of everyday activity and quality of life during leisure time All patients receive a basic check-up from a sport scientist and an orthopedic surgeon. Focus is on power and shoulder movement/mobility. A sports program will be individually developed: focusing on body regions with decreased strength and mobility. B) Increase of activity, social inclusion and quality of life at school It is planned to create an interactive guide for physical activities (e.g. warm-up games, exercises for a circle-training), which are assigned for different levels of dysostosis multiplex. Based upon the individual severity of MPS related restrictions, teachers at school can select appropriate physical activities for their students with MPS by e.g. the help of an individual “traffic-light-system” (including GOs or NO-GOs for exercises during the physical education lesson at school). Discussion: To our knowledge there is no existing sport project for children with MPS in Europe. It is our aim to collect data for save integration of physical activities for individuals with MPS. It would be helpful to implement a “coach-the-coach” concept for parents with an affected child. Second, integration of teachers at school with a close cooperation between teachers and physicians would enhance integration of children with MPS into school physical activities. Experiences from other chronic diseases (e.g. cystic fibrosis) indicate beneficial effects of physical activities. In our project the effects of physical activities will be monitored using a standardized questionnaire to investigate the quality of life 2. The daily activity will be documented by using SenseWear body-moni-toring-system 3. Close national and international cooperations will be used to develop save ways to improve physical activities for children with MPS.
References
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