Abstract
INTRODUCTION
Oculopharyngeal muscular dystrophy (OPMD) has long been characterized by a combination of bilateral ptosis, dysphagia and subsequent limb girdle weakness [1, 2]. Accumulation of expanded (exp)PABPN1 into insoluble intranuclear inclusions in skeletal muscles is the pathological hallmark of OPMD [1]. Our Dutch pilot study and a recent Danish report showed that limb girdle weakness is the first and prominent symptom in a minority of patients [3, 4]. A more detailed description of the clinical symptoms in a larger cohort is required to confirm this. Next, it is largely unknown whether the intranuclear inclusions occur before clinical onset or only during disease progression [1]. Furthermore, the impact of aggregated PABPN1 on cellular defects in OPMD is not fully understood [5, 6]. We therefore performed a clinical and histopathological study of a cohort of 40 OPMD patients (of whom 36 genetically confirmed) and 18 adult children, comprising an unique wide spectrum starting with mutation carriers at a presympomatic stage to late symptomatic patients. In addition to nuclear aggregates, biopsy specimens from OPMD patients show dystrophic changes and mitochondrial abnormalities, the latter of which are generally considered a secondary phenomenon [6]. The availability of presymptomatic muscle tissue of Ala-expanded-PABPN1 carriers in this study offered an unique opportunity to study the histopathological and biochemical changes before clinical onset. We included an autopsy in an OPMD patient, enabling a qualitative correlation of the severity of histopathological changes to the degree of muscle weakness in five different muscles.
This study in presymptomatic expPABPN1 carriers and OPMD patients enabled us to investigate the spectrum of OPMD from different perspectives: by 1) focusing on the clinical aspects of this Dutch OPMD spectrum; 2) investigating subjects from presympto-matic to progressive disease stages; and 3) by studying variably affected muscles within the same patients.
METHODS
Participants
All Dutch OPMD patients known at our national OPMD referral centre were invited to participate in this study. Adult children of genetically confirmed OPMD patients were also invited without disclosure of their individual histological and genetic results as required by the local ethical committee. The histological and genetic investigation of the children was performed for scientific purposes only. If patients desired genetic counseling this was performed separately in a clinical setting.
Genetic investigations
Genetic testing for PABPN1 expansion mutation was performed on patients and children as previously described [3].
Medical history and clinical evaluation
A structured medical history and a neurological examination including manual muscle strength testing were performed in all participants [7]. This included assessment of facial muscle force, including forced eye closure, raising the eyebrows, blowing the cheeks, showing the teeth, pursing the lips and smiling. Facial weakness was defined as weakness of any of these tests. Dysphagia, ptosis and dysphonia were assessed qualitatively. Both the initial symptoms/signs (retrospective evaluation of the start of the disease according to the patient) and the current symptoms/signs (at the time of the study) were assessed.
Muscle biopsies and autopsy
Patients and children underwent a needle biopsy of the right vastus lateralis muscle. One autopsy was performed which included specimens of the vastus lateralis, biceps brachii, diaphragm, pharyngeal and tibialis anterior muscles.
Histological analysis. The muscle samples were examined as reported previously, including COX staining [8]. The intranuclear aggregates were counted using Elecron Microscopy (EM) in 150 to 200 nuclei of each muscle sample. The samples obtained during autopsy were additionally evaluated on semi-thin sections. The extent of the histopathological changes in the autopsy muscles samples was assessed qualitatively (absence; limited presence, moderate presence, extensive presence) and related to the clinical assessment of muscle strength (MRC scores) and the occurrence of dysphagia.
Biochemical analysis Measurement of total energy-generating capacity of mitochondria and oxidative enzyme activities was performed in children of OPMD patients (asymptomatic carriers and non-carriers) as previously reported [9]. Any disturbance of the mitochondrial energy generating system, apart from complex II deficiency, results in a lower pyruvate oxidation rate and ATP production, and will be detected in this way. We thus aimed to find out whether any mitochondrial changes arose before or after the typical histopathological changes of OPMD and whether they preceded or followed clinical onset.
RESULTS
Study population
Fifty-eight persons, including 40 patients and 18 asymptomatic adult children of genetically-confirmed OPMD patients participated in this study. In 36 patients OPMD was genetically confirmed by detection of the PABPN1 repeat expansion. In three patients, genetic testing had been performed in a first degree relative. In one patient, OPMD was diagnosed based on the phenotype and the presence of intranuclear aggregates in the biopsy. Six of the 18 asymptomatic children were PABPN1 mutation carriers; the others did not have the mutation (Table 1). The Alanine expansion mutation ranges from GCN13 to GCN16, with most of the patients (63%) had a GCN14 repeat and 31% had GCN16. The small number of patients with GCN13 or GCN15 repeat does not allow to draw conclusions on the correlation between repeat expansion size and phenotype. The patient who died during the study had a GCN 14 repeat.
Medical history and clinical evaluation
The initial symptom/sign was ptosis in 17 patients (43%), dysphagia in 12 (30%), limb girdle weakness in 10 (25%), and ptosis plus dysphagia in only one patient (3%). The age at onset and the age of examination was variable in this cohort (Table 1), and was not significantly different for any of the initial symptoms. Dysphagia was the most frequent current symptom (98% of patients). Ptosis and proximal leg weakness also occurred frequently at the time of the study (88% and 85% respectively). Other common current symptoms/signs were: Dysphonia and facial weakness/limited facial expression (70 % and 60% respectively). Two patients had isolated dysphagia; all other patients had more than one symptom/sign. The weakness of the pelvic muscles varied from mild (MRC 4) to severe (MRC 2). In addition, axial muscles were also affected: Neck flexor weakness and neck extensor weakness occurred in respectively 19 (48%) and in 11 (28%) patients. None of the patients initially presented with external ophthalmoplegia and diplopia, but during progression it developed in 17 (43%) and 7 (18%) patients respectively (mean age 60 and 63 years). All 18 children had a normal neurological examination.
During the study, one patient died due to pneumonia at the age of 69 years. He did not have any pulmonary complaints before, neither dyspnoea nor orthopnoea or signs of nocturnal hypoventilation. Neurological examination performed at a check-up shortly before falling ill had shown bilateral ptosis and atrophy of proximal muscles, mostly pronounced in the legs. Muscle weakness was symmetrical: Elbow flexors (MRC 3), wrist flexors (MRC 4), wrist extensors (MRC 4), hip flexors (MRC 2), knee extensors (MRC 2), and foot extensors and flexors (MRC 4).
Muscle biopsies
Twenty-four patients and 18 children underwent a needle biopsy. The biopsy of one patient only contained fatty tissue and was excluded in this study.
Histological analysis. The biopsies in children without the PABPN1 mutation (n = 12) were normal as expected. Histopathological investigation of the six biopsies from presymptomatic carriers revealed nuclear aggregates in four, and additionally rimmed vacuoles in one. Three biopsies with aggregates showed an increased variation of fiber size (Fig. 2), one did not show any dystrophic but few atrophic fibres. The biopsies of 23 symptomatic patients showed non-specific alterations including increase in fibre size variation in all biopsies, mild changes in the checkerboard pattern without fiber type grouping (22%), and rimmed vacuoles (74%). Only two biopsies showed each one COX negative fiber. Electron microscopic examination showed subsarcolemmal aggregates of mitochondria in four biopsies, and mitochondria with crystalline inclusions in two biopsies. In 83% of the biopsies intranuclear aggregates were observed in 1.8% of the nuclei (range 0.4–4.9) (Table 2a).
The histopathological investigations of the muscle samples obtained by the autopsy showed that the severity of the dystrophic alterations varied between different muscles. The severity of muscle weakness corresponded with the severity of the dystrophic alterations of the muscles: The muscles that were most severely affected clinically in this patient - proximal limb muscles - showed the most extensive pathological alterations (Table 2b; Fig. 1). In all examined muscles nuclear aggregates were observed and their presence did not correlate to muscle weakness (Table 2b).
Biochemical analysis Measurement of the mitochondrial substrate oxidation and the ATP production rates in muscle biopsies of five children without mutations and three presymptomatic PABPN1 mutation carriers was normal (Table 3a and 3b).
DISCUSSION
The main finding of this prospective cohort study in OPMD families is the presence of intranuclear aggregates before clinical onset: Aggregates were detected in biopsies of four of the six Ala-expanded-PABPN1carriers. The youngest expanded-PABPN1 carrier showing aggregates was 35 years old, whereas clinical onset is on average at 45 years of age. Autopsy in one patient showed aggregates in most muscles, also in the clinically non-affected diaphragm. Overall this study calls for a more detailed study of the role of PABPN1 aggregates in OPMD pathology. This is of high relevance since clinical trials are now ongoing, e.g. for trehalose (http://bioblast-pharma.com/) [10]. Trehalose is a natural alpha-linked disaccharide with high water retention capabilities and an anti-oxidant that inhibits protein aggregation, that has proven to be effective in the OPMD mouse model [11].
Next, the mitochondrial energy generating capacity in fresh muscle tissue originating from presymptomatic carriers was comparable to controls. The observation that nuclear aggregates were observed in the muscle tissue from these carriers indicates that formation of expPABPN1 aggregates precedes possible mitochondrial dysfunction [12, 13]. This observation is compatible with the hypotheses that mitochondria are only secondarily involved in the pathogenesis of OPMD [6]. Alternatively, the mitochondrial alterations might occur independently later in the course of the disease. A recent study in Drosophila flies overexpressing PABPN1-17ala showed that mitochondrial function was reduced in symptomatic flies (flightless with abnormal wings) [12, 13]. Additional findings in this study shed light on the possible mechanism in which mitochondrial function becomes disturbed: Alternative polyadenylation of mitochondrial genes causes reduced expression of these genes and impairs mitochondrial function. Because the authors also found accumulation of mitochondrial proteins in sternocleidomastoid muscle biopsies of OPMD patients, which is presumably less affected, they suggested that mitochondrial activity is affected by expPABPN1 expression already early in the disease course [12, 13]. A future study with mitochondrial evaluation of OPMD patients in different stages of the disease will be helpful to clarify if and when mitochondrial dysfunction plays a role in the pathogenesis of OPMD.
The third important finding was the high variation in the clinical manifestation of OPMD in this Dutch cohort. Limb girdle weakness occurred more frequently than initially reported and even as initial symptom in some patients (25%) [14, 15]. This is higher than the prevalence of 10% reported in an OPMD cohort in the United Kingdom [16]. This might be partly related to the large average mutation size of the patients in this study, leading to a more severe disease course. Worldwide, most OPMD patients have the (GCN)13 expansion; here only two out of 40 patients (5%) have this mutation and 31% had the (GCN)16 expansion. However the two patients with the (GCN)13 expansion had limb girdle weakness as first symptom. Our findings confirm the recent observations in the Danish OPMD cohort (n = 13): Limb weakness can be common in OPMD and can be even the presenting symptom in five patients [4]. All our patients except one suffered from dysphagia, sometimes even as initial symptom. Ptosis occurred less frequently than dysphagia. The presence of diplopia did not exclude the diagnosis of OPMD. We did not find a relation between the severity of symptoms of OPMD and patients age, probably due to the large variation of age at onset and the relatively small size of the cohort. Progression over time was slow (data not shown).
Finally, we showed a remarkable correlation between MRC scores and the histopathological changes in five different muscles of an individual patient. To our knowledge this is the first report presenting results of an autopsy of a genetically-confirmed and clinically characterized OPMD patient [17, 18]. Dion et al. showed the presence of intranuclear aggregates in cerebellar neurons of brain sections of an OPMD patient [19]. Little et al. reported an autopsy of a French-Canadian not genetically-confirmed patient and found that limb muscles were less affected histopathologically than diaphragm and pharyngeal muscles [18]. We found that the quadriceps muscle was more severely affected than the diaphragm and pharyngeal muscles. This strengthens the suggestion of high variability in patterns of muscle weakness in OPMD patients.
In conclusion, our study showed the presence of intranuclear aggregates without mitochondrial changes before clinical onset in Ala-expanded-PABPN1carriers. This observation can direct future studies on the pathophysiology of this disease and the role of the intranuclear aggregates in OPMD pathology in particular. Furthermore this study confirmed our previous pilot study suggesting that limb girdle weakness occurs frequently and prominently in this Dutch OPMD cohort with relatively long repeat lengths. As such, our observations will contribute to a better recognition of the OPMD phenotype. Importantly, our study demonstrated the broad spectrum of affected muscles, and the high variation in loss of strength of affected muscles during theprogression of the disease. Since OPMD is likely to be underdiagnosed, these findings will have a direct clinical impact on disease recognition [20, 21].
ETHICAL STANDARDS
The study was approved by the local Committee on Research Involving Human Subjects, and written informed consent of all participating subjects was obtained.
CONTRIBUTIONS
All authors have approved the final article should be true and included in the disclosure.
DISCLOSURE OF CONFLICT OF INTEREST
BM van der Sluijs, V Raz, M Lammens, LP van den Heuvel, NC Voermans, BG van Engelen declare that they have no conflict of interest.
Footnotes
ACKNOWLEDGMENTS
The authors thank the patients and their families for participation in this study. We thank M. van Dijk MD (Department of Pathology, Elkerliek Hospital, Helmond, The Netherlands) for performing the autopsy and sharing the muscle samples with the Radboud University Nijmegen Medical Centre. We also thank Mrs. Kiek Verrijp for technical assistance with thebiopsies.