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Abstract

Background:

The main objective of this prospective, multicenter study (REVEAL-CP) was to test children with cerebral palsy-like signs and symptoms for raised 3-O-methyldopa (3-OMD) blood levels, a biomarker for aromatic L-amino acid decarboxylase deficiency (AADCd). A secondary objective was to characterize the molecular basis for the defective aromatic L-amino acid decarboxylase (AADC) gene product.

Methods:

Patients were identified in pediatric secondary and tertiary care hospitals through database searches and personal communication. 3-OMD concentrations from Guthrie card tests were determined using liquid chromatography/mass spectrometry. If 3-OMD was raised, cerebrospinal fluid analysis and dopa decarboxylase (DDC) gene sequencing were performed. An in-silico mutagenesis analysis was carried out to model altered AADC enzymes.

Results:

In total, 166 patients were enrolled in this study. The median age was 8 years. Sixty-six patients (39.8%) had a diagnosis of cerebral palsy, with the most common type being “mixed” (n = 42; 25.3%). One patient (0.6%), an 11-month-old boy from Italy, was diagnosed with AADCd caused by a homozygous, pathogenic DDC variant (c.749C>T; p.Ser250Phe). Three-dimensional modeling of the Ser250Phe AADC enzyme variant revealed its destabilization.

Conclusions:

A Guthrie card test for 3-OMD is a recognized screening technique for AADCd. If universal newborn screening for this metabolic disease is not available, children with signs and symptoms of a movement disorder should be investigated for AADCd.

Introduction

Aromatic L-amino acid decarboxylase deficiency (AADCd, OMIM 608643) is an autosomal recessive inborn error of metabolism first described by Hyland and Clayton in 1990 that has similarities to Parkinson’s disease (Hyland and Clayton, 1990). AADCd is caused by alterations in the dopa decarboxylase (DDC) gene (OMIM 107930) located on chromosome 7p12.2-p12.1 (genomic coordinates, 50,458,442-50,565,405; GRCh38/hg38). DDC has 15 exons and 14 introns. The exact incidence of AADCd worldwide is not known. A recent study estimated a worldwide incidence of 1/1.37 million births based on exome data from the Genome Aggregation Database (Park et al., 2023). However, there are geographical variations in incidence, with birth rates estimated to be 1/32,000 in Taiwan, 1/42,000-1/90,000 in the USA, 1/116,000 in the European Union, and 1/162,000 in Japan (Blau, 2024; Himmelreich et al., 2019). AADCd is thought to be more common in certain Asian populations, particularly Taiwan, compared with the rest of the world owing to the founder mutation IVS6 + 4A>T (c.714 + 4A>T) (Park et al., 2023; Blau, 2024). The carrier frequency ranges from 0.07% to 0.78% (Park et al., 2023). DDC encodes the enzyme aromatic L-amino acid decarboxylase (AADC, EC 4.1.1.28), which is an obligate functional homodimer and requires vitamin B6 (pyridoxal 5′-phosphate) as a cofactor. AADC is essential for the formation of the neurotransmitter serotonin and the catecholamines dopamine, noradrenaline, and adrenaline (Fig. 1) (Bisello et al., 2023). Infants with severe AADCd present with a wide range of signs and symptoms, including feeding difficulties, hypotonia, developmental delay, faltering growth, cerebral atrophy, seizures, oculogyric crisis, ptosis, hyperhidrosis, behavioral difficulties, and a reduced life expectancy (Blau et al., 2023). Milder cases with a later presentation have been reported (Cursio et al., 2023; Thys et al., 2024). DDC gene sequencing and variant analysis are the gold standards for diagnostic confirmation of patients with AADCd (Blau et al., 2023). If the AADC enzyme is defective, levodopa (L-DOPA) accumulates and is metabolized to 3-O-methyldopa (3-OMD) (Kubaski et al., 2021). Increased 3-OMD blood levels are a diagnostic biomarker for AADCd and can be efficiently measured using a dried blood spot (DBS) filter paper test (Guthrie card) (Kubaski et al., 2021). If the result of this screening test is abnormal, AADC enzyme activity assays and DDC gene analysis are performed (Kubaski et al., 2021). Cerebral palsy (CP) (spastic, dyskinetic, ataxic, or mixed) is a common childhood disability with an incidence of 1.6/1000 births (McIntyre et al., 2022). One of the many causes of CP is AADCd, which is difficult to diagnose clinically. The main aim of this international, multicenter, observational study (REVEAL-CP) was to screen patients from Western countries who presented with a movement disorder, such as CP, for AADCd using a Guthrie card test for 3-OMD. A secondary goal was to determine the molecular pathogenesis of the defect identified in new patients based on the three-dimensional structure of the AADC homodimer to predict the extent of protein loss of function.

FIG. 1.

Diagram depicting relevant steps of dopamine (A) and serotonin (B) metabolism that involve AADC and other enzymes. AADC, aromatic L-amino acid decarboxylase; ALDH, aldehyde dehydrogenase; COMT, catechol-O-methyltransferase; DBH, dopamine beta-hydroxylase; MAO, monoamine oxidase; PAH, phenylalanine hydroxylase; PNMT, phenylethanolamine-N-methyltransferase; TH, tyrosine hydroxylase; TPH, tryptophan hydroxylase.

Methods

General

This study was sponsored by PTC Therapeutics MP, Inc. (Warren, NJ; PTC-MA-AADC-402) and lasted from November 2020 to February 2022. In total, 16 study sites in the USA and Europe (France, Germany, Italy, and the UK) took part in patient recruitment.

Patient and public involvement

Patients or the public were not involved in the design, conduct, reporting, or dissemination of this research.

Ethics and consent

Before patient enrollment, research ethics committee approval was obtained in each participating country: the USA (SSU00111601), France (2019-A03014-53), Germany (EA2/057/20), Italy (219/2020; 58/20—CE; 0790/2020, 2021/489/DdP; 9/CE Medea; 2164/2020), and the UK (IRAS 274582, 20/LO/0597). Written consent was obtained from each patient and/or their legal guardian.

Patient selection

Patients of all ages who had signs and symptoms of CP or another movement disorder of unknown etiology were eligible to participate in this study. In practice, predominantly children and young people from secondary and tertiary pediatric hospital departments were recruited. Patients with periventricular leukomalacia, hypoxic-ischemic encephalopathy, cerebral infarction, encephalitis, cortical malformations, neuronal migration defects, spinal cord lesions, hydrocephalus or known AADCd, and those taking carbidopa/L-DOPA were excluded from enrollment.

Statistics

Descriptive statistics (the mean, standard deviation [SD], median, and range of patient characteristics) and the statistical analysis software IBM SPSS version 29.0.2 was used to analyze this large case series of an at-risk population.

3-OMD test

A capillary or venous blood sample was obtained from each patient and dripped onto a CentoCard (Centogene GmbH, Rostock, Germany). The filter paper cards were sent by surface mail to the Centogene laboratory in Rostock (Germany) for analysis. A detailed description of the methodology applied to measure 3-OMD blood levels with liquid chromatography/mass spectrometry has been published by Rizzi et al. (2023).

The method was validated in line with recommendations made by the College of American Pathologists and the Clinical Laboratory Improvement Amendments. A non-age-specific cut-off of >1000 nmol/L 3-OMD concentration was chosen based on blood samples from 110 healthy controls (upper limit of 95% confidence interval) and 80 patients with genetically determined AADCd.

DDC sequencing

The DDC gene was analyzed using an amplicon-based next-generation sequencing method (Illumina, San Diego, CA, USA). The amplicons cover the entire coding region and the highly conserved exon-intron splice junction (minimum coverage of >20× per amplicon). Missing regions or regions of poor quality were completed with traditional Sanger sequencing to achieve 99.9% coverage. The reference sequence was DDC NM_000790.3.

Bioinformatic analysis

Analysis of the localization of the Ser250 residue and its substitution to phenylalanine was carried out by in silico mutagenesis on the structure of human AADC bound to the substrate analogue L-DOPA methyl ester (PDB entry: 8ORA) using the program Coot 0.9.8.93 (Emsley et al., 2010). The models generated were subjected to simple molecular dynamics using the program phenix.dynamics (Burnley et al., 2012), as implemented in the program Phenix 1.21.1-5286 (Adams et al., 2010). Bond distance measurements resulting from molecular dynamics results, dimer building obtained by applying the symmetric operators, rotamer analysis satisfying the minimal energy requirements and picture drawing were performed using PyMOL 2.5 (The PyMOL, 2021).

Results

Patient cohort

In total, 166 patients were recruited during the 16-month-long study period. Eighty-nine patients (53.6%) were from Italy, 49 (29.5%) from the UK, 13 (7.8%) from France, 8 (4.8%) from Germany, and 7 (4.2%) from the USA. Ninety-seven patients (58.4%) were males and 69 (41.6%) were females. The mean age of the patient cohort was 9.5 years (SD, 11.63) and the median age was 8.0 years (25th percentile, 2.0; 75th percentile, 13.0; minimum, 0.0; maximum, 66.0). One hundred and 59 patients (95.8%) were 25 years or younger. One hundred and 32 patients (79.5%) were Caucasian; 11 (6.6%) Asian; 6 (3.6%) Black or African American; and 17 (10.2%) multiple, other, or undetermined. Sixty-six patients (39.8%) had a diagnosis of CP. Their mean age at diagnosis was 1.7 years (SD, 2.75; median, 0.5 years). The most common type of CP was “mixed” (n = 42; 25.3%), followed by spastic CP (n = 17; 10.2%) and dyskinetic CP (n = 7; 4.2%). One patient (0.6%, index case), an 11-month-old boy from Italy, had elevated 3-OMD blood levels. One hundred patients (60.2%) did not have a diagnosis of CP. The main signs and symptoms of all patients are summarized in Table 1.

Table 1.

Signs and Symptoms of REVEAL-CP Study Patients (N = 166)

Signs and symptoms, n (%)	3-OMD level		All patients(N = 166)
	Not elevated	Elevated
	(n = 165)	(n = 1)
Developmental delay	64 (38.8)	1 (100)	65 (39.2)
Hypertonia	48 (29.1)	0 (0)	48 (28.9)
Hypotonia	37 (22.4)	1 (100)	38 (22.9)
Poor head control	35 (21.2)	1 (100)	36 (21.7)
Feeding/swallowing difficulties	33 (20.0)	1 (100)	34 (20.5)
Dystonia	22 (13.3)	1 (100)	23 (13.9)
Dysarthria/speech difficulties	19 (11.5)	0 (0)	19 (11.5)
Hypokinesia	14 (8.5)	1 (100)	15 (9.0)
Oculogyric crises	14 (8.5)	1 (100)	15 (9.0)
Ptosis	10 (6.1)	1 (100)	11 (6.6)

3-OMD, 3-O-methyldopa.

Child with AADCd

This Caucasian boy was born after an uneventful pregnancy and delivery at term. He is the offspring of nonconsanguineous Italian parents. In the first few weeks of life, he was found to have remarkable irritability and generalized hypotonia. On examination, at the age of 11 months, he presented with severe global developmental delay, axial hypotonia, lack of postural reactions, mild distal dystonia of the limbs, hypokinesia, hypomimia, bilateral ptosis, oculogyric crises, and poor eye fixation. In addition, irritability, excessive salivation, hyperhidrosis, feeding and swallowing difficulties, gastroesophageal reflux, and faltering growth were observed. DBS analysis showed an increased 3-OMD concentration of 9910 nmol/L (normal range, ≤1000 nmol/L). Investigation of his cerebrospinal fluid (CSF) revealed a pattern characteristic of AADCd with neurotransmitter metabolite levels as follows (normal ranges are reported in brackets): homovanillic acid (HVA) 90 nmol/L (236-867 nmol/L); 5-hydroxyindole-3-acetic acid (5-HIAA) 26 nmol/L (97-367 nmol/L); HVA/5-HIAA ratio 3.46; 3-OMD 1843 nmol/L (<50 nmol/L); 5-hydroxytryptophan (5-HTP) 114 nmol/L (<10 nmol/L); and 3-methoxy-4-hydroxyphenylglycol (MHPG) 12 nmol/L (47-81 nmol/L). All other neurometabolic tests were within normal limits. Magnetic resonance imaging of the brain demonstrated a mild delay in white matter myelination in the frontal region bilaterally. Electroencephalography showed slow wave activity in the posterior regions. The child was initially prescribed the type-B monoamine oxidase inhibitor selegiline, pyridoxine (4-methanol form of vitamin B6), and the dopamine agonist pramipexole, which led to partial clinical improvement. Later, he underwent treatment with intraputaminal eladocagene exuparvovec (data not yet published).

Gene and protein analyses

DDC sequencing showed a homozygous pathogenic variant in exon 7 (c.749C>T; p.Ser250Phe). This means that on exon 7 in position 749, the base cytosine transitioned to thymine, which resulted in an exchange of the polar amino acid serine with the non-polar phenylalanine in position 250 of the 480-amino-acid-long synthesized AADC polypeptide chain. The exact location of this missense variant is chromosome 7p12.1, 7:50504025 (GRCh38). The amino acid substitution Ser250 to phenylalanine has been modeled in silico using the structure of the functionally homodimeric human AADC enzyme bound to the substrate analogue L-DOPA methyl ester recently solved at a high resolution (Fig. 2) (Bisello et al., 2023). With respect to previously performed bioinformatics analyses, the modeling with this human structure allowed us to precisely define the structure defect of the amino acid substitution. Ser250 is not directly present at the active site but is found in a short stretch of amino acids (243-252) between the active site and the protein surface. Ser250 establishes a stabilizing hydrogen bond with Thr245, which is known to be pathogenic when altered to isoleucine (Himmelreich et al., 2023). In the Ser250Phe variant, the interaction with Thr245 is eliminated, and modeled molecular dynamics permit the bulky phenylalanine residue to sit in two positions: (1) an “A” position toward the surface, which increases hydrophobicity and destroys the network of hydrogen bonds among a water molecule (A807) with the side chains of residues Asp252 and Ser188 and the backbone carbonyl of Phe215, decreasing stability; and (2) a “B” position toward the active site, which creates steric hindrance with structural disassembling and functional consequences. Both accommodations determine a structural defect that can have functional consequences in terms of AADC activity.

FIG. 2.

In silico mutagenesis of the human Ser250Phe AADC variant. The two subunits of the obligate functional AADC dimer are shown as ribbons in blue and cyan. The amino (N) and carboxy (C) terminals of one polypeptide chain are labeled; the other subunit is arranged in an antisymmetrical geometry. PLP bound to the substrate analogue DME is represented as CPK sticks in the two active sites. The zoom shows the Ser250 microenvironment with the interaction that Ser250 establishes with Thr245 (CPK colored in the cyan subunit) and the network of stabilizing bonds contacting the water molecule (red dot) (see the Results section for details). The “A” and “B” possible conformations of substituted Phe250 are shown in green. The same substitution Ser250Phe is present on the other subunit (blue), not shown, generating a homodimeric AADC variant. Figures of the structures were prepared and rendered using PyMOL (The PyMOL, 2021) and the dimeric structure has been generated by applying a symmetry operator to the crystallographic subunit (PDB entry: 8ORA). DME, levodopa methyl ester; PDB, Protein Data Bank; PLP, pyridoxal 5′-phosphate.

Discussion

In this prospective study among patients with signs and symptoms of CP or mimic-CP, one child with AADCd (0.6%) was identified through selective screening using a Guthrie card test for 3-OMD. Although this percentage seems low, it is significantly higher than the prevalence of AADCd reported in the general European population (1/116 000) (Himmelreich et al., 2019). Rizzi et al. performed a similar prospective, multicenter study in Italy (Rizzi et al., 2023). 3-OMD blood levels were analyzed from 390 patients with an undiagnosed neurodevelopmental disorder using Guthrie cards, and none of them were found to have AADCd (Rizzi et al., 2023). Hyland and Reott investigated 20,000 CSF samples for neurotransmitter abnormalities and detected 22 new cases of AADCd (Hyland and Reott, 2020). The prevalence of 1/900 in their at-risk population more closely resembles the figure of 1/166 from this study, which as a case series provides low-level evidence. Selective clinical screening (e.g., focused on patients with signs and symptoms of CP, like in the present study, or an early-onset movement disorder in general) may be more effective in intercepting affected patients. The current gold standard for diagnosing AADCd is a combination of DDC gene sequencing and analyzing CSF neurotransmitter metabolites and/or plasma AADC activity (Wassenberg et al., 2017). Several studies have provided evidence that 3-OMD DBS testing is a useful screening tool for the identification of patients with AADC deficiency and that it is less invasive than a lumbar puncture and more acceptable for children. If AADC activity is reduced, L-DOPA builds up and is converted to 3-OMD at an increased rate (Kubaski et al., 2021). However, a high 3-OMD plasma level is also present in pyridoxamine 5′-phosphate oxidase deficiency, characterized by early-onset seizures (Kubaski et al., 2021). Burlina et al. have established a high-throughput method for measuring 3-OMD plasma concentrations by using filter paper and applying flow-injection analysis tandem mass spectrometry (FIA-MS/MS) (Burlina et al., 2021). This test for AADCd has been added to the newborn screening program in Northeast Italy (Burlina et al., 2021). Over a period of almost 3 years, 157,371 neonates were screened for AADCd in Taiwan using a 2-tier approach for measuring 3-OMD concentrations in DBS samples (FIA-MS/MS and HPLC-MS/MS). Eight babies had raised 3-OMD levels, and six of them were subsequently diagnosed with AADCd (Chen et al., 2023).

The young patient in this investigation who presented with typical clinical features of AADCd was only diagnosed after enrollment in this study. This demonstrates the challenges clinicians in different healthcare settings face when encountering this rare disease. According to Himmelreich et al., the patient identified in the present study is the tenth patient with a homozygous, pathogenic variant (c.749C>T; p.Ser250Phe), making this the fifth most common variant of AADCd worldwide (Himmelreich et al., 2023). Overall, 5.3% of AADCd genotypes are responsible for 41.4% of all reported patients with this disorder (Fig. 3). Caine and collaborators have developed a knock-in mouse model for AADCd by introducing the same Ser250Phe missense variant in the murine Aadc gene (Caine et al., 2017). Cell culture experiments by a different group have shown that the replacement of serine with phenylalanine in position 250 leads to a 7-fold reduction in the catalytic activity of AADC that can be restored with pyridoxine (Montioli et al., 2013). A previous bioinformatic analysis of the Ser250Phe variant has been carried out with the pig AADC structure; however, low structural resolution prevented a precise structural definition. Therefore, in the present study, the structural bulky effect of the phenylalanine introduction was modeled in human AADC. The analysis of relative bond distances and rotamer stabilization evidenced a pronounced structural effect due to the bulky phenyl side chain that disrupts the hydrophilic milieu, leading to protein destabilization. Noteworthy, this could be associated with the reported functional effects: a decreased (by 3.6-fold) catalytic constant (k_cat) and an increased (by 2-fold) Michaelis-Menten constant (K_m) inversely proportional to substrate affinity (Bisello et al., 2023). Overall, the impairment of the catalytic efficiency (k_cat/K_m) is nearly 7-fold (Bisello et al., 2023), which is responsible for the loss of function of the Ser250Phe variant and possibly for the clinical phenotype of the patient.

FIG. 3.

Eight most common DDC mutations detected in patients with AADCd (Himmelreich et al., 2019). AADCd, aromatic L-amino acid decarboxylase deficiency; DDC, dopa decarboxylase; ex, exon.

Conclusion

In conclusion, this study provides evidence that 3-OMD DBS screening using Guthrie card tests targeted at patients with a movement disorder of unknown etiology can assist in identifying those with AADCd in an economic fashion and with a higher yield than in the general population. In addition, bioinformatics technology applied to one proband has enriched our understanding of the molecular pathogenesis of AADCd.

Footnotes

Acknowledgments

The authors are grateful to the young people and their carers who participated in this study. The authors would like to thank the healthcare professionals and administrative staff who were involved in this study, especially Jo-anna Allen, Kapil Arya, Guja Astrea, Eleonora Bonaventura, Peter Blomqvist, Alessandro Capuano, Carla Carducci, Claudia Carducci, Tamsin Chambers, Giovanni Cioni, Edward Dabrowski, Ian Davidson, Christian De Goede, Laura Demuth, Andrew Duckworth, Tim Ellison, Timothy Feyma, Robert Flamini, Peta Heslop, Keith Hyland, Angela Kaindl, Allen Kristensen, Michael Kruer, Warren Marks, Andrea Martinuzzi, Daniella Mascarenhas, Marie Monaghan, Thomas Opladen, Neil Parlett, Phillip Pearl, Vinay Penematsa, Mark Rance, Agathe Roubertie, Annamaria Russo, Roberta Scalise, Michael Shrader, Marie-Aude Spitz, Marc Steder, Micheal Taylor, Antonio Trabacca, and Suresh Vijay.

Authors’ Contributions

Conceptualization: S.J., P.L., and C.W. Investigation: E.-M.S., R.B. (identification of the patient with AADCd), V.G., A.K., S.A., M.B., M.P., and V.L. Supervision: S.J., P.L., and E.F. Data curation: S.A., M.B., M.P. Formal analysis: M.B., M.P., V.L. (CSF analysis for the patient with AADCd), and E.L. Writing—original draft preparation: E.-M.S. (lead), R.B., M.B., and M.P. Writing—review and editing: all authors.

Author Disclosure Statement

V.G. has participated in scientific advisory boards, scientific symposia, and teaching initiatives for Biogen, Italfarmaco, Novartis, Pfizer, PTC Therapeutics, Roche, and Wave Life Sciences; has received honoraria from Neurology Academy, Novartis, and Roche; and has been involved as principal investigator with Astria Therapeutics (formerly Catabasis Pharmaceuticals), PTC Therapeutics and Wave Life Sciences. M.B. has received a research grant from PTC Therapeutics. S.J., P.L., E.L., E.F., and C.W. are employees of PTC Therapeutics. All the other authors have no conflicts of interest to declare.

Funding Information

The study was sponsored by PTC Therapeutics MP, Inc. (Warren, NJ; PTC-MA-AADC-402). This research was supported in parts by the Italian Ministry of Health, Ricerca Corrente 2021, and the IRCCS Fondazione Stella Maris.

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