Clinical and biochemical footprints of congenital disorders of glycosylation: Proposed nosology.

We have identified 200 congenital disorders of glycosylation (CDG) caused by 189 different gene defects and have proposed a classification system for CDG based on the mode of action. This classification includes 8 categories: 1. Disorders of monosaccharide synthesis and interconversion, 2. Disorders of nucleotide sugar synthesis and transport, 3. Disorders of N-linked protein glycosylation, 4. Disorders of O-linked protein glycosylation, 5. Disorders of lipid glycosylation, 6. Disorders of vesicular trafficking, 7. Disorders of multiple glycosylation pathways and 8. Disorders of glycoprotein/glycan degradation. Additionally, using information from IEMbase, we have described the clinical involvement of 19 organs and systems, as well as essential laboratory investigations for each type of CDG. Neurological, dysmorphic, skeletal, and ocular manifestations were the most prevalent, occurring in 81%, 56%, 53%, and 46% of CDG, respectively. This was followed by digestive, cardiovascular, dermatological, endocrine, and hematological symptoms (17-34%). Immunological, genitourinary, respiratory, psychiatric, and renal symptoms were less frequently reported (8-12%), with hair and dental abnormalities present in only 4-7% of CDG. The information provided in this study, including our proposed classification system for CDG, may be beneficial for healthcare providers caring for individuals with metabolic conditions associated with CDG.

Clinical, Neuroimaging, and Metabolic Footprint of the Neurodevelopmental Disorder Caused by Monoallelic HK1 Variants.

Background and Objectives: Hexokinase 1 (encoded by HK1) catalyzes the first step of glycolysis, the adenosine triphosphate-dependent phosphorylation of glucose to glucose-6-phosphate. Monoallelic HK1 variants causing a neurodevelopmental disorder (NDD) have been reported in 12 individuals. Methods: We investigated clinical phenotypes, brain MRIs, and the CSF of 15 previously unpublished individuals with monoallelic HK1 variants and an NDD phenotype. Results: All individuals had recurrent variants likely causing gain-of-function, representing mutational hot spots. Eight individuals (c.1370C>T) had a developmental and epileptic encephalopathy with infantile onset and virtually no development. Of the other 7 individuals (n = 6: c.1334C>T; n = 1: c.1240G>A), 3 adults showed a biphasic course of disease with a mild static encephalopathy since early childhood and an unanticipated progressive deterioration with, e.g., movement disorder, psychiatric disease, and stroke-like episodes, epilepsy, starting in adulthood. Individuals who clinically presented in the first months of life had (near)-normal initial neuroimaging and severe cerebral atrophy during follow-up. In older children and adults, we noted progressive involvement of basal ganglia including Leigh-like MRI patterns and cerebellar atrophy, with remarkable intraindividual variability. The CSF glucose and the CSF/blood glucose ratio were below the 5th percentile of normal in almost all CSF samples, while blood glucose was unremarkable. This biomarker profile resembles glucose transporter type 1 deficiency syndrome; however, in HK1-related NDD, CSF lactate was significantly increased in all patients resulting in a substantially different biomarker profile. Discussion: Genotype-phenotype correlations appear to exist for HK1 variants and can aid in counseling. A CSF biomarker profile with low glucose, low CSF/blood glucose, and high CSF lactate may point toward monoallelic HK1 variants causing an NDD. This can help in variant interpretation and may aid in understanding the pathomechanism. We hypothesize that progressive intoxication and/or ongoing energy deficiency lead to the clinical phenotypes and progressive neuroimaging findings.

Does hyperphenylalaninemia induce brain glucose hypometabolism? Cerebral spinal fluid findings in treated adult phenylketonuric patients.

Despite numerous studies in human patients and animal models for phenylketonuria (PKU; OMIM#261600), the pathophysiology of PKU and the underlying causes of brain dysfunction and cognitive problems in PKU patients are not well understood. In this study, lumbar cerebral spinal fluid (CSF) was obtained immediately after blood sampling from early-treated adult PKU patients who had fasted overnight. Metabolite and amino acid concentrations in the CSF of PKU patients were compared with those of non-PKU controls. The CSF concentrations and CSF/plasma ratios for glucose and lactate were found to be below normal, similar to what has been reported for glucose transporter1 (GLUT1) deficiency patients who exhibit many of the same clinical symptoms as untreated PKU patients. CSF glucose and lactate levels were negatively correlated with CSF phenylalanine (Phe), while CSF glutamine and glutamate levels were positively correlated with CSF Phe levels. Plasma glucose levels were negatively correlated with plasma Phe concentrations in PKU subjects, which partly explains the reduced CSF glucose concentrations. Although brain glucose concentrations are unlikely to be low enough to impair brain glucose utilization, it is possible that the metabolism of Phe in the brain to produce phenyllactate, which can be transported across the blood-brain barrier to the blood, may consume glucose and/or lactate to generate the carbon backbone for glutamate. This glutamate is then converted to glutamine and carries the Phe-derived ammonia from the brain to the blood. While this mechanism remains to be tested, it may explain the correlations of CSF glutamine, glucose, and lactate concentrations with CSF Phe.

Phenylketonuria (PKU) Urinary Metabolomic Phenotype Is Defined by Genotype and Metabolite Imbalance: Results in 51 Early Treated Patients Using Ex Vivo 1H-NMR Analysis.

Phenylketonuria (PKU) is a rare metabolic disorder caused by mutations in the phenylalanine hydroxylase gene. Depending on the severity of the genetic mutation, medical treatment, and patient dietary management, elevated phenylalanine (Phe) may occur in blood and brain tissues. Research has recently shown that high Phe not only impacts the central nervous system, but also other organ systems (e.g., heart and microbiome). This study used ex vivo proton nuclear magnetic resonance (1H-NMR) analysis of urine samples from PKU patients (mean 14.9 ± 9.2 years, n = 51) to identify the impact of elevated blood Phe and PKU treatment on metabolic profiles. Our results found that 24 out of 98 urinary metabolites showed a significant difference (p < 0.05) for PKU patients compared to age-matched healthy controls (n = 51) based on an analysis of urinary metabolome. These altered urinary metabolites were related to Phe metabolism, dysbiosis, creatine synthesis or intake, the tricarboxylic acid (TCA) cycle, end products of nicotinamide-adenine dinucleotide degradation, and metabolites associated with a low Phe diet. There was an excellent correlation between the metabolome and genotype of PKU patients and healthy controls of 96.7% in a confusion matrix model. Metabolomic investigations may contribute to a better understanding of PKU pathophysiology.

Prevalence of DDC genotypes in patients with aromatic L-amino acid decarboxylase (AADC) deficiency and in silico prediction of structural protein changes.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive genetic disorder affecting the biosynthesis of dopamine, a precursor of both norepinephrine and epinephrine, and serotonin. Diagnosis is based on the analysis of CSF or plasma metabolites, AADC activity in plasma and genetic testing for variants in the DDC gene. The exact prevalence of AADC deficiency, the number of patients, and the variant and genotype prevalence are not known. Here, we present the DDC variant (n = 143) and genotype (n = 151) prevalence of 348 patients with AADC deficiency, 121 of whom were previously not reported. In addition, we report 26 new DDC variants, classify them according to the ACMG/AMP/ACGS recommendations for pathogenicity and score them based on the predicted structural effect. The splice variant c.714+4A>T, with a founder effect in Taiwan and China, was the most common variant (allele frequency = 32.4%), and c.[714+4A>T];[714+4A>T] was the most common genotype (genotype frequency = 21.3%). Approximately 90% of genotypes had variants classified as pathogenic or likely pathogenic, while 7% had one VUS allele and 3% had two VUS alleles. Only one benign variant was reported. Homozygous and compound heterozygous genotypes were interpreted in terms of AADC protein and categorized as: i) devoid of full-length AADC, ii) bearing one type of AADC homodimeric variant or iii) producing an AADC protein population composed of two homodimeric and one heterodimeric variant. Based on structural features, a score was attributed for all homodimers, and a tentative prediction was advanced for the heterodimer. Almost all AADC protein variants were pathogenic or likely pathogenic.

Complex metabolic disharmony in PMM2-CDG paves the way to new therapeutic approaches.

PMM2-CDG is the most common defect among the congenital disorders of glycosylation. In order to investigate the effect of hypoglycosylation on important cellular pathways, we performed extensive biochemical studies on skin fibroblasts of PMM2-CDG patients. Among others, acylcarnitines, amino acids, lysosomal proteins, organic acids and lipids were measured, which all revealed significant abnormalities. There was an increased expression of acylcarnitines and amino acids associated with increased amounts of calnexin, calreticulin and protein-disulfid-isomerase in combination with intensified amounts of ubiquitinylated proteins. Lysosomal enzyme activities were widely decreased as well as citrate and pyruvate levels indicating mitochondrial dysfunction. Main lipid classes such as phosphatidylethanolamine, cholesterol or alkyl-phosphatidylcholine, as well as minor lipid species like hexosylceramide, lysophosphatidylcholines or phosphatidylglycerol, were abnormal. Biotinidase and catalase activities were severely reduced. In this study we discuss the impact of metabolite abnormalities on the phenotype of PMM2-CDG. In addition, based on our data we propose new and easy-to-implement therapeutic approaches for PMM2-CDG patients.

Ex vivo proton spectroscopy (1 H-NMR) analysis of inborn errors of metabolism: Automatic and computer-assisted analyses.

There are about 1500 genetic metabolic diseases. A small number of treatable diseases are diagnosed by newborn screening programs, which are continually being developed. However, most diseases can only be diagnosed based on clinical symptoms or metabolic findings. The main biological fluids used are urine, plasma and, in special situations, cerebrospinal fluid. In contrast to commonly used methods such as gas chromatography and high performance liquid chromatography mass spectrometry, ex vivo proton spectroscopy (1 H-NMR) is not yet used in routine clinical practice, although it has been recommended for more than 30 years. Automatic analysis and improved NMR technology have also expanded the applications used for the diagnosis of inborn errors of metabolism. We provide a mini-overview of typical applications, especially in urine but also in plasma, used to diagnose common but also rare genetic metabolic diseases with 1 H-NMR. The use of computer-assisted diagnostic suggestions can facilitate interpretation of the profiles. In a proof of principle, to date, 182 reports of 59 different diseases and 500 reports of healthy children are stored. The percentage of correct automatic diagnoses was 74%. Using the same 1 H-NMR profile-targeted analysis, it is possible to apply an untargeted approach that distinguishes profile differences from healthy individuals. Thus, additional conditions such as lysosomal storage diseases or drug interferences are detectable. Furthermore, because 1 H-NMR is highly reproducible and can detect a variety of different substance categories, the metabolomic approach is suitable for monitoring patient treatment and revealing additional factors such as nutrition and microbiome metabolism. Besides the progress in analytical techniques, a multiomics approach is most effective to combine metabolomics with, for example, whole exome sequencing, to also diagnose patients with nondetectable metabolic abnormalities in biological fluids. In this mini review we also provide our own data to demonstrate the role of NMR in a multiomics platform in the field of inborn errors of metabolism.

Spectrum of DDC variants causing aromatic l-amino acid decarboxylase (AADC) deficiency and pathogenicity interpretation using ACMG-AMP/ACGS recommendations.

Pathogenic variants in dopa decarboxylase (DDC), the gene encoding the aromatic l-amino acid decarboxylase (AADC) enzyme, lead to a severe deficiency of neurotransmitters, resulting in neurological, neuromuscular, and behavioral manifestations clinically characterized by developmental delays, oculogyric crises, dystonia, and severe neurologic dysfunction in infancy. Historically, therapy has been aimed at compensating for neurotransmitter abnormalities, but response to pharmacologic therapy varies, and in most cases, the therapy shows little or no benefit. A novel human DDC gene therapy was recently approved in the European Union that targets the underlying genetic cause of the disorder, providing a new treatment option for patients with AADC deficiency. However, the applicability of human DDC gene therapy depends on the ability of laboratories and clinicians to interpret the results of genetic testing accurately enough to diagnose the patient. An accurate interpretation of genetic variants depends in turn on expert-guided curation of locus-specific databases. The purpose of this research was to identify previously uncharacterized DDC variants that are of pathologic significance in AADC deficiency as well as characterize and curate variants of unknown significance (VUSs) to further advance the diagnostic accuracy of genetic testing for this condition. DDC variants were identified using existing databases and the literature. The pathogenicity of the variants was classified using modified American College of Medical Genetics and Genomics/Association for Molecular Pathology/Association for Clinical Genomic Science (ACMG-AMP/ACGS) criteria. To improve the current variant interpretation recommendations, in silico variant interpretation tools were combined with structural 3D modeling of protein variants and applied comparative analysis to predict the impact of the variant on protein function. A total of 422 variants were identified (http://biopku.org/home/pnddb.asp). Variants were identified on nearly all introns and exons of the DDC gene, as well as the 3' and 5' untranslated regions. The largest percentage of the identified variants (48%) were classified as missense variants. The molecular effects of these missense variants were then predicted, and the pathogenicity of each was classified using a number of variant effect predictors. Using ACMG-AMP/ACGS criteria, 7% of variants were classified as pathogenic, 32% as likely pathogenic, 58% as VUSs of varying subclassifications, 1% as likely benign, and 1% as benign. For 101 out of 108 reported genotypes, at least one allele was classified as pathogenic or likely pathogenic. In silico variant pathogenicity interpretation tools, combined with structural 3D modeling of variant proteins and applied comparative analysis, have improved the current DDC variant interpretation recommendations, particularly of VUSs.

Missense variant c.1460 T > C (p.L487P) enhances protein degradation of ER mannosyltransferase ALG9 in two new ALG9-CDG patients presenting with West syndrome and review of the literature.

ALG9-CDG is a CDG-I defect within the group of Congenital Disorders of Glycosylation (CDG). We here describe the clinical symptoms of two new and unrelated ALG9-CDG patients, both carrying the novel homozygous missense variant c.1460 T > C (p.L487P) in the ALG9 gene which led to global developmental delay, psychomotor disability, facial dysmorphisms, brain and heart defects, hearing loss, hypotonia, as well as feeding problems. New clinical symptoms comprised West syndrome with hypsarrhythmia. Quantitative RT-PCR analysis revealed a significantly enhanced ALG9 mRNA transcript level, whereas the protein amount in fibroblasts was significantly reduced. This could be ascribed to a stronger degradation of the mutated ALG9 protein in patient fibroblasts. Lipid-linked oligosaccharide analysis showed an ALG9-CDG characteristic accumulation of Man6GlcNAc2-PP-dolichol and Man8GlcNAc2-PP-dolichol in patient cells. The clinical findings of our patients and of all previously published ALG9-CDG patients are brought together to further expand the knowledge about this rare N-glycosylation disorder. SYNOPSIS: Homozygosity for p.L487P in ALG9 causes protein degradation and leads to West syndrome.

Molecular and metabolic bases of tetrahydrobiopterin (BH4) deficiencies.

Tetrahydrobiopterin (BH4) deficiency is caused by genetic variants in the three genes involved in de novo cofactor biosynthesis, GTP cyclohydrolase I (GTPCH/GCH1), 6-pyruvoyl-tetrahydropterin synthase (PTPS/PTS), sepiapterin reductase (SR/SPR), and the two genes involved in cofactor recycling, carbinolamine-4α-dehydratase (PCD/PCBD1) and dihydropteridine reductase (DHPR/QDPR). Dysfunction in BH4 metabolism leads to reduced cofactor levels and may result in systemic hyperphenylalaninemia and/or neurological sequelae due to secondary deficiency in monoamine neurotransmitters in the central nervous system. More than 1100 patients with BH4 deficiency and 800 different allelic variants distributed throughout the individual genes are tabulated in database of pediatric neurotransmitter disorders PNDdb. Here we provide an update on the molecular-genetic analysis and structural considerations of these variants, including the clinical courses of the genotypes. From a total of 324 alleles, 11 are associated with the autosomal recessive form of GTPCH deficiency presenting with hyperphenylalaninemia (HPA) and neurotransmitter deficiency, 295 GCH1 variant alleles are detected in the dominant form of L-dopa-responsive dystonia (DRD or Segawa disease) while phenotypes of 18 alleles remained undefined. Autosomal recessive variants observed in the PTS (199 variants), PCBD1 (32 variants), and QDPR (141 variants) genes lead to HPA concomitant with central monoamine neurotransmitter deficiency, while SPR deficiency (104 variants) presents without hyperphenylalaninemia. The clinical impact of reported variants is essential for genetic counseling and important for development of precision medicine.

Glycosyltransferase POMGNT1 deficiency strengthens N-cadherin-mediated cell-cell adhesion.

Defects in protein O-mannosylation lead to severe congenital muscular dystrophies collectively known as α-dystroglycanopathy. A hallmark of these diseases is the loss of the O-mannose-bound matriglycan on α-dystroglycan, which reduces cell adhesion to the extracellular matrix. Mutations in protein O-mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGNT1), which is crucial for the elongation of O-mannosyl glycans, have mainly been associated with muscle-eye-brain (MEB) disease. In addition to defects in cell-extracellular matrix adhesion, aberrant cell-cell adhesion has occasionally been observed in response to defects in POMGNT1. However, specific molecular consequences of POMGNT1 deficiency on cell-cell adhesion are largely unknown. We used POMGNT1 knockout HEK293T cells and fibroblasts from an MEB patient to gain deeper insight into the molecular changes in POMGNT1 deficiency. Biochemical and molecular biological techniques combined with proteomics, glycoproteomics, and glycomics revealed that a lack of POMGNT1 activity strengthens cell-cell adhesion. We demonstrate that the altered intrinsic adhesion properties are due to an increased abundance of N-cadherin (N-Cdh). In addition, site-specific changes in the N-glycan structures in the extracellular domain of N-Cdh were detected, which positively impact on homotypic interactions. Moreover, in POMGNT1-deficient cells, ERK1/2 and p38 signaling pathways are activated and transcriptional changes that are comparable with the epithelial-mesenchymal transition (EMT) are triggered, defining a possible molecular mechanism underlying the observed phenotype. Our study indicates that changes in cadherin-mediated cell-cell adhesion and other EMT-related processes may contribute to the complex clinical symptoms of MEB or α-dystroglycanopathy in general and suggests that the impact of changes in O-mannosylation on N-glycosylation has been underestimated.

Fatal outcome after heart surgery in PMM2-CDG due to a rare homozygous gene variant with double effects.

Variants in Phosphomannomutase 2 (PMM2) lead to PMM2-CDG, the most frequent congenital disorder of glycosylation (CDG). We here describe the disease course of a ten-month old patient who presented with the classical PMM2-CDG symptoms as cerebellar hypoplasia, retinitis pigmentosa, seizures, short stature, hepato- and splenomegaly, anaemia, recurrent vomiting and inverted mamillae. A severe form of tetralogy of Fallot was diagnosed and corrective surgery was performed at the age of 10 months. At the end of the cardiopulmonary bypass, a sudden oedematous reaction of the myocardium accompanied by biventricular pump failure was observed immediately after heparin antagonization with protamine sulfate. The patient died seven days after surgery, since myocardial function did not recover on ECMO support. We here describe the first patient carrying the homozygous variant g.18313A > T in the PMM2 gene (NG_009209.1) that either can lead to c.394A > T (p.I132F) or even loss of 100 bp due to exon 5 skipping (c.348_447del; p.G117Rfs*4) which is comparable to a null allele. Proliferation and doubling time of the patient's fibroblasts were affected. In addition, we show that the induction of cellular stress by elevating the cell culture temperature to 40 °C led to a decrease of the patients' PMM2 transcript as well as PMM2 protein levels and subsequently to a significant loss of residual activity. We assume that metabolic stressful processes occurring after cardiac surgery led to the drop of the patient's PMM activity below a life-sustaining niveau which paved the way for the fatal outcome.

Molecular genetics of phenylketonuria and tetrahydrobiopterin deficiency in Jordan.

BACKGROUND: Information regarding the prevalence of PKU in the Middle East in comparison to other world regions is scarce, which might be explained by difficulties in the implementation of national newborn screening programs. OBJECTIVE: This study seeks for the first time to genotype and biochemically characterize patients diagnosed with hyperphenylalaninemia (HPA) at the Pediatric Metabolic Genetics Clinic at the King Hussein Medical Center, Amman, Jordan. METHODS: A total of 33 patients with HPA and 55 family members were investigated for pterins (neopterin and biopterin) and dihydropteridine reductase (DHPR) activity in dried blood spots. Patients with HPA were genotyped for phenylketonuria (PKU) and the genes involved in tetrahydrobiopterin (BH4) metabolism. RESULTS: In total 20 patients were diagnosed with PKU due to phenylalanine hydroxylase (PAH) deficiency, 2 with GTP cyclohydrolase I (GTPCH) deficiency, 6 with DHPR deficiency, and 3 with the 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency. Diagnosis was not possible in 2 patients. This study documents a high percentage of BH4 deficiencies within HPA patients. With one exception, all patients were homozygous for particular gene variants. CONCLUSIONS: This approach enables differentiation between PKU and BH4 deficiencies and, thus, allows for critical selection of a specific treatment strategies.

ADP-dependent glucokinase regulates energy metabolism via ER-localized glucose sensing.

Modulation of energy metabolism to a highly glycolytic phenotype, i.e. Warburg effect, is a common phenotype of cancer and activated immune cells allowing increased biomass-production for proliferation and cell division. Endoplasmic reticulum (ER)-localized ADP-dependent glucokinase (ADPGK) has been shown to play a critical role in T cell receptor activation-induced remodeling of energy metabolism, however the underlying mechanisms remain unclear. Therefore, we established and characterized in vitro and in vivo models for ADPGK-deficiency using Jurkat T cells and zebrafish. Upon activation, ADPGK knockout Jurkat T cells displayed increased cell death and ER stress. The increase in cell death resulted from a metabolic catastrophe and knockout cells displayed severely disturbed energy metabolism hindering induction of Warburg phenotype. ADPGK knockdown in zebrafish embryos led to short, dorsalized body axis induced by elevated apoptosis. ADPGK hypomorphic zebrafish further displayed dysfunctional glucose metabolism. In both model systems loss of ADPGK function led to defective N- and O-glycosylation. Overall, our data illustrate that ADPGK is part of a glucose sensing system in the ER modulating metabolism via regulation of N- and O-glycosylation.

Novel variants and clinical symptoms in four new ALG3-CDG patients, review of the literature, and identification of AAGRP-ALG3 as a novel ALG3 variant with alanine and glycine-rich N-terminus.

ALG3-CDG is one of the very rare types of congenital disorder of glycosylation (CDG) caused by variants in the ER-mannosyltransferase ALG3. Here, we summarize the clinical, biochemical, and genetic data of four new ALG3-CDG patients, who were identified by a type I pattern of serum transferrin and the accumulation of Man5 GlcNAc2 -PP-dolichol in LLO analysis. Additional clinical symptoms observed in our patients comprise sensorineural hearing loss, right-descending aorta, obstructive cardiomyopathy, macroglossia, and muscular hypertonia. We add four new biochemically confirmed variants to the list of ALG3-CDG inducing variants: c.350G>C (p.R117P), c.1263G>A (p.W421*), c.1037A>G (p.N346S), and the intron variant c.296+4A>G. Furthermore, in Patient 1 an additional open-reading frame of 141 bp (AAGRP) in the coding region of ALG3 was identified. Additionally, we show that control cells synthesize, to a minor degree, a hybrid protein composed of the polypeptide AAGRP and ALG3 (AAGRP-ALG3), while in Patient 1 expression of this hybrid protein is significantly increased due to the homozygous variant c.160_196del (g.165C>T). By reviewing the literature and combining our findings with previously published data, we further expand the knowledge of this rare glycosylation defect.

Aromatic amino acid decarboxylase deficiency: Molecular and metabolic basis and therapeutic outlook.

Aromatic-l-amino acid decarboxylase (AADC) deficiency is an ultra-rare inherited autosomal recessive disorder characterized by sharply reduced synthesis of dopamine as well as other neurotransmitters. Symptoms, including hypotonia and movement disorders (especially oculogyric crisis and dystonia) as well as autonomic dysfunction and behavioral disorders, vary extensively and typically emerge in the first months of life. However, diagnosis is difficult, requiring analysis of metabolites in cerebrospinal fluid, assessment of plasma AADC activity, and/or DNA sequence analysis, and is frequently delayed for years. New metabolomics techniques promise early diagnosis of AADC deficiency by detection of 3-O-methyl-dopa in serum or dried blood spots. A total of 82 dopa decarboxylase (DDC) variants in the DDC gene leading to AADC deficiency have been identified and catalogued for all known patients (n = 123). Biochemical and bioinformatics studies provided insight into the impact of many variants. c.714+4A>T, p.S250F, p.R347Q, and p.G102S are the most frequent variants (cumulative allele frequency = 57%), and c.[714+4A>T];[714+4A>T], p.[S250F];[S250F], and p.[G102S];[G102S] are the most frequent genotypes (cumulative genotype frequency = 40%). Known or predicted molecular effect was defined for 79 variants. Most patients experience an unrelenting disease course with poor or no response to conventional medical treatments, including dopamine agonists, monoamine oxidase inhibitors, and pyridoxine derivatives. The advent of gene therapy represents a potentially promising new avenue for treatment of patients with AADC deficiency. Clinical studies based on the direct infusion of engineered adeno-associated virus type 2 vectors into the putamen have demonstrated acceptable safety and tolerability and encouraging improvement in motor milestones and cognitive symptoms. The success of gene therapy in AADC deficiency treatment will depend on timely diagnosis to facilitate treatment administration before the onset of neurologic damage.

The Metabolic Map into the Pathomechanism and Treatment of PGM1-CDG.

Phosphoglucomutase 1 (PGM1) encodes the metabolic enzyme that interconverts glucose-6-P and glucose-1-P. Mutations in PGM1 cause impairment in glycogen metabolism and glycosylation, the latter manifesting as a congenital disorder of glycosylation (CDG). This unique metabolic defect leads to abnormal N-glycan synthesis in the endoplasmic reticulum (ER) and the Golgi apparatus (GA). On the basis of the decreased galactosylation in glycan chains, galactose was administered to individuals with PGM1-CDG and was shown to markedly reverse most disease-related laboratory abnormalities. The disease and treatment mechanisms, however, have remained largely elusive. Here, we confirm the clinical benefit of galactose supplementation in PGM1-CDG-affected individuals and obtain significant insights into the functional and biochemical regulation of glycosylation. We report here that, by using tracer-based metabolomics, we found that galactose treatment of PGM1-CDG fibroblasts metabolically re-wires their sugar metabolism, and as such replenishes the depleted levels of galactose-1-P, as well as the levels of UDP-glucose and UDP-galactose, the nucleotide sugars that are required for ER- and GA-linked glycosylation, respectively. To this end, we further show that the galactose in UDP-galactose is incorporated into mature, de novo glycans. Our results also allude to the potential of monosaccharide therapy for several other CDG.

Role of protein structure in variant annotation: structural insight of mutations causing 6-pyruvoyl-tetrahydropterin synthase deficiency.

Genetic defects on 6-pyruvoyl-tetrahydropterin synthase (PTPS) are the most prevalent cause of hyperphenylalaninaemia not due to phenylalanine hydrolyase deficiency (phenylketonuria). PTPS catalyses the second step of tetrahydrobiopterin (BH4) cofactor biosynthesis, and its deficiency represents the most common form of BH4 deficiency. Untreated PTPS deficiency results in depletion of the neurotransmitters dopamine, catecholamine and serotonin causing neurological symptoms. We archived reported missense variants of the PTS gene. Common in silico algorithms were used to predict the effects of such variants, and substantial proportions (up to 19%) of the variants were falsely classified as benign or uncertain. We have determined the crystal structure of the human PTPS hexamer, allowing another level of interpretation to understand the potential deleterious consequences of the variants from a structural perspective. The in silico and structure approaches appear to be complimentary and may provide new insights that are not available from each alone. Information from the protein structure suggested that the variants affecting amino acid residues required for interaction between monomeric subunits of the PTPS hexamer were those misclassified as benign by in silico algorithms. Our findings illustrate the important utility of 3D protein structure in interpretation of variants and also current limitations of in silico prediction algorithms. However, software to analyse mutation in the perspective of 3D protein structure is far less readily available than other in silico prediction tools.