Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism.

The SLC25A32 dysfunction is associated with neural tube defects (NTDs) and exercise intolerance, but very little is known about disease-specific mechanisms due to a paucity of animal models. Here, we generated homozygous (Slc25a32Y174C/Y174C and Slc25a32K235R/K235R) and compound heterozygous (Slc25a32Y174C/K235R) knock-in mice by mimicking the missense mutations identified from our patient. A homozygous knock-out (Slc25a32-/-) mouse was also generated. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice presented with mild motor impairment and recapitulated the biochemical disturbances of the patient. While Slc25a32-/- mice die in utero with NTDs. None of the Slc25a32 mutations hindered the mitochondrial uptake of folate. Instead, the mitochondrial uptake of flavin adenine dinucleotide (FAD) was specifically blocked by Slc25a32Y174C/K235R, Slc25a32K235R/K235R, and Slc25a32-/- mutations. A positive correlation between SLC25A32 dysfunction and flavoenzyme deficiency was observed. Besides the flavoenzymes involved in fatty acid ß-oxidation and amino acid metabolism being impaired, Slc25a32-/- embryos also had a subunit of glycine cleavage system-dihydrolipoamide dehydrogenase damaged, resulting in glycine accumulation and glycine derived-formate reduction, which further disturbed folate-mediated one-carbon metabolism, leading to 5-methyltetrahydrofolate shortage and other folate intermediates accumulation. Maternal formate supplementation increased the 5-methyltetrahydrofolate levels and ameliorated the NTDs in Slc25a32-/- embryos. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice had no glycine accumulation, but had another formate donor-dimethylglycine accumulated and formate deficiency. Meanwhile, they suffered from the absence of all folate intermediates in mitochondria. Formate supplementation increased the folate amounts, but this effect was not restricted to the Slc25a32 mutant mice only. In summary, we established novel animal models, which enabled us to understand the function of SLC25A32 better and to elucidate the role of SLC25A32 dysfunction in human disease development and progression.

Clinical, biochemical, neuroimaging and molecular findings of X-linked Adrenoleukodystrophy patients in South China.

X-linked adrenoleukodystrophy is a common X-linked recessive peroxisomal disorder caused by the mutations in the ABCD1 gene. In this study, we analyzed 19 male patients and 9 female carriers with X-linked adrenoleukodystrophy in South China. By sequencing the ABCD1 gene, 13 different mutations were identified, including 7 novel mutations, and 6 known mutations, and 1 reported polymorphism. Mutation c.1180delG was demonstrated to be de novo mutation. 26.3 % (5/19) patients carried the deletion c.1415_16delAG, which may be the mutational hot spot in South China population. In addition, 73.7 % (14/19) patients were type of childhood cerebral adrenoleukodystrophy, 26.3 %(5/19) were in Addison only. Half of the childhood cerebral adrenoleukodystrophy patients had the adrenocortical insufficiency preceded the onset of neurological symptoms. Furthermore, 5 of 19 cases underwent hematopoietic stem cell transplantation. Our data showed that hematopoietic stem cell transplantation performed at an advanced stage of the cerebral X- linked adrenoleukodystrophy would accelerate the progression of the disease. Good clinical outcome achieved when hematopoietic stem cell transplantation performed at the very early stage of the disease.

[Identification of a novel pathogenic mutation in PDHA1 gene for pyruvate dehydrogenase complex deficiency].

OBJECTIVE: To study the molecular genetic mechanism and genetic diagnosis of pyruvate dehydrogenase complex deficiency (PHD), and to provide a basis for genetic counseling and prenatal genetic diagnosis of PHD. METHODS: Polymerase chain reaction (PCR) was performed to amplify the 11 exons and exon junction of the PDHA1 gene from a child who was diagnosed with PHD based on clinical characteristics and laboratory examination results. The PCR products were sequenced to determine the mutation. An analysis of amino acid conservation and prediction of protein secondary and tertiary structure were performed using bioinformatic approaches to identify the pathogenicity of the novel mutation. RESULTS: One novel duplication mutation, c.1111_1158dup48bp, was found in the exon 11 of the PDHA1 gene of the patient. No c.1111_1158dup48bp mutation was detected in the sequencing results from 50 normal controls. The results of protein secondary and tertiary structure prediction showed that the novel mutation c.1111 _1158dup48bp led to the duplication of 16 amino acids residues, serine371 to phenylalanine386, which induced a substantial change in protein secondary and tertiary structure. The conformational change was not detected in the normal controls. CONCLUSIONS: The novel duplication mutation c.1111_1158dup48bp in the PDHA1 gene is not due to gene polymorphisms but a possible novel pathogenic mutation for PHD.

Severe Zellweger spectrum disorder due to a novel missense variant in the PEX13 gene: A case report and the literature review.

BACKGROUND: Peroxisome biogenesis disorders (PBDs) are caused by variants in PEX genes that impair peroxisome function. Zellweger spectrum disorders (ZSDs) are the most severe and common subtype of PBDs, affecting multiple organ systems due to peroxisomal involvement in various metabolic functions. PEX13 gene variants are rare causes of ZSDs, with only 21 cases reported worldwide and none in China. METHODS: We describe an infant with biochemically and molecularly confirmed ZSDs due to variants in the PEX13 gene, identified by whole exome sequencing and validated by Sanger sequencing. The patient's treatment and prognosis were followed up. We also reviewed the literature on previously reported cases with PEX13 variants. RESULTS: The patient had severe hypotonia, seizures, hepatic dysfunction, failure to thrive, and dysmorphic features. Serum analysis revealed elevated levels of very long-chain fatty acids (VLCFA), phytanic acid, and pipecolic acid. We detected a novel homozygous missense variant c.493G>C (p. Ala165Pro) in the PEX13 gene (NM_002618.3), which caused severe clinical manifestations and was inherited from the consanguineous parents. The patient died at the age of 14 months. CONCLUSION: We report the first case of ZSDs due to the PEX13 variant in China. Our findings broaden the mutational spectrum of the PEX13 gene and indicate that missense variants can lead to severe ZSDs phenotypes, which has implications for genotype-phenotype correlations and genetic counseling.

Plasma Amino Acid Profile in Children with Autism Spectrum Disorder in Southern China: Analysis of 110 Cases.

To retrospectively explore the characteristics of plasma amino acids (PAAs) in children with autism spectrum disorder and their clinical association via case-control study. A total of 110 autistic and 55 healthy children were recruited from 2014 to 2018. The clinical phenotypes included severity of autism, cognition, adaptability, and regression. Compared with the control group, autistic children had significantly elevated glutamate, γ-Amino-n-butyric acid, glutamine, sarcosine, δ-aminolevulinic acid, glycine and citrulline. In contrast, their plasma level of ethanolamine, phenylalanine, tryptophan, homocysteine, pyroglutamic acid, hydroxyproline, ornithine, histidine, lysine, and glutathione were significantly lower. Elevated neuroactive amino acids (glutamate) and decreased essential amino acids were mostly distinct characteristics of PAAs of autistic children. Increased level of tryptophan might be associated with severity of autism.

A sensitive liquid chromatography-tandem mass spectrometry method for determination biomarkers of monoamine neurotransmitter disorders in cerebrospinal fluid.

BACKGROUND: Cerebrospinal fluid (CSF) monoamine neurotransmitters, their precursors and metabolites are essential biomarkers in the diagnosis and follow-up of monoamine neurotransmitter disorders (MNDs). However, their extra low concentrations and potential instability challenge the detection method. Here, we present a method that enables simultaneous quantification of these biomarkers. METHOD: With propyl chloroformate /n-propanol, 16 biomarkers in 50 µL of CSF were derivatized in situ within seconds under an ambient temperature. The derivatives were extracted by ethyl acetate and separated by a reverse phase column followed by mass spectrometric detection. The method was fully validated. Optimal conditions for standard solution preparation and storage, as well as CSF sample handling, were investigated. CSF samples from 200 controls and 16 patients were analyzed. RESULTS: The derivatization reaction stabilized biomarkers and increased sensitivity. Most biomarkers were quantifiable in concentrations between 0.02 and 0.50 nmol/L that were sufficient to measure their endogenous concentrations. The intra- and inter-day imprecision were < 15% for most analytes, and accuracy ranged from 90.3% to 111.6%. The stability study showed that standard stock solutions were stable at -80 °C for six years when prepared in the protection solutions; Analytes in CSF samples were stable for 24 h on wet ice and at least two years at -80 °C; But repeated freeze-thaw should be avoided. With this method, age-dependent reference intervals for each biomarker in the pediatric population were established. Patients with MNDs were successfully identified. CONCLUSION: The developed method is valuable for MNDs diagnosis and research, benefiting from its advantages of sensitivity, comprehensiveness, and high throughput.

Diagnosis of an intermediate case of maple syrup urine disease: A case report.

BACKGROUND: Maple syrup urine disease (MSUD) is an autosomal recessive genetic disorder caused by defects in the catabolism of the branched-chain amino acids (BCAAs). However, the clinical and metabolic screening is limited in identifying all MSUD patients, especially those patients with mild phenotypes or are asymptomatic. This study aims to share the diagnostic experience of an intermediate MSUD case who was missed by metabolic profiling but identified by genetic analysis. CASE SUMMARY: This study reports the diagnostic process of a boy with intermediate MSUD. The proband presented with psychomotor retardation and cerebral lesions on magnetic resonance imaging scans at 8 mo of age. Preliminary clinical and metabolic profiling did not support a specific disease. However, whole exome sequencing and subsequent Sanger sequencing at 1 year and 7 mo of age identified bi-allelic pathogenic variants of the BCKDHB gene, confirming the proband as having MSUD with non-classic mild phenotypes. His clinical and laboratory data were retrospectively analyzed. According to his disease course, he was classified into an intermediate form of MSUD. His management was then changed to BCAAs restriction and metabolic monitoring conforming to MSUD. In addition, genetic counseling and prenatal diagnosis were provided to his parents. CONCLUSION: Our work provides diagnostic experience of an intermediate MSUD case, suggesting that a genetic analysis is important for ambiguous cases, and alerts clinicians to avoid missing patients with non-classic mild phenotypes of MSUD.

Clinical and biochemical characteristics of patients with ornithine transcarbamylase deficiency.

BACKGROUND: Ornithine transcarbamylase deficiency (OTCD) is pleomorphic congenital hyperammonemia, in which the prognosis of the patient is determined both by genotype and environmental factors. This study investigated the clinical and biochemical characteristics of OTCD patients with different prognosis. METHOD: Of 35 OTCD patients, six males deceased at the first disease-onset, 17 males survived and had controllable ammonia levels after treatment, and 12 females survived through the first disease-onset but had intractable hyperammonemia and high mortality. Fasting blood samples from patients collected at three disease stages were used for the analysis of amino acid (AA) profile, acylcarnitine profile, and micronutrients. Differences in profiles between patients and healthy controls and within patient groups were studied. RESULTS: All OTCD patients had accumulation of glutamine, homocitrulline, lysine, glutamate, cystathionine, and pipecolic acid, as well as deficiency of citrulline, tryptophan, threonine, and carnitine. For male non-survivors, most other AAs and long-chain acylcarnitines were elevated at disease onset, of which the levels of creatine, N-acetylaspartic acid, and homoarginine were remarkably high. Male survivors and female patients had most other AAs at low to normal levels. Compared with male survivors, female patients had much lower protein-intolerance, as indicated by significantly lower levels of protein consumption indicators, including essential AAs, 1-methylhistidine, acylcarnitines et al., but high levels of ammonia. Female patients still had significantly higher levels of citrulline, homocitrulline, and citrulline/arginine compared to male survivors. CONCLUSION: Unique profiles were observed in each group of OTCD patients, indicating specific physiological changes that happened to them.

Simultaneous quantification of 48 plasma amino acids by liquid chromatography-tandem mass spectrometry to investigate urea cycle disorders.

Urea cycle disorders (UCD) are inborn errors of ammonia detoxification in which early diagnosis and treatment are critical to prevent metabolic emergencies. Unfortunately, the diagnosis was often and pronounced delayed. To improve diagnosis, we developed herein a liquid chromatography-tandem mass spectrometry method to investigate the disturbance of amino acid profile caused by UCD. The method enabled absolute quantification of 48 amino acids (AAs) within 20 min. Only 2.5 µL plasma was required for the analysis. The lower limits of quantification for most AAs were 0.01 µmol/L. Method accuracies ranged from 89.9% to 113.4%. The within- and between-run coefficients of variation were 0.8-7.7% and 2.6-14.5%, respectively. With this method, age-specific reference values were established for 42 AAs by analyzing 150 samples from normal controls, and patients with different subtypes of UCD were successfully distinguished. The data of patients revealed that UCD not only disturbed the metabolism of urea cycle AAs and induced accumulation of ammonia detoxification AAs, but also interfered the metabolism of some nervous system related AAs, such as pipecolic acid and N-acetylaspartic acid. This data may provide new insight into pathogenesis for UCD.

A novel ABCD1 gene mutation in a Chinese patient with X-linked adrenoleukodystrophy.

BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) (OMIM: 300100) is a recessive neurodegenerative disorder caused by defects in the ABCD1 gene on chromosome Xq28. Childhood cerebral ALD (CCALD) is the most frequent phenotype. OBJECTIVE: We describe an affected boy who developed normally until he was 8 years old then suffered progressive neurological deficits that ultimately led to death. METHODS: Diagnosis was based on clinical symptoms, an abnormal very long chain fatty acid profile in plasma, typical CCALD MRI pattern, and molecular analysis. RESULTS: Direct sequencing of the ABCD1 gene in this patient identified a novel splicing mutation (IVS1+1G>A) in intron 1, which is considered to be the pathogenic mutation. CONCLUSION: We have identified a novel ABCD1 mutation as the likely cause of CCALD in a Chinese patient.