Transmembrane helix 6 of ABCD4 is indispensable for cobalamin transport.

ABCD4, which belongs to the ABC protein subfamily D, plays a role in the transport of cobalamin from lysosomes to the cytosol by cooperating with ATP-binding and ATP-hydrolysis. Pathogenic variants in the ABCD4 gene lead to an inherited metabolic disorder characterized by cobalamin deficiency. However, the structural requirements for cobalamin transport in ABCD4 remain unclear. In this study, six proteoliposomes were prepared, each containing a different chimeric ABCD4 protein, wherein each of the six transmembrane (TM) helices was replaced with the corresponding ABCD1. We analyzed the cobalamin transport activities of the ABCD mutants. In the proteoliposome with chimeric ABCD4 replacing TM helix 6, the cobalamin transport activity disappeared without a reduction in ATPase activity, indicating that TM helix 6 contributes to substrate recognition. Furthermore, the substitution of aspartic acid at position 329 or threonine at position 332 in TM helix 6 with the basic amino acid lysine led to a decrease in cobalamin-transport activity without causing a reduction in ATPase activity. The amino acids in TM helix 6 may be critically involved in substrate recognition; the charged state in the C-terminal half of TM helix 6 of ABCD4 is responsible for cobalamin transport activity.

Causes and consequences of impaired methionine synthase activity in acquired and inherited disorders of vitamin B12 metabolism.

Methyl-Cobalamin (Cbl) derives from dietary vitamin B12 and acts as a cofactor of methionine synthase (MS) in mammals. MS encoded by MTR catalyzes the remethylation of homocysteine to generate methionine and tetrahydrofolate, which fuel methionine and cytoplasmic folate cycles, respectively. Methionine is the precursor of S-adenosyl methionine (SAM), the universal methyl donor of transmethylation reactions. Impaired MS activity results from inadequate dietary intake or malabsorption of B12 and inborn errors of Cbl metabolism (IECM). The mechanisms at the origin of the high variability of clinical presentation of impaired MS activity are classically considered as the consequence of the disruption of the folate cycle and related synthesis of purines and pyrimidines and the decreased synthesis of endogenous methionine and SAM. For one decade, data on cellular and animal models of B12 deficiency and IECM have highlighted other key pathomechanisms, including altered interactome of MS with methionine synthase reductase, MMACHC, and MMADHC, endoplasmic reticulum stress, altered cell signaling, and genomic/epigenomic dysregulations. Decreased MS activity increases catalytic protein phosphatase 2A (PP2A) and produces imbalanced phosphorylation/methylation of nucleocytoplasmic RNA binding proteins, including ELAVL1/HuR protein, with subsequent nuclear sequestration of mRNAs and dramatic alteration of gene expression, including SIRT1. Decreased SAM and SIRT1 activity induce ER stress through impaired SIRT1-deacetylation of HSF1 and hypomethylation/hyperacetylation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), which deactivate nuclear receptors and lead to impaired energy metabolism and neuroplasticity. The reversibility of these pathomechanisms by SIRT1 agonists opens promising perspectives in the treatment of IECM outcomes resistant to conventional supplementation therapies.

Decreased cortical Nrf2 gene expression in autism and its relationship to thiol and cobalamin status.

Nuclear factor erythroid 2-related factor 2 (Nrf2) promotes expression of a large number of antioxidant genes and multiple studies have described oxidative stress and impaired methylation in autism spectrum disorder (ASD), including decreased brain levels of methylcobalamin(III) (MeCbl). Here we report decreased expression of the Nrf2 gene (NFE2L2) in frontal cortex of ASD subjects, as well as differences in other genes involved in redox homeostasis. In pooled control and ASD correlation analyses, hydroxocobalamin(III) (OHCbl) was inversely correlated with NFE2L2 expression, while MeCbl and total cobalamin abundance were positively correlated with NFE2L2 expression. Levels of methionine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and cystathionine were positively correlated with NFE2L2 expression, while homocysteine (HCY) was negatively correlated. The relationship between Nrf2 activity and cobalamin was further supported by a bioinformatics-based comparison of cobalamin levels in different tissues with expression of a panel of 40 Nrf2-regulated genes, which yielded a strong correlation. Lastly, Nrf2-regulated gene expression was also correlated with expression of intracellular cobalamin trafficking and processing genes, such as MMADHC and MTRR. These findings highlight a previously unrecognized relationship between the antioxidant-promoting role of Nrf2 and cobalamin status, which is dysfunctional in ASD.

Cobalamin cbiP mutant shows decreased tolerance to low temperature and copper stress in Listeria monocytogenes

BACKGROUND: Listeria monocytogenes is a foodborne pathogen that causes listeriosis in humans. This pathogen activates multiple regulatory mechanisms in response to stress, and cobalamin biosynthesis might have a potential role in bacterial protection. Low temperature is a strategy used in the food industry to control bacteria proliferation; however, L. monocytogenes can grow in cold temperatures and overcome different stress conditions. In this study we selected L. monocytogenes List2-2, a strain with high tolerance to the combination of low temperature +copper, to understand whether the cobalamin biosynthesis pathway is part of the tolerance mechanism to this stress condition. For this, we characterized the transcription level of three cobalamin biosynthesis related genes ( cbiP , cbiB, and cysG ) and the eutV gene, a transcriptional regulator encoding gene involved in ethanolamine metabolism, in L. monocytogenes strain List2-2 growing simultaneously under two environmental stressors: low temperature (8 °C) +copper (0.5 mM of CuSO4 ×5H2O). In addition, the gene cbiP , which encodes an essential cobyric acid synthase required in the cobalamin pathway, was deleted by homologous recombination to evaluate the impact of this gene in L. monocytogenes tolerance to a low temperature (8 °C) +different copper concentrations. RESULTS: By analyzing the KEGG pathway database, twenty-two genes were involved in the cobalamin biosynthesis pathway in L. monocytogenes List2-2. The expression of genes cbiP , cbiB, and cysG, and eutV increased 6 h after the exposure to low temperature +copper. The cobalamin cbiP mutant strain List2-2Δ cbiP showed less tolerance to low temperature +copper (3 mM) than the wild type L. monocytogenes List2-2. The addition of cyanocobalamin (5 nM) to the medium reverted the phenotype observed in List2-2Δ cbiP . CONCLUSION: These results indicate that cobalamin biosynthesis is necessary for L. monocytogenes growth under stress and that the cbiP gene may play a role in the survival and growth of L. monocytogenes List2-2 at low temperature +copper.

Prenatal diagnosis of combined methylmalonic acidemia and homocystinuria cobalamin C type using clinical exome sequencing and targeted gene analysis.

BACKGROUND: Combined methylmalonic acidemia and homocystinuria is a rare inherited disorder of intracellular cobalamin metabolism caused by biallelic variants in one of the following genes: MMACHC (cblC), MMADHC (cblD), LMBRD1 (cblF), ABCD4 (cblJ), THAP11 (cblX-like), and ZNF143 (cblX-like), or a hemizygous variant in HCFC1 (cblX). Prenatal diagnosis of combined methylmalonic acidemia with homocystinuria is crucial for high-risk couples since the disorder can be life-threatening for offspring. We would like to describe two infant deaths both of which are likely attributable to cblC despite not having a genetic confirmation, and subsequent pregnancy and prenatal genetic testing. METHODS: Parental clinical exome sequencing and targeted Sanger sequencing of MMACHC gene in amniotic fluid was performed to check the carrier status of the fetus. RESULTS: Parental clinical exome sequencing revealed a heterozygous pathogenic variant [NM_015506.2:c.217C>T (p.Arg73*)] in the MMACHC gene of the mother and [NM_015506.2:c.609G>A (p.Trp203*)] in the MMACHC gene of the father. Targeted Sanger sequencing of MMACHC gene in amniotic fluid revealed that the fetus carried only one nonsense variant [NM_015506.2:c.609G>A (p.Trp203*)], which was inherited from the father. The mother delivered a healthy baby and the neonate did not show any symptoms or signs of combined methylmalonic acidemia and homocystinuria after birth. CONCLUSION: We present a case of prenatal diagnosis with parental exome sequencing, which successfully diagnosed the carrier status of the fetus and parents in a combined methylmalonic acidemia and homocystinuria family.

Genetically predicted circulating B vitamins in relation to digestive system cancers.

BACKGROUND: Folate, vitamin B6 and vitamin B12 have been associated with digestive system cancers. We conducted a two-sample Mendelian randomisation study to assess the causality of these associations. METHODS: Two, one and 14 independent single nucleotide polymorphisms associated with serum folate, vitamin B6 and vitamin B12 at the genome-wide significance threshold were selected as genetic instruments. Summary-level data for the associations of the vitamin-associated genetic variants with cancer were obtained from the UK Biobank study including 367,561 individuals and FinnGen consortium comprising up to 176,899 participants. RESULTS: Genetically predicted folate and vitamin B6 concentrations were not associated with overall cancer, overall digestive system cancer or oesophageal, gastric, colorectal or pancreatic cancer. Genetically predicted vitamin B12 concentrations were positively associated with overall digestive system cancer (ORSD, 1.12; 95% CI 1.04, 1.21, p = 0.003) and colorectal cancer (ORSD 1.16; 95% CI 1.06, 1.26, p = 0.001) in UK Biobank. Results for colorectal cancer were consistent in FinnGen and the combined ORSD was 1.16 (95% CI 1.08, 1.25, p < 0.001). There was no association of genetically predicted vitamin B12 with any other site-specific digestive system cancers or overall cancer. CONCLUSIONS: These results provide evidence to suggest that elevated serum vitamin B12 concentrations are associated with colorectal cancer.

Cobalamin J disease detected on newborn screening: Novel variant and normal neurodevelopmental course.

Cobalamin J disease (CblJ) is an ultra-rare autosomal recessive disorder of intracellular cobalamin metabolism associated with combined methylmalonic acidemia and homocystinuria. It is caused by pathogenic variants in ABCD4, which encodes an ATP-binding cassette (ABC) transporter that affects the lysosomal release of cobalamin (Cbl) into the cytoplasm. Only six cases of CblJ have been reported in the literature. Described clinical features include feeding difficulties, failure to thrive, hypotonia, seizures, developmental delay, and hematological abnormalities. Information on clinical outcomes is extremely limited, and no cases of presymptomatic diagnosis have been reported. We describe a now 17-month-old male with CblJ detected by newborn screening and confirmed by biochemical, molecular, and complementation studies. With early detection and initiation of treatment, this patient has remained asymptomatic with normal growth parameters and neurodevelopmental function. To the best of our knowledge, this report represents the first asymptomatic and neurotypical patient with CblJ.

Sirt1-PPARS Cross-Talk in Complex Metabolic Diseases and Inherited Disorders of the One Carbon Metabolism.

Sirtuin1 (Sirt1) has a NAD (+) binding domain and modulates the acetylation status of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) and Fork Head Box O1 transcription factor (Foxo1) according to the nutritional status. Sirt1 is decreased in obese patients and increased in weight loss. Its decreased expression explains part of the pathomechanisms of the metabolic syndrome, diabetes mellitus type 2 (DT2), cardiovascular diseases and nonalcoholic liver disease. Sirt1 plays an important role in the differentiation of adipocytes and in insulin signaling regulated by Foxo1 and phosphatidylinositol 3'-kinase (PI3K) signaling. Its overexpression attenuates inflammation and macrophage infiltration induced by a high fat diet. Its decreased expression plays a prominent role in the heart, liver and brain of rat as manifestations of fetal programming produced by deficit in vitamin B12 and folate during pregnancy and lactation through imbalanced methylation/acetylation of PGC1α and altered expression and methylation of nuclear receptors. The decreased expression of Sirt1 produced by impaired cellular availability of vitamin B12 results from endoplasmic reticulum stress through subcellular mislocalization of ELAVL1/HuR protein that shuttles Sirt1 mRNA between the nucleus and cytoplasm. Preclinical and clinical studies of Sirt1 agonists have produced contrasted results in the treatment of the metabolic syndrome. A preclinical study has produced promising results in the treatment of inherited disorders of vitamin B12 metabolism.

[Rapid screening of a hotspot variant c.609G>A in MMACHC gene by using PCR-high-resolution melting curve analysis].

OBJECTIVE: To carry out genetic testing for two families affected with cobalamin C (cblC) and establish a rapid method for the detection of a hotspot pathogenic variant c.609G>A of the MMACHC gene by using a PCR-high-resolution melting curve (PCR-HRM) method. METHODS: Genomic DNA was extracted from peripheral blood samples of the probands and their parents. Potential variants of the MMACHC gene was analyzed by Sanger sequencing. The c.609G>A variant of the MMACHC gene was screened among 100 healthy children with the PCR-HRM method. RESULTS: Sanger sequencing revealed that proband 1 carried compound heterozygous variants c.394C>T and c.609G>A of the MMACHC gene, while proband 2 carried compound heterozygous variants c.482G>A and c.609G>A of the same gene. PCR-HRM analysis of the two probands and the 100 healthy children were consistent with the Sanger sequencing. CONCLUSION: c.609G>A is a hotspot pathogenic variant of the MMACHC gene. The diagnosis of cblC may be rapidly attained through detection by PCR-HRM.

Biological Activity of Pseudovitamin B12 on Cobalamin-Dependent Methylmalonyl-CoA Mutase and Methionine Synthase in Mammalian Cultured COS-7 Cells.

Adenyl cobamide (commonly known as pseudovitamin B12) is synthesized by intestinal bacteria or ingested from edible cyanobacteria. The effect of pseudovitamin B12 on the activities of cobalamin-dependent enzymes in mammalian cells has not been studied well. This study was conducted to investigate the effects of pseudovitamin B12 on the activities of the mammalian vitamin B12-dependent enzymes methionine synthase and methylmalonyl-CoA mutase in cultured mammalian COS-7 cells to determine whether pseudovitamin B12 functions as an inhibitor or a cofactor of these enzymes. Although the hydoroxo form of pseudovitamin B12 functions as a coenzyme for methionine synthase in cultured cells, pseudovitamin B12 does not activate the translation of methionine synthase, unlike the hydroxo form of vitamin B12 does. In the second enzymatic reaction, the adenosyl form of pseudovitamin B12 did not function as a coenzyme or an inhibitor of methylmalonyl-CoA mutase. Experiments on the cellular uptake were conducted with human transcobalamin II and suggested that treatment with a substantial amount of pseudovitamin B12 might inhibit transcobalamin II-mediated absorption of a physiological trace concentration of vitamin B12 present in the medium.

Metabolic engineering and optimization of the fermentation medium for vitamin B12 production in Escherichia coli.

Vitamin B12 is a crucial fine chemical that is widely used in the pharmaceutical, food and chemical industries, and its production solely dependents on microbial fermentation. We previously constructed an artificial vitamin B12 biosynthesis pathway in Escherichia coli, but the yield of the engineered strains was low. Here, we removed metabolic bottlenecks of the vitamin B12 biosynthesis pathway in engineered E. coli strains. After screening cobB genes from different sources, optimizing the expression of cobN and customizing the ribosome binding sites of cobS and cobT, the vitamin B12 yield increased to 152.29 µg/g dry cell weight (DCW). Optimization of the downstream module, which converts co(II)byrinic acid a,c-diamide into adenosylcobinamide phosphate, elevated the vitamin B12 yield to 249.04 µg/g DCW. A comparison of a variety of equivalent components indicated that glucose and corn steep liquor are optimal carbon and nitrogen sources, respectively. Finally, an orthogonal array design was applied to determine the optimal concentrations of glucose and nitrogen sources including corn steep liquor and yeast extract, through which a vitamin B12 yield of 530.29 µg/g DCW was obtained. The metabolic modifications and optimization of fermentation conditions achieved in this study offer a basis for further improving vitamin B12 production in E. coli and will hopefully accelerate its industrial application.

The vitamin B12 processing enzyme, mmachc, is essential for zebrafish survival, growth and retinal morphology.

Cobalamin C (cblC) deficiency, the most common inborn error of intracellular cobalamin metabolism, is caused by mutations in MMACHC, a gene responsible for the processing and intracellular trafficking of vitamin B12. This recessive disorder is characterized by a failure to metabolize cobalamin into adenosyl- and methylcobalamin, which results in the biochemical perturbations of methylmalonic acidemia, hyperhomocysteinemia and hypomethioninemia caused by the impaired activity of the downstream enzymes, methylmalonyl-CoA mutase and methionine synthase. Cobalamin C deficiency can be accompanied by a wide spectrum of clinical manifestations, including progressive blindness, and, in mice, manifests with very early embryonic lethality. Because zebrafish harbor a full complement of cobalamin metabolic enzymes, we used genome editing to study the loss of mmachc function and to develop the first viable animal model of cblC deficiency. mmachc mutants survived the embryonic period but perished in early juvenile life. The mutants displayed the metabolic and clinical features of cblC deficiency including methylmalonic acidemia, severe growth retardation and lethality. Morphologic and metabolic parameters improved when the mutants were raised in water supplemented with small molecules used to treat patients, including hydroxocobalamin, methylcobalamin, methionine and betaine. Furthermore, mmachc mutants bred to express rod and/or cone fluorescent reporters, manifested a retinopathy and thin optic nerves (ON). Expression analysis using whole eye mRNA revealed the dysregulation of genes involved in phototransduction and cholesterol metabolism. Zebrafish with mmachc deficiency recapitulate the several of the phenotypic and biochemical features of the human disorder, including ocular pathology, and show a response to established treatments.

Analysis of fibroblasts from patients with cblC and cblG genetic defects of cobalamin metabolism reveals global dysregulation of alternative splicing.

Vitamin B12 or cobalamin (Cbl) metabolism can be affected by genetic defects leading to defective activity of either methylmalonyl-CoA mutase or methionine synthase or both enzymes. Patients usually present with a wide spectrum of pathologies suggesting that various cellular processes could be affected by modifications in gene expression. We have previously demonstrated that these genetic defects are associated with subcellular mislocalization of RNA-binding proteins (RBP) and subsequent altered nucleo-cytoplasmic shuttling of mRNAs. In order to characterize the possible changes of gene expression in these diseases, we have investigated global gene expression in fibroblasts from patients with cblC and cblG inherited disorders by RNA-seq. The most differentially expressed genes are strongly associated with developmental processes, neurological, ophthalmologic and cardiovascular diseases. These associations are consistent with the clinical presentation of cblC and cblG disorders. Multivariate analysis of transcript processing revaled splicing alterations that led to dramatic changes in cytoskeleton organization, response to stress, methylation of macromolecules and RNA binding. The RNA motifs associated with this differential splicing reflected a potential role of RBP such as HuR and HNRNPL. Proteomic analysis confirmed that mRNA processing was significantly disturbed. This study reports a dramatic alteration of gene expression in fibroblasts of patients with cblC and cblG disorders, which resulted partly from disturbed function of RBP. These data suggest to evaluate the rescue of the mislocalization of RBP as a potential strategy in the treatment of severe cases who are resistant to classical treatments with co-enzyme supplements.

SIRT1 activation rescues the mislocalization of RNA-binding proteins and cognitive defects induced by inherited cobalamin disorders.

BACKGROUND: The molecular consequences of inborn errors of vitamin B12 or cobalamin metabolism are far from being understood. Moreover, innovative therapeutic strategies are needed for the treatment of neurological outcomes that are usually resistant to conventional treatments. Our previous findings suggest a link between SIRT1, cellular stress and RNA binding proteins (RBP) mislocalization in the pathological mechanisms triggered by impaired vitamin B12 metabolism. OBJECTIVES AND METHODS: The goal of this study was to investigate the effects of the pharmacological activation of SIRT1 using SRT1720 on the molecular mechanisms triggered by impaired methionine synthase activity. Experiments were performed in vitro with fibroblasts from patients with the cblG and cblC inherited defects of vitamin B12 metabolism and in vivo with an original transgenic mouse model of methionine synthase deficiency specific to neuronal cells. Subcellular localization of the RBPs HuR, HnRNPA1, RBM10, SRSF1 and Y14 was investigated by immunostaining and confocal microscopy in patient fibroblasts. RBPs methylation and phosphorylation were studied by co-immunoprecipitation and proximity ligation assay. Cognitive performance of the transgenic mice treated with SRT1720 was measured with an aquatic maze. RESULTS: Patient fibroblasts with cblC and cblG defects of vitamin B12 metabolism presented with endoplasmic reticulum stress, altered methylation, phosphorylation and subcellular localization of HuR, HnRNPA1 and RBM10, global mRNA mislocalization and increased HnRNPA1-dependent skipping of IRF3 exons. Incubation of fibroblasts with cobalamin, S-adenosyl methionine and okadaic acid rescued the localization of the RBPs and mRNA. The SIRT1 activating compound SRT1720 inhibited ER stress and rescued RBP and mRNA mislocalization and IRF3 splicing. Treatment with this SIRT1 agonist prevented all these hallmarks in patient fibroblasts but it also improved the deficient hippocampo-dependent learning ability of methionine synthase conditional knock-out mice. CONCLUSIONS: By unraveling the molecular mechanisms triggered by inborn errors of cbl metabolism associating ER stress, RBP mislocalization and mRNA trafficking, our study opens novel therapeutic perspectives for the treatment of inborn errors of vitamin B12 metabolism.

Homocysteine Serum Levels as Prognostic Marker of Hepatocellular Carcinoma with Portal Vein Thrombosis.

BACKGROUND: Portal vein thrombosis (PVT) is a common complication of endstage hepatocellular carcinoma (HCC). The aim of our study was to evaluate the role of Homocysteine (Hcy) in HCC patient with PVT. Hcy is a sulphur amino-acid involved in two pathways, trans-sulphuration and remethylation, that involve vitamins B6, B12 and folates. METHODS: We recruited 54 patients with HCC and PVT, 60 patients with HCC and without PVT and 60 control subjects. We measured serum levels of Hcy, folate, vitamins B6 and B12. RESULTS: The comparison between HCC patients with PVT versus HCC without PVT was shown that mean values of Hcy were 6.4 nmol/L (p<0.0073) higher, LDL cholesterol were 4.8 mg/dl (p<0.0079) lower, vitamin B6 were 4.6 nmol/L(p=0.0544) lower, vitamins B 12 were 22.1 pg/ml (p=0.0001) lower. CONCLUSION: High serum levels of Hcy are an established thrombotic risk factor in the general population. We found significantly higher levels of Hcy in HCC patients with PVT versus both HCC patients without PVT and controls.

Kidney disease and organ transplantation in methylmalonic acidaemia.

OBJECTIVES: MMA is associated with chronic tubulointerstitial nephritis and a progressive decline in GFR. Optimal management of these children is uncertain. Our objectives were to document the pre-, peri-, and post-transplant course of all children with MMA who underwent liver or combined liver-kidney transplant in our centers. DESIGN AND METHODS: Retrospective chart review of all cases of MMA who underwent organ transplantation over the last 10 years. RESULTS: Five children with MMA underwent liver transplant (4/5) and combined liver-kidney transplant (1/5). Three were Mut0 and two had a cobalamin B disorder. Four of five were transplanted between ages 3 and 5 years. Renal dysfunction prior to transplant was seen in 2/5 patients. Post-transplant (one liver transplant and one combined transplant) renal function improved slightly when using creatinine-based GFR formula. We noticed in 2 patients a big discrepancy between creatinine- and cystatin C-based GFR calculations. One patient with no renal disease developed renal failure post-liver transplantation. Serum MMA levels have decreased in all to <300 µmol/L. Four patients remain on low protein diet, carnitine, coenzyme Q, and vitamin E post-transplant. CONCLUSIONS: MMA is a complex metabolic disorder. Renal disease can continue to progress post-liver transplant and close follow-up is warranted. More research is needed to clarify best screening GFR method in patients with MMA. Whether liver transplant alone, continued protein restriction, or the addition of antioxidants post-transplant can halt the progression of renal disease remains unclear.

Molecular genetic characterization of cblC defects in 126 pedigrees and prenatal genetic diagnosis of pedigrees with combined methylmalonic aciduria and homocystinuria.

BACKGROUND: We sought to analyse MMACHC variants among 126 pedigrees with cobalamin (cbl) C deficiency and combined methylmalonic aciduria and homocystinuria by Sanger sequencing, characterize the spectrum of MMACHC gene variants, and perform prenatal genetic diagnosis by chorionic villus sampling among these pedigrees. METHODS: Peripheral blood was collected from 126 probands and their parents who visited the Genetic Counseling Clinic at our hospital between January 2014 and December 2017, and DNA was extracted from the blood. Then, we amplified the coding sequence and splicing regions of the MMACHC gene by PCR, and the PCR products were further sequenced to detect the variants in each pedigree. In 62 families, pregnant women were subjected to chorionic villus sampling for prenatal genetic diagnosis. RESULTS: In total, 31 distinct variants were detected in the 126 pedigrees, and the most frequent variants were c.609G > A (p.Trp203Ter), c.658_660delAAG (p.Lys220del), c.567dupT (p.Ile190Tyrfs*13) and c.80A > G (p.Gln27Arg). Two of these variants have not been previously reported in the literature. One variant [c.463_465delGGG (p.Gly155del)] is a small-scale deletion, and the other variant [c.637G>T(p.Glu213Ter)] is a nonsense mutation. Among the 62 pedigrees who received a prenatal diagnosis, 16 foetuses were normal, 34 foetuses were carriers of heterozygous variants, and the remaining 12 foetuses harboured compound heterozygous variants or homozygous variants. Couples whose foetuses were normal or carriers continued the pregnancy, whereas couples whose foetuses harboured compound heterozygous variants or homozygous variants decided to terminate the pregnancy. The follow-up results were consistent with the prenatal diagnosis. CONCLUSIONS: Two novel MMACHC variants were identified, and prenatal genetic diagnosis is an accurate and convenient method that helps avoid the delivery of combined methylmalonic aciduria and homocystinuria patients.

Late-onset cobalamin C deficiency Chinese sibling patients with neuropsychiatric presentations.

The Cobalamin C deficiency (cblC), characterized with elevated methylmalonic acidemia and homocystinuria in plasma, is an inborn error of cobalamin metabolism. The late-onset cblC siblings patients were rarely reported. In this study, we analyzed the clinical presentations and treatment outcomes of late-onset cblC in Chinese sibling patients with neuropsychiatric presentations. The clinical data of four pairs of Chinese patients were retrospectively analyzed. Serum homocysteine, urine organic acids measurements, neuroimaging exams and gene analysis were carried out in all patents. Patients were reevaluated after treatments with cobalamin, folate, betaine, L-carnitine and compound vitamin B. The mean age at disease onset was 13.7 (range 2-19) years. The neuropsychiatric disturbances including cognitive decline (3/8), psychiatric disturbances (4/8), gait instability (2/8), lower extremity weakness and numbness (3/8) and thromboembolic events (1/8). Two patients suffered nephropathy. The mean serum homocysteine when patients were diagnosed was 109.4 (range 69.5-138) µM/L. The abnormal radioimaging included scoliosis by X-ray (5/6), cerebral atrophy (4/6) and spinal cord atrophy (3/6) by MRI scan. Three pairs of siblings showed heterozygous mutations of MMACHC gene including c.482G > A (4/6), c.354G > C (2/6), c.570insT (2/6), c.445_446del (2/6) and c.656_4658del (2/6). The other two siblings showed homozygous mutation with c.452A > G in MMACHC gene. After treatments, the psychiatric symptoms were obviously relieved in all the patients. In Chinese siblings with late-onset cblC, the main clinic manifestation and abnormal radioimaging were cognitive decline and cerebral atrophy respectively. The most common gene mutation was c.482G > A of MMACHC gene. The patients responded well to the treatments.

Neuropsychological implications of Cobalamin C (CblC) disease in Hispanic children detected through newborn screening.

Cobalamin C (CblC) disease is the most common inborn error of cobalamin metabolism and recent data has indicated a higher prevalence among children of Hispanic heritage in particular. The purpose of this study was to (a) describe the neuropsychological characteristics of a pilot sample of Hispanic children with CblC disease and (b) explore potential differences in outcome based on underlying genetic mutation(s) and biochemical levels. Six Hispanic children (ages 2-10) diagnosed with CblC disease through newborn screening (NBS) underwent neuropsychological evaluation with a bilingual examiner. Biochemical levels and underlying mutation(s) were obtained through medical records. The overall sample performed below normative expectations across neuropsychological domains, including general cognition, adaptive functioning, language ability, and visual-motor integration. Underlying mutations and associative clinical phenotypes were found to significantly predict general cognitive abilities, while plasma methionine and Hcy at the time of diagnosis were significantly correlated with language outcomes. Despite limited sample size, results indicate that Hispanic children with CblC disease detected through NBS and treated early experience neuropsychological deficits even when treated with current standard treatments. However, consistent with prior research in non-Hispanic children with CblC disease, underlying mutations and early biochemical levels may predict better outcomes in this population as well.

Vitamin B12 deficiency results in severe oxidative stress, leading to memory retention impairment in Caenorhabditis elegans.

Oxidative stress is implicated in various human diseases and conditions, such as a neurodegeneration, which is the major symptom of vitamin B12 deficiency, although the underlying disease mechanisms associated with vitamin B12 deficiency are poorly understood. Vitamin B12 deficiency was found to significantly increase cellular H2O2 and NO content in Caenorhabditis elegans and significantly decrease low molecular antioxidant [reduced glutathione (GSH) and L-ascorbic acid] levels and antioxidant enzyme (superoxide dismutase and catalase) activities, indicating that vitamin B12 deficiency induces severe oxidative stress leading to oxidative damage of various cellular components in worms. An NaCl chemotaxis associative learning assay indicated that vitamin B12 deficiency did not affect learning ability but impaired memory retention ability, which decreased to approximately 58% of the control value. When worms were treated with 1mmol/L GSH, L-ascorbic acid, or vitamin E for three generations during vitamin B12 deficiency, cellular malondialdehyde content as an index of oxidative stress decreased to the control level, but the impairment of memory retention ability was not completely reversed (up to approximately 50%). These results suggest that memory retention impairment formed during vitamin B12 deficiency is partially attributable to oxidative stress.