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.

Inflammation in obsessive-compulsive disorder: A literature review and hypothesis-based potential of transcranial photobiomodulation.

Obsessive-compulsive disorder (OCD) is a disabling neuropsychiatric disorder that affects about 2%-3% of the global population. Despite the availability of several treatments, many patients with OCD do not respond adequately, highlighting the need for new therapeutic approaches. Recent studies have associated various inflammatory processes with the pathogenesis of OCD, including alterations in peripheral immune cells, alterations in cytokine levels, and neuroinflammation. These findings suggest that inflammation could be a promising target for intervention. Transcranial photobiomodulation (t-PBM) with near-infrared light is a noninvasive neuromodulation technique that has shown potential for several neuropsychiatric disorders. However, its efficacy in OCD remains to be fully explored. This study aimed to review the literature on inflammation in OCD, detailing associations with T-cell populations, monocytes, NLRP3 inflammasome components, microglial activation, and elevated proinflammatory cytokines such as TNF-α, CRP, IL-1ß, and IL-6. We also examined the hypothesis-based potential of t-PBM in targeting these inflammatory pathways of OCD, focusing on mechanisms such as modulation of oxidative stress, regulation of immune cell function, reduction of proinflammatory cytokine levels, deactivation of neurotoxic microglia, and upregulation of BDNF gene expression. Our review suggests that t-PBM could be a promising, noninvasive intervention for OCD, with the potential to modulate underlying inflammatory processes. Future research should focus on randomized clinical trials to assess t-PBM's efficacy and optimal treatment parameters in OCD. Biomarker analyses and neuroimaging studies will be important in understanding the relationship between inflammatory modulation and OCD symptom improvement following t-PBM sessions.

Treating neuropathic pain and comorbid affective disorders: Preclinical and clinical evidence.

INTRODUCTION: Neuropathic pain (NP) significantly impacts quality of life and often coexists with affective disorders such as anxiety and depression. Addressing both NP and its psychiatric manifestations requires a comprehensive understanding of therapeutic options. This study aimed to review the main pharmacological and non-pharmacological treatments for NP and comorbid affective disorders to describe their mechanisms of action and how they are commonly used in clinical practice. METHODS: A review was conducted across five electronic databases, focusing on pharmacological and non-pharmacological treatments for NP and its associated affective disorders. The following combination of MeSH and title/abstract keywords were used: "neuropathic pain," "affective disorders," "depression," "anxiety," "treatment," and "therapy." Both animal and human studies were included to discuss the underlying therapeutic mechanisms of these interventions. RESULTS: Pharmacological interventions, including antidepressants, anticonvulsants, and opioids, modulate neural synaptic transmission to alleviate NP. Topical agents, such as capsaicin, lidocaine patches, and botulinum toxin A, offer localized relief by desensitizing pain pathways. Some of these drugs, especially antidepressants, also treat comorbid affective disorders. Non-pharmacological techniques, including repetitive transcranial magnetic stimulation, transcranial direct current stimulation, and photobiomodulation therapy, modulate cortical activity and have shown promise for NP and mood disorders. CONCLUSIONS: The interconnection between NP and comorbid affective disorders necessitates holistic therapeutic strategies. Some pharmacological treatments can be used for both conditions, and non-pharmacological interventions have emerged as promising complementary approaches. Future research should explore novel molecular pathways to enhance treatment options for these interrelated conditions.

Confirming identification of the epidural space: a systematic review of electric stimulation, pressure waveform analysis, and ultrasound and a meta-analysis of diagnostic accuracy in acute pain.

To review the use of epidural electric stimulation test, pressure waveform analysis, and ultrasound assessment of injection as bedside methods for confirming identification of the epidural space in adults with acute pain, the PubMed database was searched for relevant reports between May and August 2022. Studies reporting diagnostic accuracy with conventional Touhy needles and epidural catheters were further selected for meta-analysis. Sensitivity and specificity were estimated using univariate logistic regression for electric stimulation and pressure analysis, and pooling of similar studies for ultrasound. Risk of bias and applicability was assessed using QUADAS-2. For electric stimulation, pressure waveform analysis, and ultrasound, respectively 35, 22, and 28 reports were included in the review and 9, 9, and 7 studies in the meta-analysis. Electric stimulation requires wire-reinforced catheters and an adequate nerve stimulator, does not reliably identify intravascular placement, and is affected by local anaesthetics. Sensitivity was 95% (95% CI 93-96%, N = 550) and specificity unknown (95% CI 33-94%, N = 44). Pressure waveform analysis is unaffected by local anaesthetics, but does not identify intravascular nor intrathecal catheters. Sensitivity was 90% (95% CI 72-97%, N = 694) and specificity 88% (95% CI 78-94%, N = 67). B-mode, M-mode and doppler ultrasound may be challenging, and data is still limited. Risk of bias was significant and accuracy estimates must be interpreted with caution. Electric stimulation and pressure waveform analysis seem clinically useful, although they must be interpreted cautiously. In the future, clinical trials in patients with difficult anatomy will likely be most useful. Ultrasound requires further investigation.

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.

Epimutation in inherited metabolic disorders: the influence of aberrant transcription in adjacent genes.

Epigenetic diseases can be produced by a stable alteration, called an epimutation, in DNA methylation, in which epigenome alterations are directly involved in the underlying molecular mechanisms of the disease. This review focuses on the epigenetics of two inherited metabolic diseases, epi-cblC, an inherited metabolic disorder of cobalamin (vitamin B12) metabolism, and alpha-thalassemia type α-ZF, an inherited disorder of α2-globin synthesis, with a particular interest in the role of aberrant antisense transcription of flanking genes in the generation of epimutations in CpG islands of gene promoters. In both disorders, the epimutation is triggered by an aberrant antisense transcription through the promoter, which produces an H3K36me3 histone mark involved in the recruitment of DNA methyltransferases. It results from diverse genetic alterations. In alpha-thalassemia type α-ZF, a deletion removes HBA1 and HBQ1 genes and juxtaposes the antisense LUC7L gene to the HBA2 gene. In epi-cblC, the epimutation in the MMACHC promoter is produced by mutations in the antisense flanking gene PRDX1, which induces a prolonged antisense transcription through the MMACHC promoter. The presence of the epimutation in sperm, its transgenerational inheritance via the mutated PRDX1, and the high expression of PRDX1 in spermatogonia but its nearly undetectable transcription in spermatids and spermatocytes, suggest that the epimutation could be maintained during germline reprogramming and despite removal of aberrant transcription. The epivariation seen in the MMACHC promoter (0.95 × 10-3) is highly frequent compared to epivariations affecting other genes of the Online Catalog of Human Genes and Genetic Disorders in an epigenome-wide dataset of 23,116 individuals. This and the comparison of epigrams of two monozygotic twins suggest that the aberrant transcription could also be influenced by post-zygotic environmental exposures.

Ocular manifestations in patients with inborn errors of intracellular cobalamin metabolism: a systematic review.

Inherited disorders of cobalamin (cbl) metabolism (cblA-J) result in accumulation of methylmalonic acid (MMA) and/or homocystinuria (HCU). Clinical presentation includes ophthalmological manifestations related to retina, optic nerve and posterior visual alterations, mainly reported in cblC and sporadically in other cbl inborn errors.We searched MEDLINE EMBASE and Cochrane Library, and analyzed articles reporting ocular manifestations in cbl inborn errors. Out of 166 studies a total of 52 studies reporting 163 cbl and 24 mut cases were included. Ocular manifestations were found in all cbl defects except for cblB and cblD-MMA; cblC was the most frequent disorder affecting 137 (84.0%) patients. The c.271dupA was the most common pathogenic variant, accounting for 70/105 (66.7%) cases. One hundred and thirty-seven out of 154 (88.9%) patients presented with early-onset disease (0-12 months). Nystagmus and strabismus were observed in all groups with the exception of MMA patients while maculopathy and peripheral retinal degeneration were almost exclusively found in MMA-HCU patients. Optic nerve damage ranging from mild temporal disc pallor to complete atrophy was prevalent in MMA-HCU.and MMA groups. Nystagmus was frequent in early-onset patients. Retinal and macular degeneration worsened despite early treatment and stabilized systemic function in these patients. The functional prognosis remains poor with final visual acuity < 20/200 in 55.6% (25/45) of cases. In conclusion, the spectrum of eye disease in Cbl patients depends on metabolic severity and age of onset. The development of visual manifestations over time despite early metabolic treatment point out the need for specific innovative therapies.

The Role of Ultrasonography in the Diagnosis and Decision Algorithm for the Management of Pneumothorax after Transbronchial Lung Cryobiopsy.

BACKGROUND: Pneumothorax is one of the main complications of transbronchial lung cryobiopsy (TBLC). Chest ultrasound (CUS) is a radiation-free alternative method for pneumothorax detection. OBJECTIVE: We tested CUS diagnostic accuracy for pneumothorax and assessed its role in the decision algorithm for pneumothorax management. Secondary objectives were to evaluate the post-procedure pneumothorax occurrence and risk factors. METHODS: Eligible patients underwent TBLC, followed by chest X-ray (CXR) evaluation 2 h after the procedure, as our standard protocol. Bedside CUS was performed within 30 min and 2 h after TBLC. Pneumothorax by CUS was defined by the absence of lung sliding and comet-tail artefacts and confirmed with the stratosphere sign on M-mode. Pneumothorax size was determined through lung point projection on CUS and interpleural distance on CXR and properly managed according to clinical status. RESULTS: Sixty-seven patients were included. Nineteen pneumothoraces were detected at 2 h after the procedure, of which 8 (42.1%) were already present at the first CUS evaluation. All CXR-detected pneumothoraces had a positive CUS detection. There were 3 discordant cases (κ = 0.88, 95% CI: 0.76-1.00, p < 0.001), which were detected by CUS but not by inspiration CXR. We calculated a specificity of 97.5% (95% CI: 86.8-99.9) and a sensitivity of 100% (95% CI: 87.2-100) for CUS. Pneumothorax rate was higher when biopsies were taken in 2 lobes and if histology had pleural representation. Final diagnosis was achieved in 79.1% of patients, with the most frequent diagnosis being hypersensitivity pneumonitis. Regarding patients with large-volume pneumothorax needing drainage, the rate of detection was similar between CUS and CRX. CONCLUSION: CUS can replace CXR in detecting the presence of pneumothorax after TBLC, and the lung point site can reliably indicate its size. This useful method optimizes time spent at the bronchology unit and allows immediate response in symptomatic patients, helping to choose optimal treatment strategies, while preventing ionizing radiation exposure.

A bi-allelic loss-of-function SARS1 variant in children with neurodevelopmental delay, deafness, cardiomyopathy, and decompensation during fever.

Aminoacyl-tRNA synthetases (aaRS) are ubiquitously expressed enzymes responsible for ligating amino acids to their cognate tRNA molecules through an aminoacylation reaction. The resulting aminoacyl-tRNA is delivered to ribosome elongation factors to participate in protein synthesis. Seryl-tRNA synthetase (SARS1) is one of the cytosolic aaRSs and catalyzes serine attachment to tRNASer . SARS1 deficiency has already been associated with moderate intellectual disability, ataxia, muscle weakness, and seizure in one family. We describe here a new clinical presentation including developmental delay, central deafness, cardiomyopathy, and metabolic decompensation during fever leading to death, in a consanguineous Turkish family, with biallelic variants (c.638G>T, p.(Arg213Leu)) in SARS1. This missense variant was shown to lead to protein instability, resulting in reduced protein level and enzymatic activity. Our results describe a new clinical entity and expand the clinical and mutational spectrum of SARS1 and aaRS deficiencies.

Mutations in MTHFR and POLG impaired activity of the mitochondrial respiratory chain in 46-year-old twins with spastic paraparesis.

Hereditary spastic paraplegias (HSPs) are characterized by lower extremity spasticity and weakness. HSP is often caused by mutations in SPG genes, but it may also be produced by inborn errors of metabolism. We performed next-generation sequencing of 4813 genes in one adult twin pair with HSP and severe muscular weakness occurring at the same age. We found two pathogenic compound heterozygous variants in MTHFR, including a variant not referenced in international databases, c.197C>T (p.Pro66Leu) and a known variant, c.470G>A (p.Arg157Gln), and two heterozygous pathogenic variants in POLG, c.1760C>T (p.Pro587Leu) and c.752C>T (p.Thr251Ile). MTHFR and POLG mutations were consistent with the severe muscle weakness and the metabolic changes, including hyperhomocysteinemia and decreased activity of both N(5,10)methylenetetrahydrofolate reductase (MTHFR) and complexes I and II of the mitochondrial respiratory chain. These data suggest the potential role of MTHFR and POLG mutations through consequences on mitochondrial dysfunction in the occurrence of spastic paraparesis phenotype with combined metabolic, muscular, and neurological components.

Inherited disorders of cobalamin metabolism disrupt nucleocytoplasmic transport of mRNA through impaired methylation/phosphorylation of ELAVL1/HuR.

The molecular mechanisms that underlie the neurological manifestations of patients with inherited diseases of vitamin B12 (cobalamin) metabolism remain to date obscure. We observed transcriptomic changes of genes involved in RNA metabolism and endoplasmic reticulum stress in a neuronal cell model with impaired cobalamin metabolism. These changes were related to the subcellular mislocalization of several RNA binding proteins, including the ELAVL1/HuR protein implicated in neuronal stress, in this cell model and in patient fibroblasts with inborn errors of cobalamin metabolism and Cd320 knockout mice. The decreased interaction of ELAVL1/HuR with the CRM1/exportin protein of the nuclear pore complex and its subsequent mislocalization resulted from hypomethylation at R-217 produced by decreased S-adenosylmethionine and protein methyl transferase CARM1 and dephosphorylation at S221 by increased protein phosphatase PP2A. The mislocalization of ELAVL1/HuR triggered the decreased expression of SIRT1 deacetylase and genes involved in brain development, neuroplasticity, myelin formation, and brain aging. The mislocalization was reversible upon treatment with siPpp2ca, cobalamin, S-adenosylmethionine, or PP2A inhibitor okadaic acid. In conclusion, our data highlight the key role of the disruption of ELAVL1/HuR nuclear export, with genomic changes consistent with the effects of inborn errors of Cbl metabolisms on brain development, neuroplasticity and myelin formation.

Brain Susceptibility to Methyl Donor Deficiency: From Fetal Programming to Aging Outcome in Rats.

Deficiencies in methyl donors, folate, and vitamin B12 are known to lead to brain function defects. Fetal development is the most studied but data are also available for such an impact in elderly rats. To compare the functional consequences of nutritional deficiency in young versus adult rats, we monitored behavioral outcomes of cerebellum and hippocampus circuits in the offspring of deficient mother rats and in adult rats fed a deficient diet from 2 to 8 months-of-age. We present data showing that the main deleterious consequences are found in young ages compared to adult ones, in terms of movement coordination and learning abilities. Moreover, we obtained sex and age differences in the deleterious effects on these functions and on neuronal layer integrity in growing young rats, while deficient adults presented only slight functional alterations without tissue damage. Actually, the cerebellum and the hippocampus develop and maturate according to different time lap windows and we demonstrate that a switch to a normal diet can only rescue circuits that present a long permissive window of time, such as the cerebellum, whereas the hippocampus does not. Thus, we argue, as others have, for supplements or fortifications given over a longer time than the developmental period.

Clinical or ATPase domain mutations in ABCD4 disrupt the interaction between the vitamin B12-trafficking proteins ABCD4 and LMBD1.

Vitamin B12 (cobalamin (Cbl)), in the cofactor forms methyl-Cbl and adenosyl-Cbl, is required for the function of the essential enzymes methionine synthase and methylmalonyl-CoA mutase, respectively. Cbl enters mammalian cells by receptor-mediated endocytosis of protein-bound Cbl followed by lysosomal export of free Cbl to the cytosol and further processing to these cofactor forms. The integral membrane proteins LMBD1 and ABCD4 are required for lysosomal release of Cbl, and mutations in the genes LMBRD1 and ABCD4 result in the cobalamin metabolism disorders cblF and cblJ. We report a new (fifth) patient with the cblJ disorder who presented at 7 days of age with poor feeding, hypotonia, methylmalonic aciduria, and elevated plasma homocysteine and harbored the mutations c.1667_1668delAG [p.Glu556Glyfs*27] and c.1295G>A [p.Arg432Gln] in the ABCD4 gene. Cbl cofactor forms are decreased in fibroblasts from this patient but could be rescued by overexpression of either ABCD4 or, unexpectedly, LMBD1. Using a sensitive live-cell FRET assay, we demonstrated selective interaction between ABCD4 and LMBD1 and decreased interaction when ABCD4 harbored the patient mutations p.Arg432Gln or p.Asn141Lys or when artificial mutations disrupted the ATPase domain. Finally, we showed that ABCD4 lysosomal targeting depends on co-expression of, and interaction with, LMBD1. These data broaden the patient and mutation spectrum of cblJ deficiency, establish a sensitive live-cell assay to detect the LMBD1-ABCD4 interaction, and confirm the importance of this interaction for proper intracellular targeting of ABCD4 and cobalamin cofactor synthesis.

Methionine synthase and methionine synthase reductase interact with MMACHC and with MMADHC.

An increasing number of studies indicate that each step of the intracellular processing of vitamin B12 or cobalamin (Cbl) involves protein-protein interactions. We have previously described a novel interaction between methionine synthase (MS) and MMACHC and its effect on the regulation of MMACHC activity. Our goal is to further characterize the interactions of MS with other potential partners in a so-called MS interactome. We dissected the interactions and their alterations by co-immunoprecipitation and DuoLink proximity ligation assays in fibroblasts with cblG, cblE, and cblC genetic defects affecting respectively the expression of MS, methionine synthase reductase (MSR) and MMACHC and in HepG2 cells transfected with corresponding siRNAs. We observed the known interactions of MS with MSR and with MMACHC as well as MMADHC with MMACHC, but we also observed novel interactions for MSR with MMACHC and with MMADHC and MS with MMADHC. Furthermore, we show that the absence of MS or MMACHC expression disrupts the interactions between the other interactome members, in cblC and cblG fibroblasts and in HepG2 cells transfected with siRNAs. Our data show that the processing of Cbl in cytoplasm occurs in a multiprotein complex composed of at least MS, MSR, MMACHC and MMADHC, which could contribute to shuttle safely and efficiently Cbl towards MS. Our data suggest that defective protein-protein interactions among key players of this pathway could contribute to the molecular mechanisms of the cblC, cblG and cblE genetic defects and provide novel insights into our understanding of the pathophysiology of inherited disorders of Cbl metabolism.

Mutation Update and Review of Severe Methylenetetrahydrofolate Reductase Deficiency.

Severe 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency is caused by mutations in the MTHFR gene and results in hyperhomocysteinemia and varying severity of disease, ranging from neonatal lethal to adult onset. Including those described here, 109 MTHFR mutations have been reported in 171 families, consisting of 70 missense mutations, 17 that primarily affect splicing, 11 nonsense mutations, seven small deletions, two no-stop mutations, one small duplication, and one large duplication. Only 36% of mutations recur in unrelated families, indicating that most are "private." The most common mutation is c.1530A>G (numbered from NM_005957.4, p.Lys510 = ) causing a splicing defect, found in 13 families; the most common missense mutation is c.1129C>T (p.Arg377Cys) identified in 10 families. To increase disease understanding, we report enzymatic activity, detected mutations, and clinical onset information (early, <1 year; or late, >1 year) for all published patients available, demonstrating that patients with early onset have less residual enzyme activity than those presenting later. We also review animal models, diagnostic approaches, clinical presentations, and treatment options. This is the first large review of mutations in MTHFR, highlighting the wide spectrum of disease-causing mutations.

Dose-dependent effects of siRNA-mediated inhibition of SCAP on PCSK9, LDLR, and plasma lipids in mouse and rhesus monkey.

SREBP cleavage-activating protein (SCAP) is a key protein in the regulation of lipid metabolism and a potential target for treatment of dyslipidemia. SCAP is required for activation of the transcription factors SREBP-1 and -2. SREBPs regulate the expression of genes involved in fatty acid and cholesterol biosynthesis, and LDL-C clearance through the regulation of LDL receptor (LDLR) and PCSK9 expression. To further test the potential of SCAP as a novel target for treatment of dyslipidemia, we used siRNAs to inhibit hepatic SCAP expression and assess the effect on PCSK9, LDLR, and lipids in mice and rhesus monkeys. In mice, robust liver Scap mRNA knockdown (KD) was achieved, accompanied by dose-dependent reduction in SREBP-regulated gene expression, de novo lipogenesis, and plasma PCSK9 and lipids. In rhesus monkeys, over 90% SCAP mRNA KD was achieved resulting in approximately 75, 50, and 50% reduction of plasma PCSK9, TG, and LDL-C, respectively. Inhibition of SCAP function was demonstrated by reduced expression of SREBP-regulated genes and de novo lipogenesis. In conclusion, siRNA-mediated inhibition of SCAP resulted in a significant reduction in circulating PCSK9 and LDL-C in rodent and primate models supporting SCAP as a novel target for the treatment of dyslipidemia.

Interaction between methionine synthase isoforms and MMACHC: characterization in cblG-variant, cblG and cblC inherited causes of megaloblastic anaemia.

The cblG and cblC disorders of cobalamin (Cbl) metabolism are two inherited causes of megaloblastic anaemia. In cblG, mutations in methionine synthase (MTR) decrease conversion of hydroxocobalamin  (HOCbl) to methylcobalamin, while in cblC, mutations in MMACHC disrupt formation of cob(II)alamin (detected as HOCbl). Cases with undetectable methionine synthase (MS) activity are extremely rare and classified as 'cblG-variant'. In four 'cblG-variant' cases, we observed a decreased conversion of cyanocobalamin to HOCbl that is also seen in cblC cases. To explore this observation, we studied the gene defects, splicing products and expression of MS, as well as MS/MMACHC protein interactions in cblG-variant, cblG, cblC and control fibroblasts. We observed a full-size MS encoded by MTR-001 and a 124 kDa truncated MS encoded by MTR-201 in cblG, cblC, control fibroblasts and HEK cells, but only the MTR-201 transcript and inactive truncated MS in cblG-variant cells. Co-immunoprecipitation and proximity ligation assay showed interaction between truncated MS and MMACHC in cblG-variant cells. This interaction decreased 2.2, 1.5 and 5.0-fold in the proximity ligation assay of cblC cells with p.R161Q and p.R206W mutations, and HEK cells with knock down expression of MS by siRNA, respectively, when compared with control cells. In 3D modelling and docking analysis, both truncated and full-size MS provide a loop anchored to MMACHC, which makes contacts with R-161 and R-206 residues. Our data suggest that the interaction of MS with MMACHC may play a role in the regulation of the cellular processing of Cbls that is required for Cbl cofactor synthesis.

Nutritional models of foetal programming and nutrigenomic and epigenomic dysregulations of fatty acid metabolism in the liver and heart.

Barker's concept of 'foetal programming' proposes that intrauterine growth restriction (IUGR) predicts complex metabolic diseases through relationships that may be further modified by the postnatal environment. Dietary restriction and deficit in methyl donors, folate, vitamin B12, and choline are used as experimental conditions of foetal programming as they lead to IUGR and decreased birth weight. Overfeeding and deficit in methyl donors increase central fat mass and lead to a dramatic increase of plasma free fatty acids (FFA) in offspring. Conversely, supplementing the mothers under protein restriction with folic acid reverses metabolic and epigenomic phenotypes of offspring. High-fat diet or methyl donor deficiency (MDD) during pregnancy and lactation produce liver steatosis and myocardium hypertrophy that result from increased import of FFA and impaired fatty acid ß-oxidation, respectively. The underlying molecular mechanisms show dysregulations related with similar decreased expression and activity of sirtuin 1 (SIRT1) and hyperacetylation of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α). High-fat diet and overfeeding impair AMPK-dependent phosphorylation of PGC-1α, while MDD decreases PGC-1α methylation through decreased expression of PRMT1 and cellular level of S-adenosyl methionine. The visceral manifestations of metabolic syndrome are under the influence of endoplasmic reticulum (ER) stress in overnourished animal models. These mechanisms should also deserve attention in the foetal programming effects of MDD since vitamin B12 influences ER stress through impaired SIRT1 deacetylation of HSF1. Taken together, similarities and synergies of high-fat diet and MDD suggest, therefore, considering their consecutive or contemporary influence in the mechanisms of complex metabolic diseases.

Molecular mechanisms leading to three different phenotypes in the cblD defect of intracellular cobalamin metabolism.

The cblD defect of intracellular vitamin B(12) metabolism can lead to isolated methylmalonic aciduria (cblD-MMA) or homocystinuria (cblD-HC), or combined methylmalonic aciduria and homocystinuria (cblD-MMA/HC). We studied the mechanism whereby MMADHC mutations can lead to three phenotypes. The effect of various expression vectors containing MMADHC modified to contain an enhanced mitochondrial leader sequence or mutations changing possible downstream sites of reinitiation of translation or mutations introducing stop codons on rescue of adenosyl- and methylcobalamin (MeCbl) formation was studied. The constructs were transfected into cell lines derived from various cblD patient's fibroblasts. Expression of 10 mutant alleles from 15 cblD patients confirmed that the nature and location of the mutations correlate with the biochemical phenotype. In cblD-MMA/HC cells, improving mitochondrial targeting of MMADHC clearly increased the formation of adenosylcobalamin (AdoCbl) with a concomitant decrease in MeCbl formation. In cblD-MMA cells, this effect was dependent on the mutation and showed a negative correlation with endogenous MMADHC mRNA levels. These findings support the hypothesis that a single protein exists with two different functional domains that interact with either cytosolic or mitochondrial targets. Also a delicate balance exists between cytosolic MeCbl and mitochondrial AdoCbl synthesis, supporting the role of cblD protein as a branch point in intracellular cobalamin trafficking. Furthermore, our data indicate that the sequence after Met116 is sufficient for MeCbl synthesis, whereas the additional sequence between Met62 and Met116 is required for AdoCbl synthesis. Accordingly, western blot studies reveal proteins of the size expected from the stop codon position with subsequent reinitiation of translation.

[Vitamin B12 and related genetic disorders]. / La vitamine B12 et les maladies génétiques associées.

Vitamin B12 (B12, cobalamin (Cbl)) is a water-soluble vitamin that requires complex mechanisms for its assimilation, blood transport and intracellular metabolism. Three proteins, intrinsic factor (IF), haptocorrin (HC), and transcobalamin (TC), and their specific receptors are involved in B12 absorption and transport. Acquired and inherited deficiencies can result in megaloblastic anemia and neurological manifestations. Several genetic diseases are linked to these two steps, namely inherited deficits in FI and TC, and Imerslund-Gräsbeck disease. In mammalian cells, only two enzymes depend on vitamin B12: L-methylmalonyl-CoA mutase (EC 5.4.99.2) in mitochondria, and methionine synthase (EC 2.1.1.13) in cytoplasm. Direct metabolic consequences of impaired B12 absorption and metabolism are the accumulation of methylmalonic acid (MMA) and of homocysteine (HCy), respectively. More than a dozen genes are involved in the intracellular metabolism of B12, and their defects result in several diseases designated cblA through cblJ This article reviews the steps involved in vitamin B12 absorption, transport and intracellular metabolism, and the main related genetic defects.