Multiomic analysis in fibroblasts of patients with inborn errors of cobalamin metabolism reveals concordance with clinical and metabolic variability.

BACKGROUND: The high variability in clinical and metabolic presentations of inborn errors of cobalamin (cbl) metabolism (IECM), such as the cblC/epicblC types with combined deficits in methylmalonyl-coA mutase (MUT) and methionine synthase (MS), are not well understood. They could be explained by the impaired expression/activity of enzymes from other metabolic pathways. METHODS: We performed metabolomic, genomic, proteomic, and post-translational modification (PTM) analyses in fibroblasts from three cblC cases and one epi-cblC case compared with three cblG cases with specific MS deficits and control fibroblasts. FINDINGS: CblC patients had metabolic profilings consistent with altered urea cycle, glycine, and energy mitochondrial metabolism. Metabolomic analysis showed partial disruption and increased glutamate/ketoglutarate anaplerotic pathway of the tricarboxylic acid cycle (TCA), in patient fibroblasts. RNA-seq analysis showed decreased expression of MT-TT (mitochondrial tRNA threonine), MT-TP (mitochondrial tRNA proline), OXCT1 (succinyl CoA:3-oxoacid CoA transferase deficiency), and MT-CO1 (cytochrome C oxidase subunit 1). Proteomic changes were observed for key mitochondrial enzymes, including NADH:ubiquinone oxidoreductase subunit A8 (NDUFA8), carnitine palmitoyltransferase 2 (CPT2), and ubiquinol-cytochrome C reductase, complex III subunit X (UQCR10). Propionaldehyde addition in ornithine aminotransferase was the predominant PTM in cblC cells and could be related with the dramatic cellular increase in propionate and methylglyoxalate. It is consistent with the decreased concentration of ornithine reported in 3 cblC cases. Whether the changes detected after multi-omic analyses underlies clinical features in cblC and cblG types of IECM, such as peripheral and central neuropathy, cardiomyopathy, pulmonary hypertension, development delay, remains to be investigated. INTERPRETATION: The omics-related effects of IECM on other enzymes and metabolic pathways are consistent with the diversity and variability of their age-related metabolic and clinical manifestations. PTMs are expected to produce cumulative effects, which could explain the influence of age on neurological manifestations. FUNDING: French Agence Nationale de la Recherche (Projects PREDICTS and EpiGONE) and Inserm.

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.