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.

A transgenic mice model of retinopathy of cblG-type inherited disorder of one-carbon metabolism highlights epigenome-wide alterations related to cone photoreceptor cells development and retinal metabolism.

BACKGROUND: MTR gene encodes the cytoplasmic enzyme methionine synthase, which plays a pivotal role in the methionine cycle of one-carbon metabolism. This cycle holds a significant importance in generating S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), the respective universal methyl donor and end-product of epigenetic transmethylation reactions. cblG type of inherited disorders of vitamin B12 metabolism due to mutations in MTR gene exhibits a wide spectrum of symptoms, including a retinopathy unresponsive to conventional therapies. METHODS: To unveil the underlying epigenetic pathological mechanisms, we conducted a comprehensive study of epigenomic-wide alterations of DNA methylation by NGS of bisulfited retinal DNA in an original murine model with conditional Mtr deletion in retinal tissue. Our focus was on postnatal day 21, a critical developmental juncture for ocular structure refinement and functional maturation. RESULTS: We observed delayed eye opening and impaired visual acuity and alterations in the one-carbon metabolomic profile, with a notable dramatic decline in SAM/SAH ratio predicted to impair DNA methylation. This metabolic disruption led to epigenome-wide changes in genes involved in eye development, synaptic plasticity, and retinoid metabolism, including promoter hypermethylation of Rarα, a regulator of Lrat expression. Consistently, we observed a decline in cone photoreceptor cells and reduced expression of Lrat, Rpe65, and Rdh5, three pivotal genes of eye retinoid metabolism. CONCLUSION: We introduced an original in vivo model for studying cblG retinopathy, which highlighted the pivotal role of altered DNA methylation in eye development, cone differentiation, and retinoid metabolism. This model can be used for preclinical studies of novel therapeutic targets.

Stemness of Normal and Cancer Cells: The Influence of Methionine Needs and SIRT1/PGC-1α/PPAR-α Players.

Stem cells are a population of undifferentiated cells with self-renewal and differentiation capacities. Normal and cancer stem cells share similar characteristics in relation to their stemness properties. One-carbon metabolism (OCM), a network of interconnected reactions, plays an important role in this dependence through its role in the endogenous synthesis of methionine and S-adenosylmethionine (SAM), the universal donor of methyl groups in eukaryotic cells. OCM genes are differentially expressed in stem cells, compared to their differentiated counterparts. Furthermore, cultivating stem cells in methionine-restricted conditions hinders their stemness capacities through decreased SAM levels with a subsequent decrease in histone methylation, notably H3K4me3, with a decrease in stem cell markers. Stem cells' reliance on methionine is linked to several mechanisms, including high methionine flux or low endogenous methionine biosynthesis. In this review, we provide an overview of the recent discoveries concerning this metabolic dependence and we discuss the mechanisms behind them. We highlight the influence of SIRT1 on SAM synthesis and suggest a role of PGC-1α/PPAR-α in impaired stemness produced by methionine deprivation. In addition, we discuss the potential interest of methionine restriction in regenerative medicine and cancer treatment.

Epimutations in both the TESK2 and MMACHC promoters in the Epi-cblC inherited disorder of intracellular metabolism of vitamin B12.

BACKGROUND: epi-cblC is a recently discovered inherited disorder of intracellular vitamin B12 metabolism associating hematological, neurological, and cardiometabolic outcomes. It is produced by an epimutation at the promoter common to CCDC163P and MMACHC, which results from an aberrant antisense transcription due to splicing mutations in the antisense PRDX1 gene neighboring MMACHC. We studied whether the aberrant transcription produced a second epimutation by encompassing the CpG island of the TESK2 gene neighboring CCDC163P. METHODS: We unraveled the methylome architecture of the CCDC163P-MMACHC CpG island (CpG:33) and the TESK2 CpG island (CpG:51) of 17 epi-cblC cases. We performed an integrative analysis of the DNA methylome profiling, transcriptome reconstruction of RNA-sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-Seq) of histone H3, and transcription expression of MMACHC and TESK2. RESULTS: The PRDX1 splice mutations and activation of numerous cryptic splice sites produced antisense readthrough transcripts encompassing the bidirectional MMACHC/CCDC163P promoter and the TESK2 promoter, resulting in the silencing of both the MMACHC and TESK2 genes through the deposition of SETD2-dependent H3K36me3 marks and the generation of epimutations in the CpG islands of the two promoters. CONCLUSIONS: The antisense readthrough transcription of the mutated PRDX1 produces an epigenetic silencing of MMACHC and TESK2. We propose using the term 'epi-digenism' to define this epigenetic disorder that affects two genes. Epi-cblC is an entity that differs from cblC. Indeed, the PRDX1 and TESK2 altered expressions are observed in epi-cblC but not in cblC, suggesting further evaluating the potential consequences on cancer risk and spermatogenesis.

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.

Ionizing radiations induce shared epigenomic signatures unraveling adaptive mechanisms of cancerous cell lines with or without methionine dependency.

BACKGROUND: Although radiation therapy represents a core cancer treatment modality, its efficacy is hampered by radioresistance. The effect of ionizing radiations (IRs) is well known regarding their ability to induce genetic alterations; however, their impact on the epigenome landscape in cancer, notably at the CpG dinucleotide resolution, remains to be further deciphered. In addition, no evidence is available regarding the effect of IRs on the DNA methylome profile according to the methionine dependency phenotype, which represents a hallmark of metabolic adaptation in cancer. METHODS: We used a case-control study design with a fractionated irradiation regimen on four cancerous cell lines representative of HCC (HepG2), melanoma (MeWo and MeWo-LC1, which exhibit opposed methionine dependency phenotypes), and glioblastoma (U251). We performed high-resolution genome-wide DNA methylome profiling using the MethylationEPIC BeadChip on baseline conditions, irradiated cell lines (cumulative dose of 10 Gy), and non-irradiated counterparts. We performed epigenome-wide association studies to assess the effect of IRs and methionine-dependency-oriented analysis by carrying out epigenome-wide conditional logistic regression. We looked for epigenome signatures at the locus and single-probe (CpG dinucleotide) levels and through enrichment analyses of gene ontologies (GO). The EpiMet project was registered under the ID#AAP-BMS_003_211. RESULTS: EWASs revealed shared GO annotation pathways associated with increased methylation signatures for several biological processes in response to IRs, including blood circulation, plasma membrane-bounded cell projection organization, cell projection organization, multicellular organismal process, developmental process, and animal organ morphogenesis. Epigenome-wide conditional logistic regression analysis on the methionine dependency phenotype highlighted several epigenome signatures related to cell cycle and division and responses to IR and ultraviolet light. CONCLUSIONS: IRs generated a variation in the methylation level of a high number of CpG probes with shared biological pathways, including those associated with cell cycle and division, responses to IRs, sustained angiogenesis, tissue invasion, and metastasis. These results provide insight on shared adaptive mechanisms of the epigenome in cancerous cell lines in response to IR. Future experiments should focus on the tryptic association between IRs, the initiation of a radioresistance phenotype, and their interaction with methionine dependency as a hallmark of metabolic adaptation in cancer.

Genetic, epigenetic and genomic mechanisms of methionine dependency of cancer and tumor-initiating cells: What could we learn from folate and methionine cycles.

Methionine-dependency is a common feature of cancer cells, which cannot proliferate without constant inputs of exogenous methionine even in the presence of its precursor, homocysteine. The endogenous synthesis of methionine is catalyzed by methionine synthase, which transfers the methyl group of 5-methyltetrahydrofolate (5-methylTHF) to homocysteine in the presence of vitamin B12 (cobalamin, cbl). Diverse mechanisms can produce it, including somatic mutations, aberrant DNA methylation (epimutations) and altered expression of genes. Around twenty somatic mutations have been reported as a cause of methionine dependency. Some of them are contributors but not sufficient on their own to cause methionine dependency. Epigenetic invalidation of MMACHC gene expression triggers methionine dependency of the MeWo-LC1 melanoma cancer cell line. This epimutation is generated by aberrant antisense transcription of the adjacent gene PRDX1. Methionine dependency involves the abnormal expression of 1-CM genes in cancer stem cells. It is related to an increased demand for methionine and SAM, which is not compensated by the increased production of formate by glycine decarboxylase pathway in lung cancer tumor spheres. Tumor spheres of glioblastoma U251 are methionine-dependent through disruption of folate metabolism. The rescue of the growth of glioblastoma stem cells by folate shows the considerable importance to evaluate the influence of supplements and dietary intake of folate on the risk of tumor development, in particular in countries subjected to mandatory food fortification in folic acid. Dietary methionine restriction or the use of methioninase represent promising anticancer therapeutic strategies that deserve to be explored in combination with chemotherapy.