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.

Inborn Error of Cobalamin Metabolism Associated with the Intracellular Accumulation of Transcobalamin-Bound Cobalamin and Mutations in ZNF143, Which Codes for a Transcriptional Activator.

Vitamin B12 (cobalamin, Cbl) cofactors adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl) are required for the activity of the enzymes methylmalonyl-CoA mutase (MCM) and methionine synthase (MS). Inborn errors of Cbl metabolism are rare Mendelian disorders associated with hematological and neurological manifestations, and elevations of methylmalonic acid and/or homocysteine in the blood and urine. We describe a patient whose fibroblasts had decreased functional activity of MCM and MS and decreased synthesis of AdoCbl and MeCbl (3.4% and 1.0% of cellular Cbl, respectively). The defect in cultured patient fibroblasts complemented those from all known complementation groups. Patient cells accumulated transcobalamin-bound-Cbl, a complex which usually dissociates in the lysosome to release free Cbl. Whole-exome sequencing identified putative disease-causing variants c.851T>G (p.L284*) and c.1019C>T (p.T340I) in transcription factor ZNF143. Proximity biotinylation analysis confirmed the interaction between ZNF143 and HCFC1, a protein that regulates expression of the Cbl trafficking enzyme MMACHC. qRT-PCR analysis revealed low MMACHC expression levels both in patient fibroblasts, and in control fibroblasts incubated with ZNF143 siRNA.

The Mmachc gene is required for pre-implantation embryogenesis in the mouse.

Patients with mutations in MMACHC have the autosomal recessive disease of cobalamin metabolism known as cblC. These patients are unable to convert cobalamin into the two active forms, methylcobalamin and adenosylcobalamin and consequently have elevated homocysteine and methylmalonic acid in blood and urine. In addition, some cblC patients have structural abnormalities, including congenital heart defects. MMACHC is conserved in the mouse and shows tissue and stage-specific expression pattern in midgestation stage embryos. To create a mouse model of cblC we generated a line of mice with a gene-trap insertion in intron 1 of the Mmachc gene, (Mmachc(Gt(AZ0348)Wtsi)). Heterozygous mice show a 50% reduction of MMACHC protein, and have significantly higher levels of homocysteine and methylmalonic acid in their blood. The Mmachc(Gt) allele was inherited with a transmission ratio distortion in matings with heterozygous animals. Furthermore, homozygous Mmachc(Gt) embryos were not found after embryonic day 3.5 and these embryos were unable to generate giant cells in outgrowth assays. Our findings confirm that cblC is modeled in mice with reduced levels of Mmachc and suggest an early requirement for Mmachc in mouse development.