Riassunto analitico
Background: Mitochondrial DNA (mtDNA) metabolism disorders are a heterogeneous group of disorders caused by pathogenic variants in nuclear genes encoding proteins involved in mtDNA replication and translation, and biochemically responsible for multiple OXPHOS defects in affected tissues. Patients’ cohort studies have clearly defined them as a clinical spectrum and highlighted the absence of a genotype-phenotype correlation. In fact, the same pathogenetic homozygous or compound heterozygous variants can cause an infantile, multisystemic, and severe disorder or a mild late-onset tissue-specific one, even in the same family. Identifying the mechanism responsible for the clinical variability is essential for designing new targets for therapy, finding biomarkers and prognostic factors, and thus, developing personalized medicine. There is evidence that phenotypic heterogeneity and tissue specificity of genetic diseases can be explained by mutation burden in terms of copy number variations (CNVs) and/or single nucleotide polymorphisms (SNPs) in coding and non-coding regions in the same or counteracting biological pathway. Objectives: our goal was to identify genetic and epigenetic factors regulating clinical variability in mtDNA metabolism disorders. Materials and methods: NGS studies were performed in DNA extracted from biological samples of patients with mtDNA metabolism defects. Specifically, whole genome sequencing (WGS) was applied to identify variants in the coding and non-coding regions (eg. enhancers, 3’-UTR, 5’-UTR, miRNA, lncRNA) and array comparative genomic hybridization (aCGH) to analyze copy number variations. Pathogenetic scores such as CADD and NCBoost, prediction tools, and available databases such as Franklin, Varsome, Uniprot, TargetScanHuman, FANTOM5, RegulomeDB, GnomAD, and Clinvar were used for data filtering and interpretation. Further analyses were performed on GTEx Portal and IGV. Variants were validated by direct Sanger sequencing and additional functional studies were performed in patients’ derived fibroblasts. Results: DNA samples from 3 families with 2 affected members each, presenting the same mutation in nuclear DNA (RARS2, EARS2, TK2) but different clinical phenotypes have been studied. Variants in coding and non-coding regions of proteins involved in the mitochondrial translation and replication pathways and predicted miRNA binding sites were identified and associated with the more severe phenotypes. Further analyses are currently ongoing with bulk RNA barcoding and sequencing (BRB-seq) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) for detecting chromatin accessibility and studying the transcriptome.
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