A WHOLE mtDNA NGS APPROACH TO IDENTIFY NOVEL VARIANTS IN PATIENTS AFFECTED BY MITOCHONDRIAL DISEASES

Introduction: Mitochondrial DNA (mtDNA) is a 16.6 kb, double-stranded, circular molecule that contains 37 genes coding for 13 subunits of the respiratory chain plus two rRNAs and 22 tRNAs, required to synthetize them. Mutations in these genes have been identified in patients with a variety of disorders affecting every system in the body. Mitochondrial diseases are clinically heterogeneous, so both diagnosis and treatment are extremely difficult. Traditional molecular diagnostic approach was limited to testing the most frequent mtDNA point mutations related to well-known mitochondrial diseases like MELAS, MERRF, NARP, LHON. The advent of next generation sequencing technologies has provided the whole mtDNA sequencing, allowing the identification of disease-causing pathogenic variants in a single platform. Aim of the study: The aim of this study was to develop an NGS strategy, by using an amplicon-based method to sequence the entire mitogenome, in order to identify disease-causing pathogenic variants in patients affected by mitochondrial diseases.Materials and Methods: The whole mtDNA coming from 20 patients was analyzed using an amplicon-based approach (Precision ID mtDNA Whole Genome Panel - ThermoFisher Scientific). The enriched libraries were sequenced on the Personal Genome Machine (PGM) from ThermoFisher Scientific. Primary bioinformatic analysis was carried out using the Ion Torrent suite v. 5.6. Annotation of the variants was made by the Variant Annotation Integrator from the UCSC browser. Results: After bioinformatics analysis and filtering, 100% coverage of the targeted regions was obtained with at least 500-fold mean depth. The size distribution of the amplicons ranged from 118 to 150 bp. The generated reads were 100% on target, with a uniformity of 99.8%. After variant calling, we were able to detect a mean of 35 SNVs for sample. Conclusions: The analysis of the whole mtDNA is a useful strategy for researchers studying human diseases linked to variations present in this type of genome. The NGS approach, compared to the standard methods, is a more reliable and time-cost reducing strategy to detect all the variants present in the mitogenome possibly associated with mitochondrial diseases.