Mitochondrial diseases - long read genome and transcriptome sequencing in cases unresolved after short-read genomics

• Applicant: Dr. Tobias Bernd Ludwig Haack
• Subject Area: Human Genetics ; Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 418081722

Mitochondrial disorders comprise clinically and genetically heterogeneous group of disorders arising from enzymatic defects of the pyruvate oxidation route. Broad sequencing approaches such as exome or even full genome sequencing significantly increased the diagnostic yield. However, about half of the patients remain without a firm diagnosis. This situation prevents adequate counseling of the patients and their families as well as the development of targeted therapeutic strategies.We have performed extensive efforts to identify the molecular bases of mitochondrial disease patients in a multidisciplinary team in the BMBF-funded Juniorverbund 'mitOmics'. The project included full genome trio analyses as well as RNAseq on patient-derived fibroblast cell lines displaying the biochemical signature of the disease. Although we were indeed able to pinpoint the molecular defect in an additional 5-10 % of previously unsolved cases the vast majority had remained unclear.Amongst other potential explanations (e.g. non-genetic cause) one is that we simply don’t detect the disease-causal variation because we use the wrong method. The vast majority of full exome or genome datasets is currently produced on Illumina platforms which usually allow to determine the DNA or RNA sequence of about 75-150 bp. While this technology works well to detect single nucleotide variants (SNVs) it is basically blind for other types of genetic variation such as repeat expansions or contractions as well as structural variants (SVs, e.g. inversions).Recent advances in so-called long read technologies allow the sequencing of reads with a length of several thousand base pairs. These methods resolve repetitive sequences or other genomic regions not accessible to short read genomics. In addition, the Nanopore technology has the potential to detect epigenetic marks. However, current disadvantages are higher base calling error rates and costs.In this study, we want to build on our previously established resources, data, and expertise in data interpretation. We apply for long read and bisulfite sequencing on a highly selected cohort of 30 unresolved index patients which had remained unsolved after short read whole genome and transcriptome sequencing. Data integration and analysis will be tailored to prioritize genetic and epigenetic factors previously overlooked with short read analyses. Patients’ cell lines can subsequently serve as a valuable model for confirmatory functional studies or evaluation of therapeutic interventions.Generated data will be shared within local and international rare disease networks. The results of the study are expected to provide new insights into disease mechanisms and cellular biology, to enable the evaluation of personalized therapeutic options, to improve molecular diagnostics of rare disease patients, and guide the development of tomorrow’s routine diagnostic strategies.

• DFG Programme: Research Grants