Massively parallel sequencing of enriched mitochondrial DNA in patients with clinical suspicion of mitochondrial disease

Abstract

Mitochondrial disorders constitute a clinically and genetically heterogeneous group of diseases that arise as a result of dysfunction of the mitochondrial respiratory chain. They can be caused by mutations in either the nuclear or mitochondrial DNA. In this study, three patients with clinical suspicion of mitochondrial disease were investigated by whole exome sequencing (WES), identifying the homozygous splice site mutation SURF1 c.106+1G>C in patient 1. This mutation, which was demonstrated to cause skipping of exon 2 in SURF1, was concluded to be the genetic cause of the Leigh syndrome like phenotype in the patient. Patients 2 and 3 were further investigated by massively parallel sequencing (MSP) of mtDNA from fibroblasts using a PCR free protocol for the preparation of mtDNA enriched samples. Sequencing resulted in uniform and ultra-deep coverage (>40,000) of the entire mtDNA genome, demonstrating that the PCR free approach is the method of choice when analyzing mitochondrial DNA by MSP. In parallel, analysis of mtDNA from muscle from patient 3 revealed the novel and nearly homoplastic deletion m.6211_8681del, which was concluded to be the cause of disease in the patient. This deletion was also identified in mtDNA from the patient s blood and skin tissues, but it was present in too low levels of heteroplasmy to be detected by MSP.