Detection of mitochondrial DNA (mtDNA) mutations

Abstract

The maternally inherited mitochondrial DNA (mtDNA) is a circular 16,569bp double stranded DNA that encodes 37 genes, 24 of which (2 rRNAs and 22 tRNAs) are necessary for transcription and translation of 13 polypeptides that are all subunits of respiratory chain. Pathogenic mutations in mtDNA cause respiratory chain dysfunction, and are the underlying defect in an ever-increasing number of mtDNA-related encephalomyopathies with distinct phenotypes. In this chapter, we present an overview of mtDNA mutations and describe the molecular techniques currently employed in our laboratory to detect two types of mtDNA mutations: single-large-scale rearrangements and point mutations.

Keywords: Mitochondria; Mutations; Next generation sequencing; Real-time PCR; mtDNA.

Fig 1.

The human mitochondrial genome encodes for 13 mRNAs, 22 tRNAs, and 2 rRNAs. The 7 subunits of NADH-ubiquinone oxidoreductase (ND), 3 subunits of cytochrome c oxidase (COX), cytochrome b (Cyt b), 2 subunits of ATP synthase (A), 22 tRNAs, and 2 rRNAs (12S and 16S) are shown. The origins of heavy (O_H) and light (O_L) strand replication, and the heavy (HSP) and light (LSP) strand transcriptional promoters are also shown.

Fig. 2.

Detection of a large-scale (~5kb) deletion by NGS in a patient harboring the mtDNA “common deletion” with 96% heteroplasmy (panel A patient, panel B control). The nucleotide positions (X- axis) are according to rCRS reference mitochondrial genome. Y-axis represents the coverage of each detected nucleotide.