Detecting very low levels of heteroplasmy using mtDNA ultra-deep resequencing

Mitochondrial DNA (mtDNA) is much more exposed to mutagenic events than nuclear DNA (nDNA) due to its high replication rate, lack of histone-like proteins, scarcity of repair enzymes, and production of Reactive Oxygen Species (ROS) that results from Oxidative Phosphorylation (OXPHOS) in mitochondria. The fate of heteroplasmic mutations depends on several factors, including type and location of the variation, replication rate of the cell among others in any case. Since mutations are stochastic events, mtDNA heteroplasmy tends to increase with age. High throughput sequencing technologies recently provided a reliable method to detect levels of heteroplasmy above 2%, with an average coverage of 12000X. The aim of our study is to investigate the presence of heteroplasmy in blood under a threshold of 2% using ultra-deep resequencing and to apply this approach to gather new data on mtDNA mutagenesis and to explore the heritability of this trait. At this purpose HVSII and HSVI have been sequenced, considering the following subjects: (i) 30 female centenarians (ii) 30 offspring of these centenarians sharing the same mtDNA of their mothers (iii) 27 controls. The results are innovative for two main reasons: • the technological approach, as the average coverage is the highest reached since now; • the fact that centenarian' offspring and their mothers share similar level of total heteroplasmy as well as similar heteroplasmic positions, suggesting a sort of genetic control on this trait. In conclusion our data strongly suggest that there is a non negligible hereditary mechanism in the mutagenesis of the mtDNA. This hereditary mechanism has never been taken into account in populations genetics field and could have a great impact in the study of the evolutionary history of the mitochondrial genome.