Measuring Rates of mtDNA Heteroplasmy and Assessing Transmission of Variants

As submitted by the proposer: While current techniques and technologies provide for reliable reporting of mtDNA haplotypes, the methods in place today do not effectively identify heteroplasmic variants (especially low-level variants), and even when heteroplasmy is observed at high levels, the information is typically not used during the forensic investigation. The ability to identify, report, and leverage the discrimination potential of heteroplasmy will significantly enhance the value of using mtDNA analysis in forensic casework. A next generation DNA sequencing (NGS) approach will allow the community to achieve this goal. The goals of this research are to measure the rate of observing mtDNA heteroplasmy on a per sample and per nucleotide basis; to evaluate the transmission of heteroplasmic events, both between maternal relatives and between different tissue types; and to use this information to evaluate best practices regarding the reporting of mtDNA heteroplasmy. Our initial work illustrated that the increased resolution provided by NGS produces a higher rate of detectable heteroplasmy, including across the mtDNA genome. Since then we have revealed that low-level variant differences exist within maternal lineages, and that a standardized approach to data analysis, along with software designed to evaluate NGS mtDNA data, is needed. The first part of this proposal will establish rates of heteroplasmy for the CR of the mtDNA genome through the analysis of 550 samples from unrelated individuals. The second part will evaluate transmission through whole mtDNA genome sequencing of three tissue types (blood, buccal, hair) collected from two maternal lineages consisting of multiple generations. Finally, data generated for the rate and transmission studies, as well as data previously generated, will be used to evaluate the impact of error rates on the reporting of low-level heteroplasmy. The NGS approach is clearly capable of detecting heteroplasmy, including low-levels of heteroplasmy. This will allow forensic laboratories to report out heteroplasmy on a routine basis, significantly enhancing the discrimination potential of mtDNA testing. In addition, our preliminary results suggest that a large percentage of maternal relatives can be distinguished from each other through the analysis of these heteroplasmic variants. Again, this will have a major positive impact on laboratories currently performing mtDNA analysis, and could be the impetus for other laboratories to bring mtDNA typing on line if the discrimination potential were improved, and the results of the testing become more valuable to a forensic case. ca/ncf