Rare mtDNA variants in somatic tissues and single oocytes

Mutations in mitochondrial DNA (mtDNA) are involved in a variety of diseases such as type 2 diabetes mellitus, cancer and aging. Most of these diseases are associated with heteroplasmic sites, referring to sites in mtDNA for which multiple variants coexist within a cell or among different cells within an individual. When the frequency of a disease-associated variant exceeds a certain threshold, symptoms occur. Interestingly, despite the exclusively maternal transmission of mtDNA, due to rapid shifts in heteroplasmic allele frequencies, an offspring of a healthy mother can show disease symptoms and vice versa. While for heteroplasmies at levels above 1% the transmission to the next generation has already been well studied, very little is known about variants occurring at lower frequencies. A study of these low-level heteroplasmies, as well as of mtDNA in single oocytes (through which mtDNA is passed on to the next generation), would aid in answering the following questions: How can new (potentially pathogenic) mutations arise in the germ line? How are they transmitted to the next generation? How can the level of heteroplasmy increase from one initial mutation to pathogenic levels? Therefore, the major aims of this project will be (1) to establish experimental procedures to obtain high- quality mtDNA sequences, (2) to obtain high-quality mtDNA sequences from murine somatic tissues and single oocytes, and (3) to analyze inheritance of low-level heteroplasmic sites and estimate the germline mtDNA mutation rate. Advanced sequencing technologies with very low error rates, coupled with special procedures for sample and sequencing library preparation, will be used to obtain high-quality mtDNA sequences from somatic tissues of female mice, single oocytes of these mice, and tissues and oocytes of their offspring. Detailed knowledge on formation and inheritance of mtDNA heteroplasmies becomes increasingly important since no efficient cures are currently available for mtDNA-related diseases. Furthermore, there are now new possibilities in in vitro fertilization (IVF) such as tri-parental IVF in which mtDNA of a third parent is utilized to avoid the transmission of an mtDNA-associated disease of the mother. The development of a method to obtain high-quality mtDNA sequences, in combination with the analysis of low-frequency variants in mothers and offspring, will allow me to generate a comprehensive picture of heteroplasmy transmission. It will provide important information on underlying processes affecting mtDNA mutation frequency shifts. While the proposed project focuses on the analysis of heteroplasmic sites in mouse as a model organism, the developed methodologies will be applicable to other species, including human.

Mutations - changes in DNA sequences - create genetic variation for other evolutionary forces to operate on, but also cause numerous genetic diseases. Mutations in the genome of mitochondria (mtDNA) - mitochondria are organelles that are the powerhouse of the cell - are involved in a variety of diseases such as type 2 diabetes mellitus and cancer, but also aging. Nevertheless, how new mutations in mtDNA arise remains poorly understood. When using conventional sequencing technologies - for which error rates are much higher than the mutation rate - we frequently do not know if measured changes in DNA sequence are new mutations or just errors in the sequencing. Here in this study I applied an advanced and extremely accurate DNA sequencing method - duplex sequencing - to sequence the entire mtDNA in both, female reproductive cells (oocytes - through which mtDNA is passed on to the next generation) and body cells (brain and skeletal muscle). If you think of a DNA molecule as a twisted ladder, with two rails running parallel to each other and rungs that connect the rails, you can split it down through the middle of the rungs and each side is called a strand. While most sequencing methods only look at one strand at a time, duplex sequencing reads both DNA strands and compares the two to reduce its error rate. That way, we could be confident that we are observing actual new mutations. Duplex sequencing usually requires a fairly large amount of DNA starting material, so we had to optimize the protocol to deal with tiny amounts of DNA from single oocytes. By sequencing oocytes and body cells from 36 mice belonging to two independent pedigrees, we showed that, depending on the cell type, 10-month-old mother mice had approximately two-to-three times more new mutations than their 20-day-old pups, demonstrating mutation accumulation during the period of only 9 months. Mutation frequencies and patterns differed between oocytes and body cells, and among mtDNA regions, suggestive of distinct mutagenesis mechanisms. This study deciphered for the first time the intricacies of germline de novo mutagenesis using duplex sequencing, which provided unprecedented resolution, and directly in oocytes, thus minimizing other effects present in pedigree studies (not looking at germ cells directly). Moreover, this work provided important information about the origins and accumulation of mutations with aging/maturation. The findings could have implications for understanding of human reproductive health. Compared to mice, we have a much longer amount of time before we begin reproducing and, in many societies, that time is increasing and reproduction is being delayed to later and later ages. Furthermore, the duplex sequencing method I optimized for single cells opens avenues for investigating low-frequency mutations in other studies.