Harnessing New Genomics Technologies to Assess Environmental Risk Factors That May Cause Heritable Genetic Disease

It is becoming increasingly apparent that heritable mutations play a major role in developmental disorders, yet we still know little about the role of environmental agents in the etiology of heritable mutations. This is because germ cell mutations are rare and difficult to detect. In this thesis, novel methods were developed to address data gaps in our understanding of mutation induction in sperm and offspring. To test the hypothesis that mutagens induce germline mutations that are inherited in the offspring, MutaMouse males were exposed to 100 mg/kg/day of benzo[a]pyrene (BaP), a known mutagen and common environmental pollutant, and mated to produce offspring. First, sperm from exposed males were analyzed for microsatellite mutations using a single-molecule amplification approach, and results confirmed that BaP induces microsatellite mutations. Second, the MutaMouse model, which uses a mutation reporting transgene, was adapted to facilitate mutation detection in sperm. Pairing this method with next-generation sequencing (NGS) revealed that BaP mutation spectrum in sperm differs from somatic tissues. Third, offspring of BaP-exposed males were screened for the induction of large copy number variations (CNVs). The quantity of CNVs in the BaP group was not significantly greater than controls; however, the types of CNVs were different, suggesting that BaP preferentially induces higher numbers of duplications. Fourth, genomes of the offspring were examined using NGS to detect all de novo mutations that were transmitted. Offspring of BaP-treated sires showed a 2-fold increase in genome-wide mutations compared to controls. Induced mutations observed in BaP-treated mice were consistent with the expected BaP mutation spectrum. Lastly, using data from cigarette smoke exposure studies, a common route of BaP exposure, the global impact of smoking on genetic disease burden (1.4 million aneuploidies, 2-8 million mutations) was estimated conservatively at 86 billion dollars per generation for intellectual disease alone. Overall, this work furthers our understanding of heritable mutagenesis and allows for predictions to be made on the genetic disease burden of human exposures. Furthermore, these developments will be useful for future analyses of germ cell mutagens, and will be important for making recommendations on future assessments of mutagenic hazards to the germline.