Direct Quantification of Germline Mosaicism in Humans

Congenital pediatric disease is often caused by mutations that are not inherited from the parents, but appear sporadically in the genome of the child. These diseases include life- threatening conditions, such as congenital heart disease; but also chronic fatal and non-fatal diseases, such as muscular dystrophies, intellectual disabilities, or autism spectrum disorders. The emotional and economic cost of these conditions on the affected children, families, and society is exceptionally high. Although our current knowledge and technologies have made rapid progress in identifying such mutations in affected individuals, our ability to predict and prevent them is falling short of our ambitions. While we often assume that these mutations happen just before conception, in reality, the vast majority arise in the germline of the father or the mother. In some cases, they originate during the embryonic development of the parents themselves, which results in a large proportion of cells having the exact same mutation as the one causing a disorder in the child. Consequently, this results in a higher risk of passing the same mutation on to a second child. As this phenomenon is often restricted to the germline, this risk cannot be assessed by analyzing the blood, saliva, or other somatic tissue of the parents. We previously demonstrated that the occurrence of such germline-restricted mutations has been underestimated and contributes to recurrence risk in children with autism-related disorders. More importantly, analysis of sperm can provide an accurate risk assessment for affected families and may have the potential to determine risk for certain mutations for prospective fathers. For this proposed project, we want to extend this approach to understand the basis of germline mosaicism, the phenomenon of having a specific mutation in some but not all cells of a tissue. To this end, we have chosen two complementary approaches. 1) We have collected healthy control sperm that will be analyzed by novel, advanced sequencing techniques that will allow the detection of mosaic mutations across the entire genome. This will allow us to understand the rates and extent of germline mosaicism in healthy men, informing the basal risk of recurrence for any sporadic mutation. 2) We will collect ovaries from patients with surgically indicated ovariectomies to isolate primary oocytes. We will subject these to the same analysis as for the male sperm. Taken together, these approaches will allow us unprecedented insights into the genesis and origin of mosaic mutations in the germline, both in males and females. This information will not only provide a fascinating window into this aspect of biology, but also help us to design future diagnostic tools and approaches that might reduce the impact of sporadic mutations on human health.