Probing positive selection in the human male germline

Thesis

De novo mutations (DNMs) are a significant contributor to genetic disease, accounting for over 40% of developmental disorders (DDs). The majority of DNMs are of paternal origin, and the number of transmitted DNMs increases linearly with paternal age. For a subset of DNMs associated with a defined group of dominant disorders, the relationship is nonlinear. Mutations can occur up to 1000-fold above the genome-wide rate and the incidence of disease increases supralinearly with paternal age. These mutations have been shown to arise spontaneously in spermatogonial stem cells (SSCs) and confer a selective advantage by hijacking critical signalling pathways involved in SSC regulation. Over time, through a process termed selfish spermatogonial selection, mutant SSCs clonally expand along the seminiferous tubules in which they originate, resulting in elevated mutation levels.

To date, all known selfish mutations occur in 12 genes encoding components of the receptor tyrosine kinase (RTK)-Ras signaling pathway. As positive selection is not anticipated to be limited to RTK-Ras dysregulation, this thesis investigated whether other mutations, genes, or pathways might also be subject to selfish behaviour.

To identify candidate selfish mutations for experimental inquiry, it was hypothesized that positive selection would manifest as DNM recurrence in clinically affected populations. Recurrent DNMs identified in a study of >31,000 parent-offspring trios were systematically evaluated to determine the most promising candidates, including double immunofluorescent labeling of testis sections for select candidate genes with established spermatogonial markers.

Following the prioritization of candidate mutations across 10 candidate genes, rhAmpSeq was adapted to perform a discovery screen and investigate whether these mutations were detectable in the testes of aged men. To improve assay sensitivity, unique molecular identifiers (UMIs) were incorporated, and their utility was assessed. This study detected variants in several novel genes, including those functioning outside of the RTK-Ras pathway.

Among these were two distinct variants in SMAD4 associated with Myhre syndrome (MS). To assess whether MS SMAD4 mutations are positively selected in bulk sperm, an ultra-sensitive technique was adapted to quantify MS mutations at the p.I500 codon. MS SMAD4 mutations at p.I500 were found to be elevated in sperm but not in blood, and their frequency increased with donor age, consistent with positive selection.

Taken together, this work provides direct evidence that selfish spermatogonial selection can be driven by genes operating outside of the canonical RTK-Ras pathway and expands the known landscape of positively selected genes in the male germline.

Authors: Tong, RS

• DOI: 10.5287/ora-g7z8m2ybw
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English