Analysis of De Novo Mutations in the Human Testis

It is now well recognised that de novo mutations (DNM) are important contributors to human disease. The vast majority of DNMs occur in the male germline during spermatogenesis because, unlike female gametes that do not divide after birth, the production of sperm relies on regular divisions of spermatogonial stem cells – making them more prone to accumulate DNA copy-errors as men age. Hence, the human testis represents a ‘repository’ of DNMs that can be exploited to study the process by which we all acquire new mutations at each generation.

Work in our lab has revealed a novel mechanism (called ‘selfish selection’) causing the recurrence of specific human genetic diseases, whereby the causative “selfish” mutations confer a selective advantage to spermatogonial stem cells or progenitors in the adult testis. Current data indicate that mutations affecting pathways involved in spermatogonial proliferation/survival in the testis “hijack” the normal homeostatic mechanisms to their own advantage. We have been able to link this process to the origin of testicular tumours and suggested that the mechanisms involved in selfish selection are similar to those described for tumorigenesis, but have long term implications because they are transmitted across generations .

The aim of this project will be to identify novel genes and pathways that are subject to selfish selection, by identifying rare DNMs that are enriched in the testes of elderly men. The strategy exploits the prediction that when selfish mutations occur, they will be locally enriched through clonal expansion, effectively generating a localised ‘hotspot’ of cells carrying the mutation. In hotspot regions, mutations will be present at levels in the order of ~0.1-1% and can therefore be directly detected using state-of-the-art capture techonologies and ultra-deep next-generation sequencing. Dissection of a whole human testis into discrete portions (or by separating whole tubules, the developmental units of the testis), followed by analysis of a large panel of >100 candidate genes should define the landscape of selfish mutations within the human testis and the potential impact that this process has on human disease and genome evolution.

TRAINING OPPORTUNITIES

This technically challenging project provides a unique opportunity for in-depth training in human genetics and genomics technologies; it will combine the use of novel molecular techniques (such as RainDance, molecular inversion probes, HaloPlex, LockDown probes) with ultra-deep next generation sequencing (Illumina). Once candidate genes have been identified, we will use different PCR-based technologies to validate these findings by directly measuring the levels of the associated mutations in a large number of sperm samples of men of different ages. Training will be provided both in basic molecular biology (DNA extraction, PCR, sequencing, genotyping) as well as the use of advanced technologies such as those listed above. A significant portion of the project will involve development of bioinformatic pipelines and statistical analysis, for which training will also be provided. It should be of particular value to individuals with an interest in mutation detection, genomic mechanisms of disease, genome biology and the application of state-of-the-art genomics technologies.

As well as the specific training detailed above, students will have access to a wide-range of seminars and training opportunities through the many research institutes and centres based in Oxford. Students are also able to attend the Methods and Techniques course run by the MRC Weatherall Institute of Molecular Medicine. This course runs through the year, ensuring that students have the opportunity to build a broad-based understanding of differing research techniques.

Generic skills training is offered through the Medical Sciences Division’s Skills Training Programme. This programme offers a comprehensive range of courses covering many important areas of researcher development: knowledge and intellectual abilities, personal effectiveness, research governance and organisation, and engagement, influence and impact. Students are actively encouraged to take advantage of the training opportunities available to them.

The department has a successful mentoring scheme, open to graduate students, which provides an additional possible channel for personal and professional development outside the regular supervisory framework. We hold an Athena SWAN Silver Award in recognition of our efforts to support the careers of female students and staff.