Impact of De Novo Mutations and Mosaicism in Human Disease: Applications to Non-Invasive Diagnostic Techniques

Thanks to the systematic identification of genetic variants afforded by the advent of Next Generation Sequencing (NGS), it is now well recognised that de novo mutations (DNMs) are a significant contributor to human disease, affecting ~1:300 new births. However, there are currently no methods to prevent or predict which pregnancies will be affected. Implementation of non-invasive prenatal testing (NIPT) has provided a sensitive screening method for detection of fetal aneuploidy made possible by the 1997 discovery of cell-free fetal (cff) DNA in maternal blood. This project will aim to apply similar non-invasive diagnostic techniques to the detection of pathogenic DNMs, focusing on 2 specific applications:

1. Most (>80%) DNMs originate in the paternal germline during spermatogenesis explaining why the main risk factor for DNMs is the age of the father at conception. We have previously described a mechanism contributing to the paternal age-related increase in pathogenic DNMs called ‘selfish selection’, a process equivalent to neoplasia but occurring in the unique context of the male germ cell. These ‘selfish DNMs’ become enriched with age, due to clonal expansion of mutant spermatogonia over time. Fertilization of the egg by a mutant sperm leads to serious congenital disorders in the next generation, such as Apert syndrome (caused by FGFR2 mutations), achondroplasia (FGFR3), multiple endocrine neoplasia (RET), Noonan (PTPN11) and Costello syndromes (HRAS); collectively pathogenic selfish DNMs in these 5 genes alone account for ~1:5000 births. The 1st part of this project will aim to develop non-invasive prenatal screening approaches for the most commonly recurring selfish DNMs.

2. Although most DNMs are one-off events that occur during spermatogenesis, they can also originate through a process called ‘gonadal mosaicism’. In this case, the DNM would have arisen early during one of the parent’s development and will be present in multiple eggs or sperm, leading to an increased recurrence risk (as high as 50%). Hence it is essential to be able to single-out DNMs caused by gonadal mosaicism from the more common one-off events which have no risk of recurring in another child. This project will aim to develop a novel strategy to stratify families who have already had a child with a disorder caused by a DNM, in order to offer them an evidence-based recurrence risk estimate prior to the conception of another child and allow parents to make more informed and cost-effective reproductive decisions.

TRAINING OPPORTUNITIES

This project represents a unique opportunity to gain in-depth training in Human Genetics and general principles of development. It will combine the use of novel molecular technologies (such as molecular inversion probes and LockDown probes) with the implementation of next generation sequencing (Illumina, PacBio) targeted to the study of cell free fetal (cff) DNA, in order to develop approaches to the molecular analysis of rare DNMs. Training will be provided both in basic molecular biology (DNA extraction, PCR, sequencing, genotyping, haplotyping) as well as use in 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. It should be of particular value to individuals with an interest in clinical diagnosis, analysis of rare mutation, genomic mechanisms of disease, and application of state-of-the-art genomics technologies.

The laboratory already has a strong track record of success in rare mutation detection, so there is a good chance that these approaches will lead to the implementation of the strategies developed during the project into clinical practice.

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.