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The role of functional variants in the aetiology of polycystic ovary syndrome

Smith, Rebecca (2017) The role of functional variants in the aetiology of polycystic ovary syndrome. Honours thesis, Murdoch University.

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Embargoed until August 2018.


Polycystic ovary syndrome (PCOS) is the most common endocrinopathy affecting reproductive aged women, as well as being the leading cause of anovulatory infertility. The syndrome is characterised by hyperandrogenism, anovulation, an overabundance of preovulatory follicles within the ovary and insulin resistance. Sufferers also have an increased risk of developing type 2 diabetes, obesity, endometrial cancer, and mental health sequalae.

It is well understood that PCOS has a genetic component with a heterogeneous mode of inheritance, as is evident from familial and population studies. However, despite the discovery of a number of risk alleles via genome wide association studies (GWAS), the genetic aetiology of the syndrome remains largely unknown. An alternative approach to GWAS for identifying mutations involved in genetic diseases is via whole exome sequencing. This method detects functional variants (i.e. mutations occurring in protein coding genes) that are present within the exome and are segregating within affected families. Therefore, undertaking exome sequencing on PCOS patients has the potential to identify genetic lesions involved in the aetiology of the syndrome.

The overarching aim for this project was to increase understanding of the aetiology of PCOS by identifying functional variants within families afflicted with PCOS using exome sequencing. To achieve this, bioinformatic analysis was performed on exome sequencing data files collected before the commencement of this project by the Keogh Institute. The functional variant rs143321413 within the eukaryotic elongation factor kinase 2 (EEF2K) gene was identified within one of the exome sequenced families affected by PCOS. The possible functional role of the rs143321413 variant was then investigated using an in vitro model system. This was done by producing mammalian expression vectors containing wildtype and mutant EEF2K sequences. These vectors were then transfected into a human embryonic kidney cell line containing the SV40 large T antigen (HEK293FT cells). Protein was extracted and a co-immunoprecipitation was performed to determine whether rs143321413 impacts the ability of EEF2K to bind to its substrate. Lastly, exome sequencing was also carried out on additional women affected by PCOS who were newly recruited throughout this project to identify new functional variants for future study.

The main finding from this thesis was the discovery of the novel rs143321413 variant, which has the potential to be involved with PCOS. Mammalian expression vectors containing both wildtype EEF2K and mutant EEF2K sequences were successfully created. Additionally, optimisation of the protocol was accomplished for transfection reactions to induce expression of these vectors within HEK293FT cells.Both of these experimental achievements will aid in the future research of rs143321413 and by extension PCOS. Finally, two additional variants were identified within the data from the newly exome sequenced individuals, that have the potential to be involved in aetiology of PCOS.

Publication Type: Thesis (Honours)
Murdoch Affiliation: School of Veterinary and Life Sciences
Supervisor: Wilson, S., Stuckey, B. and Mead, Robert
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