Disorders of sex development: genetic analysis and development of a novel in vitro cell model
AuthorKnarston, Ingrid May
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2021-03-26.
© 2018 Dr Ingrid May Knarston
Disorders/Differences of Sex Development (DSDs) are conditions where the chromosomal, anatomical or gonadal sex is atypical. DSDs are caused by a breakdown in the molecular pathways controlling development of the reproductive organs, such as ovarian/testicular differentiation. These conditions can carry a number of clinical complications such as an increased risk of gonadal cancer, infertility and psychosocial consequences. Importantly, the underlying genetic cause is still unknown in 60% of DSD patients, meaning clinical care is severely compromised. In the first part of this thesis, I studied a cohort of 34 patients with 46,XX (ovo)testicular DSDs. In these individuals, the testicular differentiation pathway is activated in 46,XX genetic females, resulting in the formation of testes or ovotestes. The cohort was studied using massively parallel sequencing and PCR-based approaches. This identified diagnostic findings in nine patients in two known DSD genes (NR5A1 and SOX9), as well as variants in candidate DSD genes (EMX2, FOXL2, LGR5, RXFP2 and WNT9A). In vitro analysis of the NR5A1 variants showed how these variants repress ovarian signalling pathways and factors, sufficient to switch ovarian to testicular development. In vitro and in vivo analyses of three of the candidate genes (EMX2, LGR5 and RXFP2) indicated that they are likely benign variants that don’t contribute to the phenotype. Ongoing studies of two further candidate genes (FOXL2 and WNT9A) will establish their potential role in these DSD phenotypes. In the second part of the thesis, I aimed to develop an improved in vitro model for functionally analysing DSD gene variants. Several recent studies have differentiated human induced pluripotent stem cells (iPSCs) into many different tissues, which can be used as human- and tissue-specific disease models. I developed a protocol to differentiate human iPSCs into testislike lineages. In this step-wise protocol, cells are directed through the developmental stages that give rise to the embryonic testis. Gene expression profiling has shown that at day 10-12 of iPSC differentiation, cells reach a bipotential gonad-like stage and by day 15 testis-like lineages are induced. This protocol will continue to be optimised, yet already I have shown its promising utility to study novel DSD genes. In summary, genetic analysis of a DSD cohort revealed diagnoses for a number of patients; these findings will likely improve their clinical management. It has also provided information on the most suitable genetic testing approach for 46,XX (ovo)testicular DSDs, a phenotypic group traditionally challenging to diagnose. Further, I showed functional insights into the molecular pathogenesis underlying NR5A1-mediated 46,XX (ovo)testicular DSD. Finally, development of a stem cell-based model of the human testis will help us to establish how novel DSD genes and variants affect human gonad development.
Keywordsgenomics; differences of sex development; testis development; induced pluripotent stem cells
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