NR5A1 gene variants repress the ovarian-specific WNT signaling pathway in 46,XX disorders of sex development patients

Several recent reports have described a missense variant in the gene NR5A1 (c.274C>T; p.Arg92Trp) in a significant number of 46,XX ovotesticular or testicular disorders of sex development (DSDs) cases. The affected residue falls within th

Disorders of sex development : insights from targeted gene sequencing of a large international patient cohort

Background: Disorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously. Results: We analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46, XY DSD and 48 with 46, XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46, XY DSD. In patients with 46, XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management. Conclusions: Our massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes

Disorders of sex development: genetic analysis and development of a novel in vitro cell model

© 2018 Dr Ingrid May KnarstonDisorders/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

Disorders of sex development: Insights from targeted gene sequencing of a large international patient cohort

Disorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously. Results: We analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46,XY DSD and 48 with 46,XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46,XY DSD. In patients with 46,XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management. Conclusions: Our massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes

Author Correction: Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9

SRY and its target SOX9 are known key determinants in testis development. Here the authors by studying duplications and deletions upstream of SOX9 from patient samples with disorders of sex development (DSD) reveal enhancers for SOX9 critical for human sex development and DSD

Disorders of sex development: insights from targeted gene sequencing of a large international patient cohort

Background: Disorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously. Results: We analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46,XY DSD and 48 with 46,XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46,XY DSD. In patients with 46,XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management. Conclusions: Our massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes

Additional file 2: Figure S1. of Disorders of sex development: insights from targeted gene sequencing of a large international patient cohort

DSD gene variants in different global regions. DSD gene variants among the international cohort of 46,XY DSD patients. For ease of analysis, countries were grouped together into regions: Asia comprises Indonesia (97), Pakistan (25), Vietnam (35), Cambodia (16), India (1), a total of 174 patients ; Europe comprises the Netherlands (38), Austria (15), Belgium (6), and Italy (2), a total of 61 patients; and AUS & NZL comprises Australia (83) and New Zealand (7), a total of 90 patients. All curated variants are shown; those which have been curated and called pathogenic, likely pathogenic, and VUS. In the cohort from Asia, 35% of the patients were found to have a diagnostic variant (pathogenic or likely pathogenic), while this was 44% for Europe and 45% for AUS/NZL. Two patients from Canada were not included in the diagram. (PPTX 158 kb

Disorders of sex development: Insights from targeted gene sequencing of a large international patient cohort

Background: Disorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively