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 the DNA-binding domain of the NR5A1 protein, however the exact mechanism by which it causes testicular development in 46,XX individuals remains unclear. We have screened a cohort of 26 patients with 46,XX (ovo)testicular DSD and identified three unrelated individuals with this NR5A1 variant (p.Arg92Trp), as well as one patient with a novel NR5A1 variant (c.779C>T; p.Ala260Val). We examined the functional effect of these changes, finding that while protein levels and localization were unaffected, variant NR5A1 proteins repress the WNT signaling pathway and have less ability to upregulate the anti-testis gene NR0B1. These findings highlight how NR5A1 variants impact ovarian differentiation across multiple pathways, resulting in a switch from ovarian to testis development in genetic females.

Molecular mechanisms associated with 46,XX disorders of sex development.

In the female gonad, distinct signalling pathways activate ovarian differentiation while repressing the formation of testes. Human disorders of sex development (DSDs), such as 46,XX DSDs, can arise when this signalling is aberrant. Here we review the current understanding of the genetic mechanisms that control gonadal development, with particular emphasis on those that drive or inhibit ovarian differentiation. We discuss how disruption to these molecular pathways can lead to 46,XX disorders of ovarian development. Finally, we look at recently characterized novel genes and pathways that contribute and speculate how advances in technology will aid in further characterization of normal and disrupted human ovarian development.

Democratizing clinical-genomic data: How federated platforms can promote benefits sharing in genomics.

Since the first sequencing of the human genome, associated sequencing costs have dramatically lowered, leading to an explosion of genomic data. This valuable data should in theory be of huge benefit to the global community, although unfortunately the benefits of these advances have not been widely distributed. Much of today's clinical-genomic data is siloed and inaccessible in adherence with strict governance and privacy policies, with more than 97% of hospital data going unused, according to one reference. Despite these challenges, there are promising efforts to make clinical-genomic data accessible and useful without compromising security. Specifically, federated data platforms are emerging as key resources to facilitate secure data sharing without having to physically move the data from outside of its organizational or jurisdictional boundaries. In this perspective, we summarize the overarching progress in establishing federated data platforms, and highlight critical considerations on how they should be managed to ensure patient and public trust. These platforms are enabling global collaboration and improving representation of underrepresented groups, since sequencing efforts have not prioritized diverse population representation until recently. Federated data platforms, when combined with advances in no-code technology, can be accessible to the diverse end-users that make up the genomics workforce, and we discuss potential strategies to develop sustainable business models so that the platforms can continue to enable research long term. Although these platforms must be carefully managed to ensure appropriate and ethical use, they are democratizing access and insights to clinical-genomic data that will progress research and enable impactful therapeutic findings.

An In Vitro Differentiation Protocol for Human Embryonic Bipotential Gonad and Testis Cell Development.

Currently an in vitro model that fully recapitulates the human embryonic gonad is lacking. Here we describe a fully defined feeder-free protocol to generate early testis-like cells with the ability to be cultured as an organoid, from human induced pluripotent stem cells. This stepwise approach uses small molecules to mimic embryonic development, with upregulation of bipotential gonad markers (LHX9, EMX2, GATA4, and WT1) at day 10 of culture, followed by induction of testis Sertoli cell markers (SOX9, WT1, and AMH) by day 15. Aggregation into 3D structures and extended culture on Transwell filters yielded organoids with defined tissue structures and distinct Sertoli cell marker expression. These studies provide insight into human gonadal development, suggesting that a population of precursor cells may originate from a more lateral region of the mesoderm. Our protocol represents a significant advance toward generating a much-needed human gonad organoid for studying disorders/differences of sex development.

Analysis of NR5A1 in 142 patients with premature ovarian insufficiency, diminished ovarian reserve, or unexplained infertility.

Ovarian deficiency, including diminished ovarian reserve and premature ovarian insufficiency, represents one of the main causes of female infertility. Little is known of the genetic basis of diminished ovarian reserve, while premature ovarian insufficiency often has a genetic basis, with genes affecting various processes. NR5A1 is a key gene required for gonadal function, and variants are associated with a wide phenotypic spectrum of disorders of sexual development, and are found in 0.26-8% of patients with premature ovarian insufficiency. As there is some debate about the extent of involvement of NR5A1 in the pathogenesis of ovarian deficiency, we performed an in-depth analysis of NR5A1 variants detected in a cohort of 142 patients with premature ovarian insufficiency, diminished ovarian reserve, or unexplained infertility associated with normal ovarian function. We identified rare non-synonymous protein-altering variants in 2.8 % of women with ovarian deficiency and no such variants in our small cohort of women with infertility but normal ovarian function. We observed previously reported variants associated with premature ovarian insufficiency in patients with diminished ovarian reserve, highlighting a genetic relationship between these conditions. We confirmed functional impairment resulting from a p.Val15Met variant, detected for the first time in a patient with premature ovarian insufficiency. The remaining variants were associated with preserved transcriptional activity and localization of NR5A1, indicating that rare NR5A1 variants may be incorrectly curated if functional studies are not undertaken, and/or that NR5A1 variants may have only a subtle impact on protein function and/or confer risk of ovarian deficiency via oligogenic inheritance.

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.

Disorders of sex development (DSDs) are conditions affecting development of the gonads or genitalia. Variants in two key genes, SRY and its target SOX9, are an established cause of 46,XY DSD, but the genetic basis of many DSDs remains unknown. SRY-mediated SOX9 upregulation in the early gonad is crucial for testis development, yet the regulatory elements underlying this have not been identified in humans. Here, we identified four DSD patients with overlapping duplications or deletions upstream of SOX9. Bioinformatic analysis identified three putative enhancers for SOX9 that responded to different combinations of testis-specific regulators. All three enhancers showed synergistic activity and together drive SOX9 in the testis. This is the first study to identify SOX9 enhancers that, when duplicated or deleted, result in 46,XX or 46,XY sex reversal, respectively. These enhancers provide a hitherto missing link by which SRY activates SOX9 in humans, and establish SOX9 enhancer mutations as a significant cause of DSD.

Comparative assessment of DNA methylation patterns between reduced representation bisulfite sequencing and Sequenom EpiTyper methylation analysis.

AIM: Validation of sequencing-based DNA methylation data is an important step for meaningful translation of findings. However, there has been limited assessment of different platforms to validate methylation data from next generation sequencing. METHODS: We performed a comparative methylation analysis between the genome-wide platform of reduced representation bisulfite sequencing with a targeted, Sequenom EpiTyper platform (four genes were analyzed in 15 cell lines covering 52 CpG sites). RESULTS: We show that the accuracy of validation substantially improves if results from multiple and adjacent CpG sites are combined rather than at single CpG sites. We demonstrate increased read number improves accuracy of reduced representation bisulfite sequencing results. Further, by using series of replicates, we document variation in samples analyzed by Sequenom EpiTyper, indicating the importance of including replicates to increase precision. CONCLUSION: The results reveal potential sources of bias and provide a guideline for refining study design for DNA methylation analysis.

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.