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Sex determination refers to the developmental processes by which the bipotential gonads develop as either testes or ovaries. Disorders of sexual development (DSD) are rare congenital conditions affecting more than 1 in 4500 newborns. Children with DSD face considerable challenges including surgical correction and gender assignment, as well as associated complications such as infertility and predisposition to gonadal tumors. Unfortunately, causative genetic mutations are found only in a minority of affected patients due to an incomplete understanding of the genetic programs and molecular pathways involved in sex determination and DSD.
We use state-of-the-art next generation sequencing and bioinformatics approaches to identify gene variants likely implicated in DSD patient phenotypes.
Our group described three new clinical entities leading to DSD in patients, the WNT4 deficiency (the Biason-Lauber syndrome), CBX2 defects and defects in the androgen backdoor pathway (1-3).
1. Biason-Lauber A, Konrad D, Meyer M, DeBeaufort C, Schoenle EJ. Ovaries and female phenotype in a girl with 46,XY karyotype and mutations in the CBX2 gene. American journal of human genetics 2009; 84:658-663
2. Biason-Lauber A, Konrad D, Navratil F, Schoenle EJ. A WNT4 mutation associated with Mullerian-duct regression and virilization in a 46,XX woman. The New England journal of medicine 2004; 351:792-798
3. Fluck CE, Meyer-Boni M, Pandey AV, Kempna P, Miller WL, Schoenle EJ, Biason-Lauber A. Why boys will be boys: two pathways of fetal testicular androgen biosynthesis are needed for male sexual differentiation. American journal of human genetics 2011; 89:201-218
Currently, around 50% of patients presenting with disorders or differences of sexual development (DSD), do not have a diagnosis. Not only is this a hindrance in the management of the cases, but it is also stressful for the families and the patients concerned. In order to identify the genetic and molecular basis of disease, Whole Exome Sequencing (WES) has become one of the foremost methods of choice. In collaboration with national and international partners, we gathered samples of a large cohort of patients with unexplained DSD (n > 150) and used WES to identify genetic variants. We are currently analyzing the WES data obtained in order to make connections between the phenotype and the genotype of the undiagnosed DSD patients and to gain novel insights into the underlying process of human sex determination and differentiation.
Human genome wide next generation sequencing (NGS) assays, such as whole exome sequencing (WES) have successfully identified thousands of variants in human disease. These insights could lead the way to breakthrough treatments; however, several challenges hinder progress, making innovative approaches to accelerate the follow-up of results from WES an urgent priority.
Mouse models are often not entirely appropriate for investigating the functional consequences of the identified variant. Together with more traditional approaches, such as cell-based studies, we exploit the largely untapped and rather unconventional potential of the fruit fly, Drosophila melanogaster, for functional investigation of findings from human WES (4,5).
This newly acquired expertise will appeal to both human geneticists seeking innovative strategies for experimental validation of findings from WES, as well as the Drosophila research community, by whom ongoing investigations of the implicated genes will powerfully inform our understanding of human disease.
Ivan Domenech Mercadè
4. Wangler MF, Hu Y, Shulman JM. Drosophila and genome-wide association studies: a review and resource for the functional dissection of human complex traits. Dis Model Mech 2017; 10:77-88
5. Weinberg-Shukron A, Renbaum P, Kalifa R, Zeligson S, Ben-Neriah Z, Dreifuss A, Abu-Rayyan A, Maatuk N, Fardian N, Rekler D, Kanaan M, Samson AO, Levy-Lahad E, Gerlitz O, Zangen D. A mutation in the nucleoporin-107 gene causes XX gonadal dysgenesis. The Journal of clinical investigation 2015; 125:4295-4304
Human sex development relies on differentiation of the gonads, in which the Sertoli and granulosa cells play a key role respectively for men and women. Many differences of sex development (DSD) are due to alteration of these two cell types. The study of the mechanisms underlying these conditions is crucial for optimal clinical management of DSDs. The primary collection of these cells is painful for the patient and their culture extremely difficult due to their short lifespan and the loss of their unique characteristics when cultured in vitro. Additionally, the available cell models cannot reproduce the mechanism leading to disease in single DSD patients.
Human induced-pluripotent stem cells (iPSCs) are developing as exciting cell sources for applications in regenerative medicine and drug discovery, primarily based on their extensive similarities to their human embryonic stem cell counterparts and shared properties of self-renewal and multilineage differentiation capabilities. iPSCs can be derived from somatic cells like fibroblasts, peripheral blood mononuclear cells (PBMCs) or urinary progenitors cells (UPs) via ectopic expression of transcription factors SOX2, OCT4, KLF4, NANOG, C-MYC and LIN28. Further differentiation into male (Sertoli-like) and female (granulosa-like) cells is obtained by exposure of the patient-derived iPSCs to sex-specific factors, such as FGF9 or estradiol.
Daniel Rodriguez-Gutierrez & Dirk Hart