Characterization of 35 novel NR5A1/SF-1 variants identified in individuals with atypical sexual development: The SF1next study.

CONTEXT: Steroidogenic factor 1 (NR5A1/SF-1) is a nuclear receptor that regulates sex development, steroidogenesis and reproduction. Genetic variants in NR5A1/SF-1 are common among differences of sex development (DSD) and associate with a wide range of phenotypes, but their pathogenic mechanisms remain unclear. OBJECTIVE: Novel, likely disease-causing NR5A1/SF-1 variants from the SF1next cohort of individuals with DSD were characterized to elucidate their pathogenic effect. METHODS: Different in silico tools were used to predict the impact of novel NR5A1/SF-1 variants on protein function. An extensive literature review was conducted to compare and select the best functional studies for testing the pathogenic effect of the variants in a classic cell culture model. The missense NR5A1/SF-1 variants were tested on the promoter luciferase reporter vector -152CYP11A1_pGL3 in HEK293T cells and assessed for their cytoplasmic/nuclear localization by Western blot. RESULTS: Thirty-five novel NR5A1/SF-1 variants were identified in the SF1next cohort. Seventeen missense NR5A1/SF-1 variants were functionally tested. Transactivation assays showed reduced activity for 40% of the variants located in the DNA binding domain and variable activity for variants located elsewhere. Translocation assessment revealed three variants (3/17) with affected nuclear translocation. No clear genotype-phenotype, structure-function correlation was found. CONCLUSIONS: Genetic analyses and functional assays do not explain the observed wide phenotype of individuals with these novel NR5A1/SF-1 variants. In nine individuals, additional likely disease-causing variants in other genes were found, strengthening the hypothesis that the broad phenotype of DSD associated with NR5A1/SF-1 variants may be caused by an oligogenic mechanism.

Clinical and genetic characteristics of a large international cohort of individuals with rare NR5A1/SF-1 variants of sex development.

BACKGROUND: Steroidogenic factor 1 (SF-1/NR5A1) is essential for human sex development. Heterozygous NR5A1/SF-1 variants manifest with a broad range of phenotypes of differences of sex development (DSD), which remain unexplained. METHODS: We conducted a retrospective analysis on the so far largest international cohort of individuals with NR5A1/SF-1 variants, identified through the I-DSD registry and a research network. FINDINGS: Among 197 individuals with NR5A1/SF-1 variants, we confirmed diverse phenotypes. Over 70% of 46, XY individuals had a severe DSD phenotype, while 90% of 46, XX individuals had female-typical sex development. Close to 100 different novel and known NR5A1/SF-1 variants were identified, without specific hot spots. Additionally, likely disease-associated variants in other genes were reported in 32 individuals out of 128 tested (25%), particularly in those with severe or opposite sex DSD phenotypes. Interestingly, 48% of these variants were found in known DSD or SF-1 interacting genes, but no frequent gene-clusters were identified. Sex registration at birth varied, with <10% undergoing reassignment. Gonadectomy was performed in 30% and genital surgery in 58%. Associated organ anomalies were observed in 27% of individuals with a DSD, mainly concerning the spleen. Intrafamilial phenotypes also varied considerably. INTERPRETATION: The observed phenotypic variability in individuals and families with NR5A1/SF-1 variants is large and remains unpredictable. It may often not be solely explained by the monogenic pathogenicity of the NR5A1/SF-1 variants but is likely influenced by additional genetic variants and as-yet-unknown factors. FUNDING: Swiss National Science Foundation (320030-197725) and Boveri Foundation Zürich, Switzerland.

Genetic reanalysis of patients with a difference of sex development carrying the NR5A1/SF-1 variant p.Gly146Ala has discovered other likely disease-causing variations.

NR5A1/SF-1 (Steroidogenic factor-1) variants may cause mild to severe differences of sex development (DSD) or may be found in healthy carriers. The NR5A1/SF-1 c.437G>C/p.Gly146Ala variant is common in individuals with a DSD and has been suggested to act as a susceptibility factor for adrenal disease or cryptorchidism. Since the allele frequency is high in the general population, and the functional testing of the p.Gly146Ala variant revealed inconclusive results, the disease-causing effect of this variant has been questioned. However, a role as a disease modifier is still possible given that oligogenic inheritance has been described in patients with NR5A1/SF-1 variants. Therefore, we performed next generation sequencing (NGS) in 13 DSD individuals harboring the NR5A1/SF-1 p.Gly146Ala variant to search for other DSD-causing variants and clarify the function of this variant for the phenotype of the carriers. Panel and whole-exome sequencing was performed, and data were analyzed with a filtering algorithm for detecting variants in NR5A1- and DSD-related genes. The phenotype of the studied individuals ranged from scrotal hypospadias and ambiguous genitalia in 46,XY DSD to opposite sex in both 46,XY and 46,XX. In nine subjects we identified either a clearly pathogenic DSD gene variant (e.g. in AR) or one to four potentially deleterious variants that likely explain the observed phenotype alone (e.g. in FGFR3, CHD7). Our study shows that most individuals carrying the NR5A1/SF-1 p.Gly146Ala variant, harbor at least one other deleterious gene variant which can explain the DSD phenotype. This finding confirms that the NR5A1/SF-1 p.Gly146Ala variant may not contribute to the pathogenesis of DSD and qualifies as a benign polymorphism. Thus, individuals, in whom the NR5A1/SF-1 p.Gly146Ala gene variant has been identified as the underlying genetic cause for their DSD in the past, should be re-evaluated with a NGS method to reveal the real genetic diagnosis.

Oligogenic Causes of Human Differences of Sex Development: Facing the Challenge of Genetic Complexity.

BACKGROUND: Deviations of intrauterine sex determination and differentiation and postnatal sex development can result in a very heterogeneous group of differences of sex development (DSD) with a broad spectrum of phenotypes. Variants in genes involved in sexual development cause different types of DSD, but predicting the phenotype from an individual's genotype and vice versa remains challenging. SUMMARY: Next Generation Sequencing (NGS) studies suggested that oligogenic inheritance contributes to the broad manifestation of DSD phenotypes. This review will focus on possible oligogenic inheritance in DSD identified by NGS studies with a special emphasis on NR5A1variants as an example of oligogenic origin associated with a broad range of DSD phenotypes. We thoroughly searched the literature for evidence regarding oligogenic inheritance in DSD diagnosis with NGS technology and describe the challenges to interpret contribution of these genes to DSD phenotypic variability and pathogenicity. KEY MESSAGES: Variants in common DSD genes like androgen receptor (AR), mitogen-activated protein kinase kinase kinase 1 (MAP3K1), Hydroxy-Delta-5-Steroid Dehydrogenase 3 Beta- And Steroid Delta-Isomerase 2 (HSD3B2), GATA Binding Protein 4 (GATA4), zinc finger protein friend of GATA family member 2 (ZFPM2), 17b-hydroxysteroid dehydrogenase type 3 (HSD17B3), mastermind-like domain-containing protein 1 (MAMLD1), and nuclear receptor subfamily 5 group A member 1 (NR5A1) have been detected in combination with additional variants in related genes in DSD patients with a broad range of phenotypes, implying a role of oligogenic inheritance in DSD, while still awaiting proof. Use of NGS approach for genetic diagnosis of DSD patients can reveal more complex genetic traits supporting the concept of oligogenic cause of DSD. However, assessing the pathomechanistic contribution of multiple gene variants on a DSD phenotype remains an unsolved conundrum.