<b><i>Background:</i></b> 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. <b><i>Summary:</i></b> 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 <i>NR5A1</i>variants 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. <b><i>Key Messages:</i></b> Variants in common DSD genes like androgen receptor (<i>AR</i>), mitogen-activated protein kinase kinase kinase 1 (<i>MAP3K1</i>), Hydroxy-Delta-5-Steroid Dehydrogenase 3 Beta- And Steroid Delta-Isomerase 2 (<i>HSD3B2</i>), GATA Binding Protein 4 (<i>GATA4</i>), zinc finger protein friend of GATA family member 2 (<i>ZFPM2</i>), 17b-hydroxysteroid dehydrogenase type 3 (<i>HSD17B3</i>), mastermind-like domain-containing protein 1 (<i>MAMLD1</i>), and nuclear receptor subfamily 5 group A member 1 (<i>NR5A1</i>) 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.

Steroidogenic factor 1 (SF-1, NR5A1) plays an important role in human sex development. Variants of NR5A1/SF-1 may cause mild to severe differences of sex development (DSD) or may be found in healthy carriers. So far, the broad DSD phenotypic variability associated NR5A1/SF-1 variants remains a conundrum. The NR5A1/SF-1 variant c.437G>C/p.Gly146Ala is common in individuals with a DSD and has been suggested to act as a susceptibility factor for adrenal disease or cryptorchidism. However, as the allele frequency in the general population is high, and as functional testing of the p.Gly146Ala variant in vitro revealed inconclusive results, the disease-causing effect of this variant has been questioned. However, a role as a disease modifier in concert with other gene variants is still possible given that oligogenic inheritance has been described in patients with NR5A1/SF-1 gene variants. Therefore, we performed next generation sequencing in DSD individuals harboring the NR5A1/SF-1 p.Gly146Ala variant to search for other DSD-causing variants. Aim was to clarify the function of this variant for the phenotype of the carriers. We studied 14 pediatric DSD individuals who carried the p.Gly146Ala variant. Panel and whole-exome sequencing was performed, and data were analyzed with a specific data 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 typical male external genitalia and ovotestes in 46,XX DSD patients. Patients were of African, Spanish, and Asian origin. Of the 14 studied subjects, five were homozygous and nine heterozygous for the NR5A1/SF-1 p.Gly146Ala variant. In ten subjects we identified either a clearly pathogenic DSD gene variant (e.g. in AR, LHCGR) or one to four potentially deleterious variants that likely explain the observed phenotype alone (e.g. in FGFR3, CHD7, ADAMTS16). 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 p.Gly146Ala variant of NR5A1/SF-1 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 next-generation sequencing method to reveal the real genetic diagnosis.

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.