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.

The authors have declared no competing interest.

This work has been supported by the University of the Basque Country (UPV-EHU) (Spain) (IT1739-22) and by the Swiss National Science Foundation (320030-197725). IM is supported by a Postdoctoral Fellowship Grant from the Education Department of Basque Government (Spain). JA is supported by ASONMEC (Asociacion de Oncologia Medica del Hospital de Cruces) (Spain).

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The study was approved by the ethics committee for clinical research of Euskadi (CEIC-E), Spain

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