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

Abstract

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.

Fig 1. Algorithm of genetic workup.

A. Mode of genetic analysis, e.g. panel and whole exome sequencing (WES). Two patients were identified with pathogenic variants in LHCGR and AR by panel analysis and were not further analyzed by whole-exome sequencing (WES). B. Filtering algorithm of genetic data. Steps used for variant filtering after WES of eleven DSD patients harboring the NR5A1/SF-1 p.Gly146Ala variant are depicted (letters a to f). Final candidates and their possible impact are listed and characterized in Table 2 and S3 Table.

Fig 2. External genital phenotype of the 13 DSD patients harboring the NR5A1/SF-1 p.Gly146Ala variant shown with respect to their karyotype.

Fig 3. Genetic variants identified in 13 DSD patients harboring the NR5A1/SF-1 p.Gly146Ala variant illustrated with respect to the known pathways of male and female sex determination and differentiation.

The scheme shows an overview of involved genes and their currently assumed relationship to sexual development. Genes with variants identified by whole exome sequencing in the patients have specific colors. In dark blue: Candidate genes in patient 1; in brown: Candidate genes in patient 3; in green: Candidate genes in patient 6; in yellow: Candidate genes in patient 8; in red: Candidate genes in patient 9; in pink: Candidate genes in patient 10; in light blue: Candidate genes in patient 11; in purple: Candidate genes in patient 12; in orange: Candidate genes in patient 13; in dark grey: Known genes involved in sexual development. Interrogation mark (?): Function/timing/location is not clear; arrows: Activation; inhibitors: Inhibition; lines: Interaction/partnership; dashed lines/arrows: Hormone production.