Variants of STAR, AMH and ZFPM2/FOG2 May Contribute towards the Broad Phenotype Observed in 46,XY DSD Patients with Heterozygous Variants of NR5A1

Abstract

Variants of NR5A1 are often found in individuals with 46,XY disorders of sex development (DSD) and manifest with a very broad spectrum of clinical characteristics and variable sex hormone levels. Such complex phenotypic expression can be due to the inheritance of additional genetic hits in DSD-associated genes that modify sex determination, differentiation and organ function in patients with heterozygous NR5A1 variants. Here we describe the clinical, biochemical and genetic features of a series of seven patients harboring monoallelic variants in the NR5A1 gene. We tested the transactivation activity of novel NR5A1 variants. We additionally included six of these patients in a targeted diagnostic gene panel for DSD and identified a second genetic hit in known DSD-causing genes STAR, AMH and ZFPM2/FOG2 in three individuals. Our study increases the number of NR5A1 variants related to 46,XY DSD and supports the hypothesis that a digenic mode of inheritance may contribute towards the broad spectrum of phenotypes observed in individuals with a heterozygous NR5A1 variation.

Keywords: AMH; DSD; FOG2; NR5A1/SF1; STAR; disorder/difference of sex development; genotype–phenotype correlation; oligogenic disorders; steroidogenic factor 1.

Figure 1

Scheme of the structure of the NR5A1 protein and localization of studied variations. Critical functional domains comprise a DNA-binding domain (DBD) at the amino terminal (from amino acids 13 to 112) containing two zinc finger domains (ZNI and ZNII) and an A box, the flexible hinge region (amino acids 112–225) and a ligand-binding domain (LBD) (amino acids 225–458) with activation function 1 (AF1) and 2 (AF2). A close-up loop of the first zinc finger domain in DBD is shown where the His24Leu and Cys30Ser alterations are located. Locations of the NR5A1 variations described in this work are indicated by arrow heads.

Figure 2

Functional studies of the three novel NR5A1 variants. The ability of wild-type (WT) and mutant NR5A1 to activate promoter luciferase reporter constructs was tested in HEK293 cells (A–C). Cells were transiently transfected with NR5A1 expression vectors and promoter reporter constructs: 227CYP17A1_Δluc (A), -301HSD17B3_pGL3 (B) and -152CYP11A1_pGL3 (C) [7]. Results are shown as the mean ± standard error of the mean (SEM) of five independent experiments, all performed in duplicate. (D) Western blot showing expression of WT and mutant NR5A1/SF1 proteins. The HA antibody recognized hemagglutinin-tagged NR5A1/SF1 (band at 53 kDa). β-actin was used as a control (band at 42 kDa). ** p-value  ≤  0.01. HA, hemagglutinin; RLU, relative light units; Ve, empty vector; WT, wild type.