Mutational analysis of compound heterozygous mutation p.Q6X/p.H232R in SRD5A2 causing 46,XY disorder of sex development

Abstract

Background: Over 100 mutations in the SRD5A2 gene have been identified in subjects with 46,XY disorder of sex development (DSD). Exploration of SRD5A2 mutations and elucidation of the molecular mechanisms behind their effects should reveal the functions of the domains of the 5α-reductase 2 enzyme and identify the cause of 46,XY DSD. Previously, we reported a novel compound heterozygous p.Q6X/p.H232R mutation of the SRD5A2 gene in a case with 46,XY DSD. Whether the compound heterozygous p.Q6X/p.H232R mutation in this gene causes 46,XY DSD requires further exploration.

Methods: The two 46,XY DSD cases were identified and sequenced. In order to identify the source of the compound heterozygous p.Q6X/p.H232R mutation, the parents, maternal grandparents, and maternal uncle were sequenced. Since p.Q6X mutation is a nonsense mutation, p.H232R mutation was transfected into HEK293 cells and dihydrotestosterone (DHT) production were analyzed by liquid chromatography-mass spectrometry (LC-MS) for 5α-reductase 2 enzyme activities test. Apparent michaelis constant (Km) were measured of p.H232R mutation to analyze the binding ability change of 5α-reductase 2 enzyme with testosterone (T) or NADPH.

Results: The sequence results showed that the two 46,XY DSD cases were the compound heterozygous p.Q6X/p.H232R mutation, of which the heterozygous p.Q6X mutation originating from maternal family and heterozygous p.H232R mutation originating from the paternal family. The function analysis confirmed that p.H232R variant decreased the DHT production by LC-MS test. The Km analysis demonstrated that p.H232R mutation affected the binding of SRD5A2 with T or NADPH.

Conclusions: Our findings confirmed that the compound heterozygous p.Q6X/p.H232R mutation in the SRD5A2 gene is the cause of 46,XY DSD. p.H232R mutation reduced DHT production while attenuating the catalytic efficiency of the 5α-reductase 2 enzyme.

Keywords: 46,XY DSD; 5α-reductase 2 catalytic efficiency; Dihydrotestosterone; SRD5A2; p.Q6X/p.H232R mutation.

Fig. 1

Clinical features of the case 1 (III-1). a. B-ultrasound analysis of the case’s internal reproductive organs showed the absence of a uterus and ovaries, but testes on the left labia and right groin. b. Karyotype analysis revealed that the case’s karyotype was 46,XY. c. Pedigree of the case’s family; Males, females and the patient are indicated by squares, circles, filled circle, respectively. the case 1 (III-1) and her sister (case 2, III-2) were 46,XY DSD patients, while the other relatives did not suffer from 46,XY DSD. d. Sequencing analysis of the SRD5A2 gene. The heterozygous mutation 16C > T found in the patient lead to a stop codon. e. Sequencing analysis of the SRD5A2 gene. The heterozygous mutation 695A > G was found, causing amino acid 232 to change from histidine to arginine. Red arrows indicate mutated nucleotide

Fig. 2

Clinical features of the case 2 (III-2). a. Prenatal B-ultrasound testing of the case 2 at 21 and 25 weeks revealed no abnormalities. b. Chromosome karyotype analysis showed a normal 46, XY karyotype. c. Fluorescence in situ hybridization (FISH) prenatally diagnosed 13/16/18/21/22/X/Y chromosomes in the case 2. d. The physical examination of the fetus aborted at 25 weeks’ gestation displayed female external genitalia, but with blind ending vagina. e. HE staining showed the fetus had epididymis tissue and testis tissue. f. Sequencing analysis of the SRD5A2 gene and the p.Q6X (c.16C > T) mutation were found. g. Sequencing analysis of the SRD5A2 gene and the p.H232R (c.695A > G) mutation were found. Red arrows indicate mutated nucleotide

Fig. 3

Sequence analysis of SRD5A2 in the cases’ father II-1, mother II-2 and younger sister III-3. a. The heterozygous mutation p.Q6X (c.16C > T) in SRD5A2 gene was only found in their mother, not found in their father and younger sister. b. The heterozygous mutation p.H232R (c.695A > G) in SRD5A2 gene was found in their patient's father and younger sister, not found in their mother. Red arrows indicate mutated nucleotide. Green arrows indicate unmutated nucleotide

Fig. 4

Sequencing analysis of SRD5A2 gene in the cases’ grandfather I-1, grandmother I-2 and maternal uncle II-3. a. The heterozygous mutation p.Q6X (c.16C > T) in SRD5A2 gene was found in the cases’ grandfather and maternal uncle, but not found in their grandmother. b. The heterozygous mutation p.H232R (c.695A > G) in SRD5A2 gene was not found in the cases’ grandfather, grandmother and maternal uncle. Red arrows: mutated nucleotides. Green arrows: unmutated nucleotides

Fig. 5

Validation of SRD5A2 wild-type (WT) and p.H232R mutant HEK293 cell models. a. The HEK293 cells were transiently transfected with WT or p.H232R (c.695A > G) mutant SRD5A2 plasmids. Sequencing analysis showed the mutation c.695A > G in SRD5A2 in HEK-293 cells transfected with p.H232R mutant plasmids, causing amino acid 232 to change from histidine to arginine. b. The transcription of the SRD5A2 gene in HEK-293 cells transfected with WT and p.H232R mutant plasmids using qRT-PCR. The β-actin gene was used as an internal control. The amounts of SRD5A2 transcripts were calculated by the standard 2 − ΔΔCt method and were made into a histogram. c. Western blot results of SRD5A2 protein in HEK-293 cells transfected with WT-Flag and p.H232R-Flag mutant plasmids. β-Actin was used as an internal control. d. Enzyme activity analysis of SRD5A2 WT and mutants H232R binding with T by LC–MS. e. Enzyme activity analysis of SRD5A2 WT and mutants H232R binding with NADPH by LC–MS

Fig. 6

Conservation of the mutation site. a Conservation analysis of SRD5A2 p.Q6X (c.16C > T). b. Conservation analysis of SRD5A2 p.H232R (c.695A > G)