Molecular Characterization of Two Known SRD5A2 Gene Variants in Mexican Patients With Disorder of Sexual Development

Abstract

Background: The 5α-reductase type 2 deficiency (5α-RD2) is a specific form of disorder of sexual development (DSD). Pathogenic variants in the SRD5A2 gene, which encodes this enzyme, are responsible for 46,XY DSD. Objective: The objective of the study was to investigate the genetic etiology of 46,XY DSD in two Mexican families with affected children. Materials and methods: The SRD5A2 gene of the parents and affected children was screened in both families via polymerase chain reaction amplification and DNA direct sequencing. The role of genetic variants in enzymatic activity was tested by site-directed mutagenesis. Results: Subject 1 presented two variants: p.Glu197Asp and p.Pro212Arg. Subject 2 was homozygous for the variant p.Glu197Asp. The two variants were reported in early studies. The directed mutagenesis study showed that the p.Glu197Asp and p.Pro212Arg variants lead to a total loss of enzymatic activity and, consequently, abnormal genitalia development in the patients. Conclusion: These results suggest that p.Glu197Asp and p.Pro212Arg are pathogenic variants that lead to the phenotypic expression of DSD. 5α-RD2 is of extreme importance not only because of its frequency (it is rare) but also because of its significance in understanding the mechanism of androgen action, the process of sexual differentiation, and the factors that influence normal sexual behavior.

Keywords: 46.XY; 5-alpha-reductase deficiency; dihydrotestosterone; testosterone; undefined genitalia.

FIGURE 1

(A) Pedigree family 1 and (B) Pedigree family 2.

FIGURE 2

(A) Direct sequencing of wild type. (B) Heterozygous (Subject 1) for two mutations (p.Glu197Asp and p.Pro212Arg). (C) Homozygous individuals (Subject 2) for mutation P.Glu197Asp.

FIGURE 3

Steriod 5α-reductase 2 activity expressed in HEK293 cells. Saturation curves of mutants were determined by measuring the conversion of T [1, 2, 6, 7-3H(N)] to DHT with increasing concentrations of unlabeled T (0.125–8.0 mmil/L). All experiments were performed in triplicate. p < 0.05 *vs. WT; † vs. Control; § vs. m197, m212.

FIGURE 4

Comparison between multiple sequence alignment of human steriod 5α-reductase type 2 and other sequence proteins vertebrate reductases. Mutations reported in this study are shaded blue. Alignment was performed with ClustalW program (http://clustalw.genome.jp/).

FIGURE 5

(A) SRD5A2 protein structure. (B) Mutation p.Glu197Asp close-up. (C) Mutation p.Pro212Arg close-up. In dark blue wild protein and in light blue mutated protein. The protein is in complex with finasteride orange.