ESPE Abstracts

Background: 5-α-reductase type 2 enzyme catalyzes the conversion of testosterone into dihydrotestosterone, a potent androgen responsible for male sexual development during the fetal period. From 2017 to 2019, a homozygous hg38_chr2:31529414 C>T variant in SRD5A2 gene have been identified in 3 unrelated patients with DSD 46,XY of Buryat origin. The variant has been previously reported in one patient from China (Song et al, 2019) and listed as a missense substitution (NM_000348.4:c.589G>A p.E197K) in HGMD database (CM1922241). According to ACMG criteria the variant is rated as a variant of uncertain significance (PM2;PP3;PP4).

Aims: To characterize the variant c.589G>A. To study carrier frequency of the variant c.589G>A among Buryats.

Methods: A potential effect of c.589G>A substitution on splicing was evaluated in silico using SpliceAI tool (https://spliceailookup.broadinstitute.org). For a minigene assay a genomic region containing exon 4 with the flanking introniс sequences of SRD5A2 from one of the patients’ parent was cloned into pSpl3-Flu2-TK vector. The obtained plasmids with wild-type sequence (SRD5A2_E197) and the variant (SRD5A2_K197) were then transfected into HEK293T cells. 48h after transfection, total RNA was isolated and RT-PCR analysis was performed. Genotyping of the nucleotide variant c.589G>A was performed by Real-time PCR. 300 healthy individuals of Buryats origin were included in the study. Allele frequencies, Fisher’s confidence intervals were calculated using the WinPepi v.11.65 software.

Results: In silico SpliceAI analysis revealed that the variant can influence splicing by activating a cryptic donor site in the exon. The minigene assay confirmed that the c.589G>A variant leads to activation of a cryptic donor splicing site, resulting in a shortening of exon 4 by 112 nucleotides. This alteration in mRNA structure disrupts the open reading frame and produces a truncated SRD5A2 protein lacking two transmembrane domains. Among 300 studied samples the nucleotide variant c.589G>A in SRD5A2 was detected in 6 cases in a heterozygous state. The allele frequency of the studied variant was 0,01 (95%CI=0.0028-0.018). The frequency of heterozygous carriers was 0.02 (95%CI=0.0056-0.036). The frequency of DSD 46,XY due to the variant c.589G>A among Buryats was 1:20000 or 5 per 100,000 (95%CI=1:2482–1:176582 or 0.6-40 per 100,000). The frequency of heterozygous carriers of c.589G>A variant was 1:51 subjects (95%CI=1:28–1:179).

Conclusions: The results reclassify the NM_000348.4:c.589G>A substitution as a pathogenic variant (PM2, PVS1, PP4) affecting splicing. The study demonstrates high carrier frequency of the NM_000348.4:c.589G>A variant among Buryats, which is most likely attributed to a founder effect.