Functional assessment of DMRT1 variants and their pathogenicity for isolated male infertility.

Emich, Jana; Gaikwad, Avinash S; Stallmeyer, Birgit; Fietz, Daniela; Schuppe, Hans-Christian; Oud, Manon S; Kliesch, Sabine; Gromoll, Jörg; Friedrich, Corinna; Tüttelmann, Frank

Abstract

Objective: To study the impact of Doublesex and mab-3-related transcription factor 1 (DMRT1) gene variants on the encoded protein's function and the variants' pathogenic relevance for isolated male infertility caused by azoospermia. Design: This study established a novel luciferase assay for DMRT1 missense variants using 2 different target promotors and validated the assay by analyzing previously published variants associated with differences in sex development. Setting: University genetics research institute and tertiary referral center for couples' infertility. Patient(s): Eleven infertile men with severely impaired spermatogenesis resulting in crypto- or azoospermia and carrying rare heterozygous missense variants in DMRT1 were identified within the Male Reproductive Genomics study. Main outcome measure(s): Luciferase assays with human DMRT1 variants to test functional effects on the CYP19A1 and Stra8 target promoters. Result(s): We first developed and refined luciferase assays to reliably test the functional impact of DMRT1 missense variants. Next, the assay was validated by analyzing 2 DMRT1 variants associated with differences in sex development, of which c.240G>C p.(Arg80Ser) displayed highly significant effects on both target promoters compared with the wild-type protein (-40% and +100%, respectively) and c.331A>G p.(Arg111Gly) had a significant effect on the Stra8 promoter (-76%). We then systematically characterized 11 DMRT1 variants identified in infertile men. The de novo variant c.344T>A p.(Met115Lys) showed a pronounced loss of function in both DMRT1 target promoters (-100% and -86%, respectively). Variants c.308A>G p.(Lys103Arg) and c.991G>C p.(Asp331His) showed a significant gain of function exclusively for the CYP19A1 promoter (+15% and +19%, respectively). Based on these results, 3 variants were reclassified according to clinical guidelines. Conclusion(s): The present study highlights the importance of functionally characterizing DMRT1 variants of uncertain clinical significance. Using luciferase assays for diagnostic purposes enables an improved causal diagnosis for isolated male infertility.