Abstract
ObjectiveTo 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. DesignThis 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. SettingUniversity 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.
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