P-015 Pathogenic mutations in DMRTB1 cause human male infertility with non-obstructive azoospermia by impairing transcription regulation in spermatogenesis

Abstract Study question What is the role of DMRTB1 in the etiology of non-obstructive azoospermia (NOA) and spermatogenesis in humans? Summary answer DMRTB1 pathogenic mutation leads to spermatogenesis by disrupting transcriptional regulation, ultimately resulting in NOA in humans. What is known already DMRTB1 is a member of the DM domain family. Dmrtb1 knockout mice showed male infertility, which indicated that Dmrtb1 is associated with spermatogenesis. The specific role of DMRTB1 in regulating spermatogenesis is unclear, and clinical evidence for the relationship between DMRTB1 and the pathogenesis of human male infertility is still unknown. Study design, size, duration We performed DMRTB1 mutation screening by whole-exome sequencing(WES) from 626 infertile men with NOA, and 392 fertile individuals (as control). We conducted pedigree analysis and a series of bioinformatics analyses on two patients identified as carrying DMRTB1 mutations. DMRTB1-specific antibodies were used to detect the expression of DMRTB1 in patients, and Immunoprecipitation-Mass spectrometry(IP-MS) was performed in mouse testes for subsequent mechanism exploration. The entire study was conducted over 2 years. Participants/materials, setting, methods WES was performed in participants of this cohort, followed by Sanger sequencing validation. Bioinformatics predictions were performed to evaluate the pathogenicity of candidate disease-causing variants. Hematoxylin and eosinstaining, and immunofluorescence assays (IF) were performed to detect the spermatogenesis of testis. Dual-luciferase assay, IP-MS, co-immunoprecipitation (CO-IP), western blotting (WB), and IF were performed to identify the mutations' impact on function in-vivo and in-vitro. Main results and the role of chance Following a large-scale sequencing screening from clinical centers, our study identified two infertile men with NOA who were carriers of different DMRTB1 mutations (P1:c.446_449dupGAGC[p.A151Sfs*58]; P2:c.154G>A[p.A52T] and c.793C>T [p.P265S] ) from unrelated families. All are co-segregating with the disease according to a recessive pattern of inheritance. Functional evidence based on in-vivo and in-vitro has shown that these mutations cause abnormal expression of DMRTB1 protein and impair the transcription regulation of the promoter of PIWIL2 and PIWIL1. Overall, our results support the hypothesis that DMRTB1 expression was affected by pathogenic mutations identified in patients and it inhibits PIWIL1 and PIWIL2 transcription, thereby arresting normal spermatogenesis. Limitations, reasons for caution This is a preliminary report suggesting that defects in DMRTB1 can lead to NOA in humans. The pathogenic mechanism could be further validated in the knock-in mouse model in the future. Wider implications of the findings Our study of DMRTB1 in humans provides new information on the transcription regulatory mechanism of spermatogenesis, suggesting that these pathogenic mutations in DMRTB1 are etiological factors in patients with NOA and providing a valuable clue for genetic counselling on the DMRTB1 mutants associated with infertility in human males. Trial registration number No