Novel mutation leading to splice donor loss in a conserved site of DMD causes cryptorchidism

Abstract Background As one of the most common congenital abnormalities in male births, cryptorchidism has been found to have a polygenic etiology according to previous studies of common variants. However, little is known about genetic predisposition of rare variants for cryptorchidism, since rare variants have larger effective size on diseases than common variants. Methods In this study, a cohort of 115 Chinese probands with cryptorchidism was analyzed using whole-genome sequencing (WGS), alongside 19 parental controls and 2136 unaffected men. Additionally, CRISPR-Cas9 editing of a conserved variant was performed in a mouse model, with MRI screening utilized to observe the phenotype. Results In 30 of 115 patients (26.1%), we identified four novel genes (ARSH, DMD, MAGEA4, and SHROOM2) affecting at least five unrelated patients and four known genes (USP9Y, UBA1, BCORL1, and KDM6A) with the candidate rare pathogenic variants affecting at least two cases. Burden tests of rare variants revealed the genome-wide significances for newly identified genes (p < 2.5x10-6) under the Bonferroni correction. Surprisingly, novel and known genes were mainly from X chromosome (seven on X and one on Y) and all rare X-chromosomal segregating variants exhibited a maternal inheritance rather than de novo origin. CRISPR-Cas9 mouse modeling of a splice donor loss variant in DMD (NC_000023.11:g.32454661C>G), which resides in a conserved site across vertebrates, replicated bilateral cryptorchidism phenotypes, confirmed by Magnetic resonance imaging (MRI) at 4 and 10 weeks. Conclusion Our results revealed the role of the DMD gene mutation in causing cryptorchidism. The results also suggest that maternal-X inheritance of pathogenic defects could have a predominant role in the development of cryptorchidism. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was supported by the fifth batch of technological innovation research projects in Chengdu (2021-YF05 -01331-SN), the Postdoctoral Research and Development Fund of West China Hospital of Sichuan University (2020HXBH087), the Short-Term Expert Fund of West China Hospital (139190032), Fund of Sichuan Provincial Department of Science and Technology (2021YFS0244), and Fund of Sichuan Provincial Department of Science and Technology (2021YFS0026). We also acknowledge the computing support from the West China Biomedical Big Data Center and the Med-X Center for Informatics of Sichuan University. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All authors declare that they have no Conflict of Interest. This study conformed with the Helsinki Declaration of 1975 (as revised in 2008) concerning Human and Animal Rights. All participating parents provided informed consent, and this study was formally approved by the ethics committees of WCH (Registration number: 2021389) and WCSUH (Registration number: 2021389). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors