Association And Pathogenesis Of Male Sterility Induced By WDR63 Gene Mutation

Background: Infertility is a major health problem worldwide and is estimated to affect 8–12% of couples in the reproductive age group with a male factor being a primary or contributing cause in approximately 50% of couples.A systematic review by Levine and colleagues reported that sperm counts decreased by 50–60% between 1973 and 2011.Infertility causes substantial psychological and social distress.Thus,early detection of male subfertility offers the opportunity for identification and correction of medical conditions affecting not only fertility,but also general health and wellbeing.A multitude of causes and risk factors contribute to the increasing incidence of male infertility,which can be stratified as congenital,acquired,and idiopathic.The primary known genetic causes of male infertility are congenital bilateral absence of the vas deferens associated with cystic fibrosis gene mutations,Kallmann syndrome,chromosomal abnormalities leading to deterioration of testicular function,and Y chromosome microdeletions resulting in isolated spermatogenic defects.Among acquired factors,varicocele is the most common and correctable cause of infertility in men,with a prevalence of 40%.About 30–50% of male infertility cases are idiopathic,with no discernible cause or contributory female infertility.Male oxidative stress infertility involves altered semen characteristics and oxidative stress,and affects about 37 million men with idiopathic male infertility.Environmental or occupational exposure to toxic chemicals and various lifestyle factors（eg,smoking,alcohol consumption,recreational drug use,obesity and psychological stress）are all potential risk factors for male infertility.In most cases,male infertility is clinically diagnosed if semen parameters are reduced.Descriptive diagnoses are “oligozoospermia”（reduced sperm count）,“asthenozoospermia”（reduced sperm motility）,“teratozoospermia”（reduced percentage of sperm with normal morphology）.Combinations are common;most frequently “oligoasthenoteratozoospermia” or “OAT syndrome” are found.The advent of high-throughput genomic technologies and the completion of international genomic projects,such as gnom ADand the 1000 Genomes Project,stimulated largescale genomic studies in MMAF diseases.Multiple morphological abnormalities of the sperm flagellum（MMAF）is one of the severest asthenoteratozoospermia which is usual y combined with reduced sperm count and is characterized by abnormal flagellar formation,including absent,short,coiled,bent flagellar,and/or irregular caliber.Whole Exome Sequencing（WES）is an effective method of selective Sequencing of coding regions of the genome（exons）,often used to find rare or common variants associated with diseases or phenotypes.At present,25 pathogenic genes of MMAF have been reported,including DNAH1,DNAH2,DNAH10,CFAP43 and CFAP44.Genetic research on male infertility caused by MMAF has greatly promoted the diagnosis and treatment of MMAF patients in clinic.However,there are still many unknown problems in this field: ⅰ.The genetic etiology of about 40% of MMAF patients is still unknown,suggesting that there are still many potential disease-causing genes to be discovered.ⅱ.Many mutations of unknown significance have been identified in MMAF patients.Due to the lack of rapid and reliable techniques to verify the pathogenicity of these mutations,these mutations of unknown significance have rarely been reported;ⅲ.Thanks to the development and application of CRISPR-Cas9 technology,the pathogenicity of MMAF-related genes has been preliminarily verified in mouse models,but most studies remain at the phenotypic stage,and the molecular mechanism is still unknown.Results: To identify the potential MMAF associated variants in male infertility,we conducted whole-exome sequencing（WES）analyses in a distinct MMAF cohort depend on variants frequency and functional annotation（minor al ele frequency [MAF] < 0.001 in the gnom AD database and combined annotation-dependent depletion [CADD] score of ≥ 15）.In the cohort of 243 MMAF-affected Chinese men,we identified a harboring homozygous stop-gain variant（M1: c.163C>T [p.Arg55*],CADD=37）in WDR63（MIM: 617968;NCBI: NM₁45172.5）.Semen parameters of men harboring bi-allelic WDR63 variant was analyzed in the source laboratories according to WHO guidelines.The typical MMAF characteristic was observed in the spermatozoa from the men harboring bi-allelic WDR63 variant.Notably,the sperm counts were dramatically lower than the normal reference values.In addition,we identified an additional bi-allelic variant of WDR63 in 121 non-obstructive azoospermia（NOA）male（M2: c.1075C>T [p.Arg359*],CADD=35）using Sanger sequencing,indicating that variants of WDR63 might cause the severely decrease in sperm counts.Human WDR63 contains 23 exons and encodes a predicted 891-aminoacid protein that comprises four Trp-Asp（WD40）repeat domains.In this study,all of variants in WDR63 are located before the WD40 domain,leading to the loss of WD40 domains in WDR63 protein.We over-expressed ful-length wild-type and mutant c DNA constructs in HEK293 T cel s and found the significantly absence of mutant c DNA constructs of WDR63.Besides,in addition to those homozygous stop-gain variants,WDR63 gene harboring a lot of heterozygous missense variants with low frequency and high effect in males.Association study indicated that these variants significant relative to male infertility（OR = 20.7,P = 3.7×10-06）.Considering that mouse WDR63 is highly homologous to human WDR63 protein and primarily expressed in mice testes,we generated Wdr63-KO mice by using CRISPR-Cas9 technology.Sanger sequencing of Wdr63 homozygous mutated mice confirmed the presence of a nonsense mutation plus a 2-bp deletion in exon 10,which was predicted to cause premature translational termination（p.Arg362*）Remarkably,Wdr63-knockout（Wdr63-KO）induced decreased sperm number,abnormal flagellar morphology and male infertility.In addition,transmission electron microscopy assay showed severely disorganized “9+2” axoneme and absent inner dynein arms in the spermatozoa from Wdr63-KO male mice.In mechanism,we found that WDR63 interacted with WDR78 mainly via WD40 repeat domain and is necessary for IDA assembly.Furthermore,intracytoplasmic sperm injection（ICSI）treatment revealed both MMAF-affected men with WDR63 mutations and Wdr63-KO mice acquired successful clinical pregnancy and obtained biological children.Conclusions: Here,we identified two bi-allelic variants of WDR63 in both MMAF and NOA affected cohorts.Furthermore,WDR63 forms a complex with the IDA component WDR78,and these two proteins co-localized in the sperm flagella.Wdr63-KO impaired the assembly of IDA and caused severely decreased sperm number,sperm motility and abnormal flagellar morphology.Comfortingly,intracytoplasmic sperm injection（ICSI）treatment revealed both Wdr63-KO mice and men harboring WDR63 variants acquired successful clinical pregnancy.Our study suggests that bi-allelic variants of WDR63 can be used as an inherited pathogenic factor and a genetic diagnostic indicator for infertility males.