Computationally predicted pathogenic USP9X mutation identified in infertile men does not affect spermatogenesis in mice

Infertility is a major health issue,affecting approximately 15%of couples of child-bearing age.Although nearly half of idiopathic infertility cases are assumed to have a genetic basis,the underlying causes remain largely unknown in most infertile men.

TOL6BL突变导致减数分裂程序性DNA双链断裂无法产生和人类不孕

我们找到一个近亲婚配后代不育家系,该家系有3位非梗阻性无精子症患者和1位原因不明的女性不孕患者.全外显子组测序结合Sanger测序分析发现TOP6BL的c.483dup T突变在该家系中与不孕呈隐性共分离.正常的TOP6BL可与SPO11β结合进而导致减数分裂程序性DNA双链断裂(DSBs)产生,而该突变破坏了二者的结合.对家系中一位男患者的精母细胞进行分析,发现其同源染色体联会异常,减数分裂不能到达粗线期,且染色体轴上缺少减数分裂重组蛋白RPA和DMC1信号,表明其减数分裂程序性DSBs未能产生.我们制备了携带类似患者突变的小鼠,发现突变雄鼠具有与男患者相同的减数分裂异常,突变雌鼠不孕,其卵母细胞中程序性DSBs和减数分裂重组也不能发生,卵母细胞不能成熟.这些发现表明TOP6BL突变可导致人类不孕,为相关不孕不育患者的病因诊断和人工辅助生殖胚胎的遗传检查提供了分子标靶.

The molecular control of meiotic double-strand break (DSB) formation and its significance in human infertility

Meiosis is an essential step in gametogenesis which is the key process in sexually reproducing organisms as meiotic aberrations may result in infertility. In meiosis, programmed DNA double-strand break (DSB) formation is one of the fundamental processes that are essential for maintaining homolog interactions and correcting segregation of chromosomes. Although the number and distribution of meiotic DSBs are tightly regulated, still abnormalities in DSB formation are known to cause meiotic arrest and infertility. This review is a detailed account of molecular bases of meiotic DSB formation, its evolutionary conservation, and variations in different species. We further reviewed the mutations of DSB formation genes in association with human infertility and also proposed the future directions and strategies about the study of meiotic DSB formation.

Novel biallelic loss-of-function mutations in CFAP43 cause multiple morphological abnormalities of the sperm flagellum in Pakistani families

Multiple morphological abnormalities of the sperm flagella(MMAF)is a specific type of asthenoteratozoospermia,presenting with multiple morphological anomalies in spermatozoa,such as absent,bent,coiled,short,or irregular caliber flagella.Previous genetic studies revealed pathogenic mutations in genes encoding cilia and flagella-associated proteins(CFAPs;e.g.,CFAP43,CFAP44,CFAP65,CFAP69,CFAP70,and CFAP251)responsible for the MMAF phenotype in infertile men from different ethnic groups.However,none of them have been identified in infertile Pakistani males with MMAF.In the current study,two Pakistani families with MMAF patients were recruited.Whole-exome sequencing(WES)of patients and their parents was performed.WES analysis reflected novel biallelic loss-of-function mutations in CFAP43 in both families(Family 1:ENST00000357060.3,p.Arg300Lysfs*22 and p.Thr526Serfs*43 in a compound heterozygous state;Family 2:ENST00000357060.3,p.Thr526Serfs*43 in a homozygous state).Sanger sequencing further confirmed that these mutations were segregated recessively in the families with the MMAF phenotype.Semiquantitative reverse-transcriptase polymerase chain reaction(qRT-PCR)was carried out to detect the effect of the mutation on mRNA of the affected gene.Previous research demonstrated that biallelic loss-of-function mutations in CFAP43 accounted for the majority of all CFAP43-mutant MMAF patients.To the best of our knowledge,this is the first study to report CFAP43 biallelic loss-of-function mutations in a Pakistani population with the MMAF phenotype.This study will help researchers and clinicians to understand the genetic etiology of MMAF better.

A recurrent homozygous missense mutation in CCDC103 causes asthenoteratozoospermia due to disorganized dynein arms

Asthenoteratozoospermia is one of the most severe types of qualitative sperm defects.Most cases are due to mutations in genes encoding the components of sperm flagella,which have an ultrastructure similar to that of motile cilia.Coiled-coil domain containing 103(CCDC103)is an outer dynein arm assembly factor,and pathogenic variants of CCDC103 cause primary ciliary dyskinesia(PCD).However,whether CCDC103 pathogenic variants cause severe asthenoteratozoospermia has yet to be determined.Whole-exome sequencing(WES)was performed for two individuals with nonsyndromic asthenoteratozoospermia in a consanguineous family.A homozygous CCDC103 variant segregating recessively with an infertility phenotype was identified(ENST00000035776.2,c.461A>C,p.His154Pro).CCDC103 p.His154Pro was previously reported as a high prevalence mutation causing PCD,though the reproductive phenotype of these PCD individuals is unknown.Transmission electron microscopy(TEM)of affected individuals’spermatozoa showed that the mid-piece was severely damaged with disorganized dynein arms,similar to the abnormal ultrastructure of respiratory ciliary of PCD individuals with the same mutation.Thus,our findings expand the phenotype spectrum of CCDC103 p.His154Pro as a novel pathogenic gene for nonsyndromic asthenospermia.

The deubiquitinating gene Usp29 is dispensable for fertility in male mice

The balanced actions between ubiquitination and deubiquitination precisely control the levels of various proteins vital for spermatogenesis. Ubiquitin-specific processing proteases(USPs) are the largest family of deubiquitinatingenzymes(DUBs),containing more than 50 members. So far, the functions of only a few USPs in male fertility have been studied, the roles of the majority are yet unknown. The present study aimed to explore the function of Usp29(ubiquitin-specific protease 29) in male fertility. We found that Usp29 showed predominant expression in mouse testis, and its m RNA expression started to increase at 14 days postpartum(dpp), with a peak at 28 and 35 dpp. Using CRISPR/Cas9 technology, we generated Usp29 knockout mice(Usp29^(–/–)). Usp29^(–/–)mice exhibited no overt developmental anomalies. Further examination revealed that Usp29^(–/–)mice had normal fertility and showed no detectable difference in the testis/body weight ratio, testicular and epididymal histology as well as epididymal sperm count from the wild-type littermates. Moreover, Usp29 is not a pseudogene in mice. Taken together, our study first reported that though Usp29 is predominantly expressed in the testis, it is not essential for male fertility in mice.

CFTR mutations causing congenital unilateral absence of the vas deferens(CUAVD)and congenital absence of the uterus(CAU)in a consanguineous family

Cystic fibrosis(CF)is one of the most common recessive genetic diseases,with a wide spectrum of phenotypes,ranging from infertility to severe pulmonary disease.Mutations in the cystic fibrosis transmembrane conductance regulator(CFTR)gene are considered the main genetic cause for CF.In this study,we recruited a consanguineous Iranian pedigree with four male patients diagnosed with congenital unilateral absence of the vas deferens(CUAVD),and one female patient diagnosed with congenital absence of the uterus(CAU).Testicular biopsy of one patient was performed,and hematoxylin and eosin(H and E)staining of testis sections displayed the presence of germ cell types ranging from spermatogonia to mature spermatids,indicating obstructive azoospermia.To explore the underlying genetic factor in this familial disorder,we therefore performed whole-exome sequencing(WES)on all available family members.WES data filtration and CFTR haplotype analysis identified compound heterozygous mutations in CFTR among four patients(two CUAVD patients carried p.H949Y and p.L997F,and one CUAVD and the female CAU patient carried p.H949Y and p.I148T).All these mutations were predicted to be deleterious by at least half of the prediction software programs and were confirmed by Sanger sequencing.Our study reported that CFTR compound heterozygous mutations in a consanguineous Iranian family cause infertility in both sexes.