Mutation Screening And Genetic Study Of Multiple Morphological Abnormalities In The Sperm Flagella

Background: Infertility has become a primary human health concern,affecting 10-15 % of the global childbearing population.Asthenoteratospermia is a main cause of male infertility,which is characterised by decreased sperm motility and obvious morphological abnormalities in sperm head,neck or flagella.As early as 2003,homozygous partial deletion in CATSPER2(MIM: 607249)was first described to be associated with infertility phenotype with abnormal sperm motility and morphology in humans.Till now,several genes have been identified,which responsible for different types of asthenoteratospermia.For example,biallelic SUN5(MIM: 613942)mutations cause severe acephalic spermatozoa.DNAH1(MIM: 603332),CFAP family members and some other genes have been reported to induce human multiple morphological abnormalities of the flagella(MMAF).Mutations in AURKC and DYP19L2 account for most cases of macrozoospermia and globozoospermia,respectively.These findings demonstrated that asthenoteratospermia has strong genetic heterogeneity and diverse phenotypes.MMAF associated asthenoteratospermia is a main cause of male infertility,and exhibits strong genetic heterogeneity and thus,unidentified genes may be related to its development.Previous studies have shown that gene mutations related to axoneme and its surrounding structure,centrosome structure and function,and protein transport system may lead to the disorder of axoneme formation,which may lead to MMAF associated asthenoteratospermia in both human and mice.Object: Whole exome sequencing was used to study severe asthenospermia with MMAF primary infertility patients,to find the pathogenic gene of asthenospermia MMAF,and the outcome of ICSI treatment in MMAF patients was observed,so as to provide further genetic counseling and theoretical basis for clinical genetic diagnosis of MMAF patients.Methods: Three unrelated Han Chinese men preliminarily diagnosed with MMAF were recruited from the reproductive medicine centre of First Affiliated Hospital of Anhui Medical University.All of them in this study were from consanguineous families.All of them were diagnosed as asthenoteratospermia with MMAF.One of the proband(AP1)was characterized by typical MMAF phenotypic features,including short flagella,coiled flagella,absent flagella,angulation and flagella of irregular calibre.The other two proband(A029 IV-1 and A033 IV-1)were characterized by absent flagella(> 90%).Detailed histories of the onset and progression of infertility were obtained from the patients directly.Examination revealed normal development of male external genitalia,normal bilateral testicular size,normal hormone levels and secondary sexual characteristics.The chromosomal karyotypes of all patients were normal(46;XY),and no microdeletion was identified in the Y chromosome.Moreover,primary ciliary dyskinesia(PCD)-associated symptoms(such as sinusitis,bronchitis,pneumonia and otitis media)and other ciliopathies(such as polycystic kidney disease or Bardet–Biedl syndrome)were excluded by olfactory testing with smelling bottles and computed tomography(CT)imaging.Peripheral whole blood samples of the proband and his parents were collected for subsequent genetic analysis.Whole exome sequencing(WES)was performed on the three probands and two new genes/mutations were identified that could be the cause of the disease.Sperm immunofluorescence test and real-time fluorescence quantitative PCR test on human samples were used to detect the changes of proteins and RNA in sperm.Scanning electron microscopy and transmission electron microscopy were used to observe the ultrastructural changes of spermatozoa.After plasmid transfection,immunofluorescence and Western Blot were performed to observe whether gene mutations caused changes in protein levels in vitro.In addition,we constructed a knockout mouse model for one of the genes and observed the effect of specific gene mutations on sperm morphology and function in mice. It was further confirmed that this mutation can cause MMAF in both humans and mice.Results: We identified a homozygous missense mutation c.A3811G(p.K1271E)in WDR19 in proband AP1 and two homozygous mutations c.188G>A(p.Arg63Gln)and c.690T>G(p.Tyr230*)of DZIP1 in A029 IV-1 and A033 IV-1,respectively.These mutations were neither detected in data from the 1000 Genomes Project,nor in the Exome Aggregation Consortium databases.,nor in our control cohort of 875 Han Chinese individuals.For the WDR19 mutated patient,semen analysis showed that the progressive rate decreased to zero and flagella were short and coiled.Sperm scanning electron microscopy and transmission electron microscopy indicated that the axoneme structure was completely destroyed,and the microtubule structure disappeared,while immunofluorescence revealed that WDR19 was absent from the sperm flagellum.Moreover,flagella-related WDR19 interacting proteins(IFT88,IFT140 and SPAG6)were significantly downregulated in WDR19 mutated sperm.For the DZIP1 mutated patients,the spermatozoa of both probands consistently presented as immotile.Various morphological abnormalities of spermatozoa from DZIP1-mutated men were observed,such as short and absent flagella.Notably,the major flagellar malformation in DZIP1-mutated men was absent in flagella,accounting for more than 90% spermatozoa.The spermatozoa of proband A033 IV-1 presented the abnormalities including two centriolar dots with abnormal angle,no concentrated dot,or more than two centriolar dots.None of the DZIP1-mutated spermatozoa had well-shaped axonemes.In vitro effects of these two homozygous DZIP1 mutations were also investigated in HEK293 T cells transfected with WT or DZIP1-mutated constructs.The expression level of DZIP1 was obviously reduced for the p.Arg63 Gln mutation,and DZIP1 was truncated for the p.Tyr230* mutation.The consistent asthenoteratospermia phenotypes between Dzip1-knockout male mice and human furtherly confirmed the pathogenicity of DZIP1 deficient.Conclusions:(1)WDR19 homozygous missense mutation is a novel pathogenic gene variant for male infertility caused by asthenoteratospermia without any ciliopathies.Furthermore,WDR19 cooperates with specific proteins to cause flagellar microtubules disorganization and sperm immotility.(2)DZIP1 deficiency caused by homozygous DZIP1 mutations results in male infertility characterised by asthenoteratospermia with the damage of both flagellar formation and sperm centrioles,thus establishing DZIP1 mutation as being responsible for asthenoteratospermia with severe MMAF.