Genetic Study And Pathogenetic Mechanism Exploration Of CFAP58 And CFAP47 Mutations In Multiple Morphological Abnormalities Of The Sperm Flagella

Genetic Study and Pathogenetic Mechanism Exploration of CFAP58 and CFAP47 Mutations in Multiple Morphological Abnormalities ofthe Sperm Flagella Background: Infertility has become a primary human health concern,affecting about15% of the global childbearing population.Male infertility has been reported as a complex multifactorial pathological condition accounting for about 50% of the infertile cohort.Asthenoteratozoospermia with decreased sperm motility and elevated sperm malformation represents one of the main causes of male infertility.Multiple morphological abnormalities of the sperm flagella(MMAF)is a specific subtype of asthenoteratozoospermia and is characterized by the following five types of flagellar malformations: absent,short,bent,coiled,and irregular flagella.Owing to the severe impairment of sperm motility,MMAF consistently causes male infertility.As a genetically heterogeneous disorder,the previously reported pathogenic genes can only responsible for approximately 60% of human MMAF cases.Numerous studies have confirmed that defects in axoneme and peripheral axoneme could disrupt the biogenesis of axoneme,result in MMAF associated asthenoteratospermia.Cilium and flagellum associated proteins(CFAPs)play an important role in the assembly and function of cilia and flagella.Several CFAP genes were demonstrated to be associated with MMAF,such as CFAP43,CFAP47,CFAP69,CFAP70 and CFAP251.Object: Whole exome sequencing was performed to identified the candidate causative genes in 105 Chinese MMAF patients.We also generated the knockout models in the corresponding mouse orthologue genes via CRISPR-Cas9 technology to further confirmed the association of these genes with MMAF.Furthermore,according to the explorative studies in these patients and the corresponding KO mice,we primarily elucidated the molecular mechanism of these genes in sperm flagellogenesis.Results: We identified bi-allelic loss-of-function variants in CFAP58 by whole-exome sequencing(WES)in five unrelated MMAF patients.Homozygous CFAP58 stop-gain variants c.2092C>T.p.Arg698*,c.1696C>T.p.Gln566*,and c.2274C>A.p.Tyr758*were identified in three cases.Compound heterozygous variants of CFAP58(c.2092C>T.p.Arg698*,c.1429 del.p.lle477*)and a homozygous frameshift variant in CFAP58(c.2052 del.p.His685Thrfs*7)were identified in another two cases,respectively.CFAP58(cilia-and flagella-associated protein 58)is preferentially expressed in human testis and encodes a predicted 872-amino-acid protein.Furthermore,we identified three hemizygous missense variants in the X-linked CFAP47(c.7154T>A.p.Ile2385 Asn,c.5224A>G.p.Ser1742 Gly,c.8668C>A.p.Pro2890Thr)in another three unrelated MMAF patients.CFAP47(ciliaand flagella-associated protein 47)is also highly expressed in human testis and encodes a predicted 3187-amino-acid protein.Defects in CFAP58 and CFAP47 cause male infertility with severe asthenoteratozoospermia characterized by multiple flagellar malformations(absent,short,and coiled flagella).We performed transmission electron microscopy(TEM)analysis to investigate the sperm flagellar ultrastructure of patients carrying CFAP58 and CFAP47 mutations.The results revealed various axonemal malformations,including absence and the complicated disorganization of central pair of microtubule doublets and/or mitochondrial sheath.Immunofluorescence(IF),quantitative real-time PCR and Western Blotting(WB)were employed to assess the pathogenicity of these mutations.CFAP58 was almost absent in the sperm flagella of CFAP58-mutated patients.In addition,CFAP47 was dramatically reduced,or absent,in the spermatozoa from patients carrying hemizygous CFAP47 mutations.To further confirm the association of CFAP58 and CFAP47 defects with MMAF,we generated Cfap58-and Cfap47-knockout(KO)mouse model by CRISPR-Cas9 technology.The male KO mice were infertile and presented with severe flagellar defects,consistent with the sperm phenotypes in MMAF-affected patients.Overall,the phenotype of KO mice strongly suggest that CFAP58 and CFAP47 are recurrent causative genes of MMAF.According to the TEM results,IF,WB,and Co-IP were performed to further explore the pathogenesis of mutations in CFAP58 and CFAP47 in MMAF.WB and IF assays showed that the abundances of axoneme ultrastructure markers SPAG6 and SPEF2 and a mitochondrial sheath protein,HSP60,were significantly reduced in the CFAP58-mutated sperm,consisting with the TEM results.Moreover,IF analysis revealed that ODF2 signal was obviously increased in the CFAP58 defected sperm.Therefore,we speculated that CFAP58 could be important for ODF transportation and the deficiency might result in an abnormal location of ODF2,leading to the abnormalities in the assembly of flagellum.The interacting proteins of CFAP47 were conducted by Co-IP.Notably,the connection between CFAP47 and centrosome protein CFAP65 were observed in both human spermatozoa and mouse testis,suggesting that CFAP47 might interact with CFAP65 during spermiogenesis.This hypothesis is strongly supported by the following IF assays.CFAP65 signal was localized mainly at the equatorial segment of sperm head and the base of flagella in normal spermatozoa from control individuals,whereas the CFAP65 signal was only diffusely clustered in the acrosome of spermatozoa from MMAF-affected individuals harboring hemizygous CFAP47 mutations.However,further studies are required to elucidate the precise molecular function of CFAP47 in the biogenesis of axoneme.Conclusions: Our findings in humans and mice strongly suggest that CFAP58 and CFAP47 play vital roles in sperm flagellogenesis and demonstrate that mutations in CFAP58 and CFAP47 can lead to MMAF-related asthenoteratospermia.CFAP58 mutations cause striking flagellar defects with axonemal and mitochondrial sheath malformations.The CC domains of CFAP58 might play a vital role in sperm flagellogenesis;furthermore,CFAP58 might be important for ODF transportation and that its deficiency might result in an abnormal location of ODF2,leading to the disorder of flagellar structure.The inter-regulation between CFAP47 and CFAP65 might be important for sperm flagellogenesis.