Biallelic mutations in CFAP65 cause male infertility with multiple morphological abnormalities of the sperm flagella in humans and mice.

Male infertility is a prevalent issue worldwide, mostly due to the impaired sperm motility. Multiple morphological abnormalities of the sperm flagella (MMAF) present aberrant spermatozoa with absent, short, coiled, bent and irregular-calibre flagella resulting in severely decreased motility. Previous studies reported several MMAF-associated genes accounting for approximately half of MMAF cases. We conducted genetic analysis using whole-exome sequencing in 88 Han Chinese MMAF probands. CFAP65 homozygous mutations were identified in four unrelated consanguineous families, and CFAP65 compound heterozygous mutations were found in two unrelated cases with MMAF. All these CFAP65 mutations were null, including four frameshift mutations (c.1775delC [p.Pro592Leufs*8], c.3072_3079dup [p.Arg1027Profs*41], c.1946delC [p.Pro649Argfs*5] and c.1580delT [p.Leu527Argfs*31]) and three stop-gain mutations (c.4855C>T [p.Arg1619*], c.5270T>A [p.Leu1757*] and c.5341G>T [p.Glu1781*]). Additionally, two homozygous CFAP65 variants likely affecting splicing were identified in two MMAF-affected men of Tunisian and Iranian ancestries, respectively. These biallelic variants of CFAP65 were verified by Sanger sequencing and were absent or very rare in large data sets aggregating sequence information from various human populations. CFAP65, encoding the cilia and flagella associated protein 65, is highly and preferentially expressed in the testis. Here we also generated a frameshift mutation in mouse orthologue Cfap65 using CRISPR-Cas9 technology. Remarkably, the phenotypes of Cfap65-mutated male mice were consistent with human MMAF. Our experimental observations performed on both human subjects and on Cfap65-mutated mice demonstrate that the presence of biallelic mutations in CFAP65 causes the MMAF phenotype and impairs sperm motility.

Li W ，Wu H ，Li F ，Tian S ，Kherraf ZE ，Zhang J ，Ni X ，Lv M ，Liu C ，Tan Q ，Shen Y ，Amiri-Yekta A ，Cazin C ，Zhang J ，Liu W ，Zheng Y ，Cheng H ，Wu Y ，Wang J ，Gao Y ，Chen Y ，Zha X ，Jin L ，Liu M ，He X ，Ray PF ，Cao Y ，Zhang F ... -  《-》

Biallelic Mutations in CFAP43 and CFAP44 Cause Male Infertility with Multiple Morphological Abnormalities of the Sperm Flagella.

Sperm motility is vital to human reproduction. Malformations of sperm flagella can cause male infertility. Men with multiple morphological abnormalities of the flagella (MMAF) have abnormal spermatozoa with absent, short, coiled, bent, and/or irregular-caliber flagella, which impair sperm motility. The known human MMAF-associated genes, such as DNAH1, only account for fewer than 45% of affected individuals. Pathogenic mechanisms in the genetically unexplained MMAF remain to be elucidated. Here, we conducted genetic analyses by using whole-exome sequencing and genome-wide comparative genomic hybridization microarrays in a multi-center cohort of 30 Han Chinese men affected by MMAF. Among them, 12 subjects could not be genetically explained by any known MMAF-associated genes. Intriguingly, we identified compound-heterozygous mutations in CFAP43 in three subjects and a homozygous frameshift mutation in CFAP44 in one subject. All of these recessive mutations were parentally inherited from heterozygous carriers but were absent in 984 individuals from three Han Chinese control populations. CFAP43 and CFAP44, encoding two cilia- and flagella-associated proteins (CFAPs), are specifically or preferentially expressed in the testis. Using CRISPR/Cas9 technology, we generated two knockout models each deficient in mouse ortholog Cfap43 or Cfap44. Notably, both Cfap43- and Cfap44-deficient male mice presented with MMAF phenotypes, whereas the corresponding female mice were fertile. Our experimental observations on human subjects and animal models strongly suggest that biallelic mutations in either CFAP43 or CFAP44 can cause sperm flagellar abnormalities and impair sperm motility. Further investigations on other CFAP-encoding genes in more genetically unexplained MMAF-affected individuals could uncover novel mechanisms underlying sperm flagellar formation.

Tang S ，Wang X ，Li W ，Yang X ，Li Z ，Liu W ，Li C ，Zhu Z ，Wang L ，Wang J ，Zhang L ，Sun X ，Zhi E ，Wang H ，Li H ，Jin L ，Luo Y ，Wang J ，Yang S ，Zhang F ... -  《-》

A novel homozygous CFAP65 mutation in humans causes male infertility with multiple morphological abnormalities of the sperm flagella.

Multiple morphological abnormalities of the sperm flagella (MMAF) is a rare autosomal recessive inherited disorder associated with male infertility. To date, 14 genetic causative genes have been identified in MMAF, which can only explain the genetic causes of ~60% of MMAF cases. Here, we report a man with primary infertility, who had a typical MMAF phenotype. Whole-exome sequencing (WES) was performed on the patient and a homozygous mutation (c. 2675 G>A [p. Trp892*]) was identified in cilia and flagella-associated protein 65 (CFAP65) gene, which is primarily expressed in the testis. Another loss-of-function mutation of CFAP65 has been detected in a MMAF patient, and the orthologue of CFAP65 also plays a vital role in sperm motility in chickens. Our experimental observations on human subjects suggested that CFAP65 is involved in sperm flagellum structure and assembly and that loss-of-function mutations could lead to male infertility in humans by causing the MMAF phenotype.

Zhang X ，Shen Y ，Wang X ，Yuan G ，Zhang C ，Yang Y ... -  《-》

Biallelic mutations in Sperm flagellum 2 cause human multiple morphological abnormalities of the sperm flagella (MMAF) phenotype.

Male patients with multiple morphological abnormalities of the sperm flagella (MMAF) are infertile and exhibit absent, short, coiled, bent and/or irregular sperm flagella. Mutations in the SPEF2 gene reduce sperm motility and cause sperm tail defects in animal models and humans. In the present study, we performed a genetic analysis on an MMAF patient and identified novel biallelic mutations in the SPEF2 gene. The biallelic mutations were confirmed by Sanger sequencing and in silico analysis revealed that, these variations were deleterious. The expression of truncated SPEF2 protein was reduced significantly in the patient's spermatozoa. The spermatozoa harbored biallelic mutations and showed severe ultrastructural defects in the axoneme and mitochondrial sheath. Our data suggest that biallelic mutations in SPEF2 can cause severe sperm flagellum defects, thus providing a novel candidate genetic pathogen for the human MMAF phenotype.

Sha Y ，Liu W ，Wei X ，Zhu X ，Luo X ，Liang L ，Guo T ... -  《-》

Bi-allelic DNAH8 Variants Lead to Multiple Morphological Abnormalities of the Sperm Flagella and Primary Male Infertility.

Sperm malformation is a direct factor for male infertility. Multiple morphological abnormalities of the flagella (MMAF), a severe form of asthenoteratozoospermia, are characterized by immotile spermatozoa with malformed and/or absent flagella in the ejaculate. Previous studies indicated genetic heterogeneity in MMAF. To further define genetic factors underlying MMAF, we performed whole-exome sequencing in a cohort of 90 Chinese MMAF-affected men. Two cases (2.2%) were identified as carrying bi-allelic missense DNAH8 variants, variants which were either absent or rare in the control human population and were predicted to be deleterious by multiple bioinformatic tools. Re-analysis of exome data from a second cohort of 167 MMAF-affected men from France, Iran, and North Africa permitted the identification of an additional male carrying a DNAH8 homozygous frameshift variant. DNAH8 encodes a dynein axonemal heavy-chain component that is expressed preferentially in the testis. Hematoxylin-eosin staining and electron microscopy analyses of the spermatozoa from men harboring bi-allelic DNAH8 variants showed a highly aberrant morphology and ultrastructure of the sperm flagella. Immunofluorescence assays performed on the spermatozoa from men harboring bi-allelic DNAH8 variants revealed the absent or markedly reduced staining of DNAH8 and its associated protein DNAH17. Dnah8-knockout male mice also presented typical MMAF phenotypes and sterility. Interestingly, intracytoplasmic sperm injections using the spermatozoa from Dnah8-knockout male mice resulted in good pregnancy outcomes. Collectively, our experimental observations from humans and mice demonstrate that DNAH8 is essential for sperm flagellar formation and that bi-allelic deleterious DNAH8 variants lead to male infertility with MMAF.

Liu C ，Miyata H ，Gao Y ，Sha Y ，Tang S ，Xu Z ，Whitfield M ，Patrat C ，Wu H ，Dulioust E ，Tian S ，Shimada K ，Cong J ，Noda T ，Li H ，Morohoshi A ，Cazin C ，Kherraf ZE ，Arnoult C ，Jin L ，He X ，Ray PF ，Cao Y ，Touré A ，Zhang F ，Ikawa M ... -  《-》