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 ... -  《-》

Loss-of-function mutations in SPEF2 cause multiple morphological abnormalities of the sperm flagella (MMAF).

Multiple morphological abnormalities of the sperm flagella (MMAF) is a kind of severe teratozoospermia. Patients with the MMAF phenotype are infertile and present aberrant spermatozoa with absent, short, coiled, bent and/or irregular flagella. Mutations in several genes can explain approximately 30%-50% of MMAF cases and more genetic pathogenies need to be explored. SPEF2 was previously demonstrated to play an essential role in sperm tail development in mice and pig. Dysfunctional mutations in SPEF2 impair sperm motility and cause a short-tail phenotype in both animal models. Based on 42 patients with severe infertility and MMAF phenotype, we explored the new genetic cause of human MMAF phenotype. By screening gene variants in 42 patients with MMAF using whole exome sequencing, we identified the c. 12delC, c. 1745-2A > G, c. 4102 G > T and c. 4323dupA mutations in the SPEF2 gene from two patients. Both of these mutations are rare and potentially deleterious. Transmission electron microscope (TEM) analysis showed a disrupted axonemal structure with mitochondrial sheath defects in the patients' spermatozoa. The SPEF2 protein level was significantly decreased in the spermatozoa of the patients revealed by Western blot (WB) and immunofluorescence (IF) analyses. Our experimental findings indicate that loss-of-function mutations in the SPEF2 gene can cause the MMAF phenotype in human.

Liu W ，Sha Y ，Li Y ，Mei L ，Lin S ，Huang X ，Lu J ，Ding L ，Kong S ，Lu Z ... -  《-》

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 ... -  《-》

Novel mutations in SPEF2 causing different defects between flagella and cilia bridge: the phenotypic link between MMAF and PCD.

Tu C ，Nie H ，Meng L ，Wang W ，Li H ，Yuan S ，Cheng D ，He W ，Liu G ，Du J ，Gong F ，Lu G ，Lin G ，Zhang Q ，Tan YQ ... -  《-》

Bi-allelic Loss-of-function Variants in CFAP58 Cause Flagellar Axoneme and Mitochondrial Sheath Defects and Asthenoteratozoospermia in Humans and Mice.

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. Although recent studies have revealed several MMAF-associated genes and demonstrated MMAF to be a genetically heterogeneous disease, at least one-third of the cases are still not well understood for their etiology. Here, we identified bi-allelic loss-of-function variants in CFAP58 by using whole-exome sequencing in five (5.6%) unrelated individuals from a cohort of 90 MMAF-affected Chinese men. Each of the men harboring bi-allelic CFAP58 variants presented typical MMAF phenotypes. Transmission electron microscopy demonstrated striking flagellar defects with axonemal and mitochondrial sheath malformations. CFAP58 is predominantly expressed in the testis and encodes a cilia- and flagella-associated protein. Immunofluorescence assays showed that CFAP58 localized at the entire flagella of control sperm and predominantly concentrated in the mid-piece. Immunoblotting and immunofluorescence assays showed that the abundances of axoneme ultrastructure markers SPAG6 and SPEF2 and a mitochondrial sheath protein, HSP60, were significantly reduced in the spermatozoa from men harboring bi-allelic CFAP58 variants. We generated Cfap58-knockout mice via CRISPR/Cas9 technology. The male mice were infertile and presented with severe flagellar defects, consistent with the sperm phenotypes in MMAF-affected men. Overall, our findings in humans and mice strongly suggest that CFAP58 plays a vital role in sperm flagellogenesis and demonstrate that bi-allelic loss-of-function variants in CFAP58 can cause axoneme and peri-axoneme malformations leading to male infertility. This study provides crucial insights for understanding and counseling of MMAF-associated asthenoteratozoospermia.

He X ，Liu C ，Yang X ，Lv M ，Ni X ，Li Q ，Cheng H ，Liu W ，Tian S ，Wu H ，Gao Y ，Yang C ，Tan Q ，Cong J ，Tang D ，Zhang J ，Song B ，Zhong Y ，Li H ，Zhi W ，Mao X ，Fu F ，Ge L ，Shen Q ，Zhang M ，Saiyin H ，Jin L ，Xu Y ，Zhou P ，Wei Z ，Zhang F ，Cao Y ... -  《-》