Loss of Calmodulin- and Radial-Spoke-Associated Complex Protein CFAP251 Leads to Immotile Spermatozoa Lacking Mitochondria and Infertility in Men

Abstract

Flagella and motile cilia share a 9 + 2 microtubule-doublet axoneme structure, and asthenozoospermia (reduced spermatozoa motility) is found in 76% of men with primary ciliary dyskinesia (PCD). Nevertheless, causal genetic variants in a conserved axonemal component have been found in cases of isolated asthenozoospermia: 30% of men with multiple morphological anomalies of sperm flagella (MMAF) carry bi-allelic mutations in DNAH1, encoding one of the seven inner-arm dynein heavy chains of the 9 + 2 axoneme. To further understand the basis for isolated asthenozoospermia, we used whole-exome and Sanger sequencing to study two brothers and two independent men with MMAF. In three men, we found bi-allelic loss-of-function mutations in WDR66, encoding cilia- and flagella-associated protein 251 (CFAP251): the two brothers were homozygous for the frameshift chr12: g.122359334delA (p.Asp42Metfs4), and the third individual was compound heterozygous for chr12: g.122359542G>T (p.Glu111) and chr12: g.122395032_122395033delCT (p.Leu530Valfs4). We show that CFAP251 is normally located along the flagellum but is absent in men carrying WDR66 mutations and reveal a spermatozoa-specific isoform probably generated during spermatozoon maturation. CFAP251 is a component of the calmodulin- and radial-spoke- associated complex, located adjacent to DNAH1, on the inner surface of the peripheral microtubule doublets of the axoneme. In Tetrahymena, the CFAP251 ortholog is necessary for efficient coordinated ciliary beating. Using immunofluorescent and transmission electron microscopy, we provide evidence that loss of CFAP251 affects the formation of the mitochondrial sheath. We propose that CFAP251 plays a structural role during biogenesis of the spermatozoon flagellum in vertebrates.

Whole exome sequencing of men with multiple morphological abnormalities of the sperm flagella reveals novel homozygous QRICH2 mutations

“…The genetic characterization of this phenotype began in 2014 with the identification of DNAH1 (dynein axonemal heavy chain 1) as a major gene implicated in MMAF . After the development and the introduction of high throughput sequencing (HTS) techniques such as whole exome sequencing (WES), deleterious mutations were formally identified in eight additional MMAF genes ( AK7 , ARMC2 , CFAP43 , CFAP44 , CFAP69 , FSIP2 , TTC21A , and WDR66 ) showing the high genetic heterogeneity of this phenotype. Despite these important advances in gene identification, about half of the MMAF cases remain idiopathic without a genetic diagnosis.…”

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

“…Many researchers have shown that the MMAF phenotype is associated with genetic mutations. Previous studies revealed that mutations in DNAH1 (MIM: 603332), DNAH2 (MIM: 603333), AKAP4 (MIM: 300185), CCDC39 (MIM: 613798), AK7 (MIM: 615364), CFAP251 (MIM: 618146), CEP135 (MIM: 611423), FSIP2 (MIM: 615796), ARMC2 (MIM: 611226), QRICH2 (MIM: 618304), SPEF2 (MIM: 610172), CFAP69 (MIM: 617949), CFAP43 (MIM: 617558) and CFAP44 (MIM:617559) could cause the human MMAF phenotype . However, more research is needed to reveal the etiology of the remaining 30%–40% of MMAF cases.…”

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

“…It has been reported that loss‐of‐function mutations in several cilia‐ and flagella‐associated protein‐(CFAP)‐encoding genes are identified to cause MMAF in human and mouse . In this study, a homozygous nonsense mutation in another CFAP‐encoding gene, CFAP65 , was detected in a MMAF patient.…”

“…Since 2014, when MMAF was defined, studies have shown that mutations in genes associated with sperm flagellum development may lead to MMAF . Here, another candidate gene, CFAP65 , was identified in an infertile male with MMAF by WES.…”

“…The genetic basis of male infertility is still a work in progress, more than 4000 genes are expressed in germ cells during sperm production, and variations in these genes may hamper spermatogenesis . Recent studies of families with positive history related to male infertility have reported that mutations in cilia‐related genes are responsible for MMAF (eg, DNAH1 , CFAP43 , CFAP44 , CFAP69 , CFAP251 , QRICH2 , AK7 , CEP135 , FSIP2 , SPEF2 , TTC21A , ARMC2 , AKAP4 , CCDC39 ) . However, the known genetic defects can only explain ~60% of the individuals affected by MMAF …”