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

Abstract

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. <i>CFAP65</i> homozygous mutations were identified in four unrelated consanguineous families, and <i>CFAP65</i> compound heterozygous mutations were found in two unrelated cases with MMAF. All these <i>CFAP65</i> 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 <i>CFAP65</i> variants likely affecting splicing were identified in two MMAF-affected men of Tunisian and Iranian ancestries, respectively. These biallelic variants of <i>CFAP65</i> were verified by Sanger sequencing and were absent or very rare in large data sets aggregating sequence information from various human populations. <i>CFAP65</i>, 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 <i>Cfap65</i> using CRISPR-Cas9 technology. Remarkably, the phenotypes of <i>Cfap65</i>-mutated male mice were consistent with human MMAF. Our experimental observations performed on both human subjects and on <i>Cfap65</i>-mutated mice demonstrate that the presence of biallelic mutations in <i>CFAP65</i> causes the MMAF phenotype and impairs sperm motility.