Identification and splicing assessment of a novel homozygous CFAP65 splice-site variant associated with oligozoospermia and complete asthenozoospermia

Abstract

Pathogenic variants in CFAP65 have been reported in male infertility with sperm motility defects. This study investigated a novel homozygous CFAP65 splice-site variant identified in a consanguineous family in which the proband presented with oligozoospermia and complete asthenozoospermia. Whole-exome sequencing was performed to identify candidate variants, followed by population- and disease-database annotation. Kinship and runs of homozygosity analyses were used to assess familial relatedness and autozygosity. RNA Splicer prediction and a minigene assay in HEK-293T cells were performed to evaluate the splicing effect of the identified variant. Semen analysis showed a sperm concentration of 8.1 × 10⁶/mL, with total and progressive motility of 0%. Whole-exome sequencing identified a novel homozygous canonical splice-site variant in CFAP65 (NM_194302.4:c.645 + 1G > A), which was classified as pathogenic according to ACMG guidelines. ROH analysis showed increased autosomal homozygosity in the proband (FROH = 7.88%), supporting parental consanguinity. The minigene assay demonstrated complete exon 6 skipping, resulting in a 103-bp transcript deletion and a predicted frameshifted truncated protein p.(Pro182ArgfsTer29). This study expands the CFAP65 mutational spectrum and supports exon 6 skipping-mediated loss of function as the likely pathogenic mechanism. These findings highlight the value of genetic screening and functional splicing assessment in male infertility with sperm motility defects.