A novel homozygous frameshift mutation in CFAP65 is associated with multiple morphological abnormalities of sperm flagella in a consanguineous Pakistani family

Abstract

Multiple morphological abnormalities of the sperm flagella (MMAF) represent a severe form of asthenozoospermia that leads to male infertility, and it is commonly associated with genetic defects affecting flagellar components. Although the gene CFAP65 has been implicated in MMAF, its full mutational spectrum and clinical relevance within highly consanguineous populations remain poorly characterized. To elucidate the genetic basis of this condition, whole-exome sequencing followed by Sanger sequencing was performed in two infertile individuals from a consanguineous Pakistani family. Sperm morphology was assessed using hematoxylin-eosin (H&E) staining; while scanning and transmission electron microscopy (SEM and TEM) were utilized to evaluate ultrastructural defects. A novel homozygous frameshift mutation in CFAP65 (c.582_587delinsCG; p.Q194Hfs*4) was identified. This mutation introduces a premature stop codon within the transmembrane helix domain. Morphological analysis revealed classic MMAF features, including absent, short, bent, and coiled flagella in over 97% of sperm. TEM further demonstrated severe axonemal disorganization, with absent or disrupted microtubule doublets observed in 85% of the cross-sections. Based on clinical history and chest X-ray, the affected individuals reported no chronic respiratory symptoms and had no situs inversus, suggesting an isolated sperm-specific phenotype. In conclusion, this study identifies a novel domain-specific CFAP65 mutation associated with MMAF and male infertility in a consanguineous Pakistani family, thereby expanding the known genetic mutational landscape of MMAF. These findings reinforce the crucial role of CFAP65 in sperm flagellar morphogenesis and underscore its significance in the genetic diagnosis of male infertility.