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.