CFAP251

CFAP251 (WDR66) is an axonemal protein that functions within the calmodulin- and radial-spoke-associated complex on the inner surface of peripheral microtubule doublets and is essential for sperm flagellum assembly and motility. It localizes along the sperm flagellum, where it physically interacts with TUBB4B to support spermiogenesis and ciliary function and with the mitochondrial carrier SLC25A4, linking axonemal integrity to mitochondrial sheath formation and energy transport (PMID:30122541, PMID:41943837). Loss of the C-terminal region impairs flagellar beating, as demonstrated by ortholog deletion in Trypanosoma (PMID:30122540). Biallelic loss-of-function mutations in CFAP251—including deletions, frameshifts, nonsense, and splice-site variants—cause multiple morphological abnormalities of the sperm flagella (MMAF) and male infertility (PMID:30122541, PMID:30122540, PMID:35087568).

1. 2018 High

   Identification of CFAP251 as a disease gene resolved the question of whether axonemal WDR-repeat proteins could independently cause MMAF: biallelic loss-of-function mutations abolished CFAP251 from the flagellum and disrupted both axonemal ultrastructure and mitochondrial sheath formation.

   Evidence Whole-exome sequencing in independent MMAF patient cohorts with immunofluorescence, TEM, and Western blot validation; Tetrahymena ortholog studies

   PMID:30122540 PMID:30122541 PMID:30310178

   Open questions at the time

   • Direct binding partners within the calmodulin- and radial-spoke-associated complex were not identified
   • Mechanism by which CFAP251 loss disrupts the mitochondrial sheath was unknown
   • No knockout animal model existed to confirm cell-autonomous function
2. 2018 High

   Cross-species deletion of the WDR66 C-terminal region in Trypanosoma demonstrated that the conserved C-terminus is specifically required for flagellar beating, establishing functional conservation and pinpointing a critical domain.

   Evidence Targeted deletion of TbWDR66 C-terminal exons in Trypanosoma brucei with flagellar motility assay

   PMID:30122540

   Open questions at the time

   • Structural basis for C-terminal domain function remains undefined
   • Whether the C-terminal region mediates protein–protein interactions within the radial spoke complex was untested
3. 2022 Medium

   Discovery of a splicing mutation (c.1192-3C>G) causing exon skipping and reduced CFAP251 protein established that even partial reduction of CFAP251 expression is sufficient to produce MMAF, broadening the mutational spectrum.

   Evidence Minigene splicing assay, Western blot, and immunofluorescence in patient spermatozoa

   PMID:35087568

   Open questions at the time

   • Threshold of CFAP251 protein required for normal flagellar function was not quantified
   • Whether heterozygous carriers have subclinical motility defects was not examined
4. 2026 Medium

   A Cfap251 knockout mouse model identified TUBB4B and SLC25A4 as direct interacting partners, linking axonemal structural integrity to tubulin homeostasis and mitochondrial energy transport and explaining the earlier observation of mitochondrial sheath defects.

   Evidence Cfap251 KO mouse with co-immunoprecipitation, immunofluorescence, and whole-exome sequencing

   PMID:41943837

   Open questions at the time

   • TUBB4B and SLC25A4 interactions await reciprocal validation and structural mapping
   • Whether CFAP251–SLC25A4 interaction directly regulates ATP transport or is indirect remains untested
   • Role of CFAP251 in motile cilia outside the male germline (e.g., respiratory cilia) has not been characterized in the KO model

• The precise position of CFAP251 within the radial spoke 3 base architecture, its full interactome within the calmodulin- and radial-spoke-associated complex, and whether its loss affects motile cilia beyond spermatozoa remain unresolved.
• No high-resolution structural data (cryo-ET or cryo-EM) placing CFAP251 within the 96-nm axonemal repeat
• Systematic interactome of CFAP251 in ciliated cells has not been performed
• Potential role in primary ciliary dyskinesia or respiratory ciliopathy phenotypes is unexplored