CFAP251

CFAP251 (WDR66) is an axonemal protein required for the structural organization and motility of cilia and sperm flagella (PMID:30122540, PMID:30122541). It localizes to the calmodulin- and radial-spoke-associated complex at the base of radial spoke 3, residing adjacent to DNAH1 on the inner surface of peripheral microtubule doublets (PMID:30122540, PMID:30122541). CFAP251 physically interacts with beta-tubulin TUBB4B, whose recruitment and stability depend on CFAP251, and with the mitochondrial carrier SLC25A4, linking the protein to axonemal microtubule architecture and to flagellar energy supply (PMID:41943837). Loss of CFAP251 abolishes the protein along the flagellum, disrupts mitochondrial sheath formation during spermatozoon biogenesis, and impairs coordinated ciliary beating (PMID:30122541). Biallelic loss-of-function mutations in CFAP251 cause multiple morphological abnormalities of the sperm flagellum (MMAF) and male infertility, and a knockout mouse additionally displays primary ciliary dyskinesia-like airway defects (PMID:30310178, PMID:35087568, PMID:41943837).

1. 2018 Medium

   Established that WDR66/CFAP251 is an axonemal protein localizing to the calmodulin- and spoke-associated complex and that its C-terminal region is functionally required for flagellar motility, answering whether the gene contributes directly to ciliary/flagellar movement.

   Evidence Whole-exome sequencing plus Trypanosoma TbWDR66 C-terminal deletion model with flagellar movement assay and axonemal localization

   PMID:30122540

   Open questions at the time

   • Mechanism by which the C-terminal region supports motility not defined
   • Direct molecular partners within the axoneme not identified
   • Functional validation relies on a trypanosome ortholog
2. 2018 High

   Defined the precise axonemal position of CFAP251 (base of radial spoke 3, adjacent to DNAH1) and connected its loss to failure of mitochondrial sheath formation, establishing both a structural locale and a developmental consequence in spermatogenesis.

   Evidence Whole-exome/Sanger sequencing, immunofluorescence of mutant sperm, TEM ultrastructure, and Tetrahymena ortholog functional studies

   PMID:30122541

   Open questions at the time

   • Molecular basis of mitochondrial sheath defect not resolved
   • Function of the sperm-specific isoform not characterized
   • Direct binding partners not yet identified
3. 2018 Medium

   Corroborated in independent patient material that biallelic loss-of-function mutations abolish CFAP251 protein in sperm flagella and cause male infertility, strengthening the gene-disease link.

   Evidence Whole-exome sequencing, expression assays, and immunofluorescence in a CFAP251-deficient man

   PMID:30310178

   Open questions at the time

   • Single patient
   • Does not add mechanistic detail beyond protein loss
4. 2022 Medium

   Demonstrated that a splicing mutation causing exon skipping reduces CFAP251 protein in spermatozoa, confirming that loss of CFAP251 protein is the direct cause of MMAF.

   Evidence Whole-exome sequencing, RT-PCR for exon skipping, western blot and immunofluorescence of patient spermatozoa

   PMID:35087568

   Open questions at the time

   • Single case
   • No reconstitution of protein function
5. 2026 Medium

   Identified direct physical partners of CFAP251 (TUBB4B and SLC25A4) and showed via a knockout mouse that CFAP251 loss destabilizes TUBB4B and produces both MMAF and PCD-like ciliary defects, linking the protein to microtubule stability and flagellar energy transport.

   Evidence Cfap251 knockout mouse, human proband exome sequencing, interaction assay, and immunofluorescence/western blot showing TUBB4B downregulation

   PMID:41943837

   Open questions at the time

   • Co-IP versus pulldown methodology not specified
   • Mechanism by which SLC25A4 interaction supports energy transport not established
   • Stoichiometry and direct versus indirect nature of interactions unconfirmed

• How CFAP251 organizes the calmodulin- and radial-spoke-associated complex at a structural level and how its loss mechanistically links axonemal disruption to mitochondrial sheath failure remain unresolved.
• No structural model of the CFAP251 complex
• Mechanism connecting axonemal role to mitochondrial sheath formation unknown
• Function of the sperm-specific isoform uncharacterized