{"gene":"CFAP251","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":2018,"finding":"CFAP251 (WDR66) is a component of the calmodulin- and radial-spoke-associated complex, located adjacent to DNAH1 on the inner surface of the peripheral microtubule doublets of the axoneme, and loss of CFAP251 affects formation of the mitochondrial sheath during spermatozoon flagellum biogenesis.","method":"Immunofluorescence, transmission electron microscopy, whole-exome sequencing of loss-of-function mutations in human patients; Tetrahymena ortholog functional studies","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (IF, TEM, patient mutations) replicated across two independent studies in the same year","pmids":["30122541","30122540"],"is_preprint":false},{"year":2018,"finding":"Homozygous deletion of the two penultimate exons of WDR66/CFAP251 causes MMAF; the Trypanosoma WDR66 ortholog (TbWDR66) localizes to the axoneme, and reproduction of the human deletion in TbWDR66 impaired flagellar movement, confirming the importance of the WDR66 C-terminal region for flagellar function.","method":"Whole-exome sequencing, SVA breakpoint analysis, Trypanosoma TbWDR66 genetic deletion and motility assay","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 1–2 — genetic deletion in model organism with direct motility readout, confirmed in human patient cohort","pmids":["30122540"],"is_preprint":false},{"year":2018,"finding":"Biallelic loss-of-function mutations in CFAP251 result in absence of CFAP251 protein along the sperm flagellum, demonstrating that CFAP251 is required for flagellar development; a spermatozoa-specific isoform was identified that is likely generated during spermatozoon maturation.","method":"Whole-exome sequencing, immunofluorescence staining of patient spermatozoa, Western blot","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 — reciprocal human genetic and protein localization evidence with multiple patients","pmids":["30122541"],"is_preprint":false},{"year":2018,"finding":"Biallelic loss-of-function mutations in CFAP251 identified in MMAF patients abolish CFAP251 protein expression in sperm flagella, supporting its role in flagellar function.","method":"Whole-exome sequencing, immunofluorescence staining of patient spermatozoa","journal":"Journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 — single lab, patient-based evidence with IF confirmation","pmids":["30310178"],"is_preprint":false},{"year":2022,"finding":"A novel splicing mutation in CFAP251 (c.1192-3C>G) causes exon skipping, leading to significantly reduced CFAP251 protein expression in spermatozoa and MMAF phenotype.","method":"Splicing assay, Western blot, immunofluorescence in patient spermatozoa","journal":"Frontiers in genetics","confidence":"Medium","confidence_rationale":"Tier 2 — direct splicing mechanism demonstrated with protein expression validation","pmids":["35087568"],"is_preprint":false},{"year":2026,"finding":"CFAP251 interacts with TUBB4B, and its disruption leads to downregulation of TUBB4B, impairing spermiogenesis and ciliary function; CFAP251 also interacts with the mitochondrial protein SLC25A4, suggesting a role in regulating energy transport.","method":"Cfap251 knockout mouse model, co-immunoprecipitation/interaction assays, whole-exome sequencing, immunofluorescence","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 — KO mouse model replicating human phenotype plus interaction data with TUBB4B and SLC25A4, single study","pmids":["41943837"],"is_preprint":false}],"current_model":"CFAP251 (WDR66) is an axonemal protein residing in the calmodulin- and radial-spoke-associated complex at the base of radial spoke 3 on the inner surface of peripheral microtubule doublets; it physically interacts with TUBB4B (required for spermiogenesis and ciliary function) and the mitochondrial carrier SLC25A4, and its loss-of-function—via homozygous deletion, frameshift, nonsense, or splice mutations—abolishes CFAP251 protein from the sperm flagellum, disrupts mitochondrial sheath formation and flagellar beating, and causes multiple morphological abnormalities of sperm flagella (MMAF) and male infertility."},"narrative":{"teleology":[{"year":2018,"claim":"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","pmids":["30122541","30122540","30310178"],"confidence":"High","gaps":["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"]},{"year":2018,"claim":"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","pmids":["30122540"],"confidence":"High","gaps":["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"]},{"year":2022,"claim":"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","pmids":["35087568"],"confidence":"Medium","gaps":["Threshold of CFAP251 protein required for normal flagellar function was not quantified","Whether heterozygous carriers have subclinical motility defects was not examined"]},{"year":2026,"claim":"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","pmids":["41943837"],"confidence":"Medium","gaps":["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"]},{"year":null,"claim":"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.","evidence":"","pmids":[],"confidence":"Low","gaps":["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"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1,5]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[0,1,2,3]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,1]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,1,2]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[0,2,5]}],"complexes":["Calmodulin- and radial-spoke-associated complex (CSC)"],"partners":["TUBB4B","SLC25A4","DNAH1"],"other_free_text":[]},"mechanistic_narrative":"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]."},"prefetch_data":{"uniprot":{"accession":"Q8TBY9","full_name":"Cilia- and flagella-associated protein 251","aliases":["WD repeat-containing protein 66"],"length_aa":1149,"mass_kda":130.0,"function":"Involved in spermatozoa motility (PubMed:30122540, PubMed:30122541). May also regulate cilium motility through its role in the assembly of the axonemal radial spokes (By similarity)","subcellular_location":"Cytoplasm, cytoskeleton, cilium axoneme; Cell projection, cilium, flagellum","url":"https://www.uniprot.org/uniprotkb/Q8TBY9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CFAP251","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CFAP251","total_profiled":1310},"omim":[{"mim_id":"621376","title":"CILIA- AND FLAGELLA-ASSOCIATED PROTEIN 206; CFAP206","url":"https://www.omim.org/entry/621376"},{"mim_id":"620381","title":"CILIA- AND FLAGELLA-ASSOCIATED PROTEIN 61; CFAP61","url":"https://www.omim.org/entry/620381"},{"mim_id":"618152","title":"SPERMATOGENIC FAILURE 33; SPGF33","url":"https://www.omim.org/entry/618152"},{"mim_id":"618146","title":"CILIA- AND FLAGELLA-ASSOCIATED PROTEIN 251; CFAP251","url":"https://www.omim.org/entry/618146"},{"mim_id":"609910","title":"CILIA- AND FLAGELLA-ASSOCIATED PROTEIN 91; CFAP91","url":"https://www.omim.org/entry/609910"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Mid piece","reliability":"Supported"},{"location":"End piece","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"choroid plexus","ntpm":19.0},{"tissue":"fallopian tube","ntpm":20.9},{"tissue":"testis","ntpm":18.6}],"url":"https://www.proteinatlas.org/search/CFAP251"},"hgnc":{"alias_symbol":["MGC33630","CaM-IP4"],"prev_symbol":["WDR66"]},"alphafold":{"accession":"Q8TBY9","domains":[{"cath_id":"2.130.10.10","chopping":"801-934","consensus_level":"medium","plddt":91.3002,"start":801,"end":934},{"cath_id":"1.10.238.10","chopping":"941-1038","consensus_level":"high","plddt":87.23,"start":941,"end":1038},{"cath_id":"-","chopping":"1046-1136","consensus_level":"high","plddt":77.7204,"start":1046,"end":1136}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TBY9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TBY9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TBY9-F1-predicted_aligned_error_v6.png","plddt_mean":76.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CFAP251","jax_strain_url":"https://www.jax.org/strain/search?query=CFAP251"},"sequence":{"accession":"Q8TBY9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8TBY9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8TBY9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TBY9"}},"corpus_meta":[{"pmid":"30122540","id":"PMC_30122540","title":"A Homozygous Ancestral SVA-Insertion-Mediated Deletion in WDR66 Induces Multiple Morphological Abnormalities of the Sperm Flagellum and Male Infertility.","date":"2018","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/30122540","citation_count":76,"is_preprint":false},{"pmid":"23514407","id":"PMC_23514407","title":"Wdr66 is a novel marker for risk stratification and involved in epithelial-mesenchymal transition of esophageal squamous cell carcinoma.","date":"2013","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/23514407","citation_count":74,"is_preprint":false},{"pmid":"30122541","id":"PMC_30122541","title":"Loss of Calmodulin- and Radial-Spoke-Associated Complex Protein CFAP251 Leads to Immotile Spermatozoa Lacking Mitochondria and Infertility in Men.","date":"2018","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/30122541","citation_count":74,"is_preprint":false},{"pmid":"30310178","id":"PMC_30310178","title":"Biallelic mutations of CFAP251 cause sperm flagellar defects and human male infertility.","date":"2018","source":"Journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/30310178","citation_count":60,"is_preprint":false},{"pmid":"31225659","id":"PMC_31225659","title":"LINC00324 accelerates the proliferation and migration of osteosarcoma through regulating WDR66.","date":"2019","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/31225659","citation_count":33,"is_preprint":false},{"pmid":"31140103","id":"PMC_31140103","title":"LIN28A-stabilized FBXL19-AS1 promotes breast cancer migration, invasion and EMT by regulating WDR66.","date":"2019","source":"In vitro cellular & developmental biology. Animal","url":"https://pubmed.ncbi.nlm.nih.gov/31140103","citation_count":21,"is_preprint":false},{"pmid":"29717028","id":"PMC_29717028","title":"Long non-coding RNA AK096174 promotes cell proliferation and invasion in gastric cancer by regulating WDR66 expression.","date":"2018","source":"Bioscience reports","url":"https://pubmed.ncbi.nlm.nih.gov/29717028","citation_count":14,"is_preprint":false},{"pmid":"35087568","id":"PMC_35087568","title":"Successful Results of Intracytoplasmic Sperm Injection of a Chinese Patient With Multiple Morphological Abnormalities of Sperm Flagella Caused by a Novel Splicing Mutation in CFAP251.","date":"2022","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/35087568","citation_count":13,"is_preprint":false},{"pmid":"38230259","id":"PMC_38230259","title":"CircPOFUT1 fosters colorectal cancer metastasis and chemoresistance via decoying miR-653-5p/E2F7/WDR66 axis and stabilizing BMI1.","date":"2023","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/38230259","citation_count":8,"is_preprint":false},{"pmid":"41943837","id":"PMC_41943837","title":"CFAP251 Deficiency Induces Male Infertility and PCD-like Ciliary Defects by Disrupting TUBB4B and SLC25A4 Recruitment in Humans and Mice.","date":"2026","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/41943837","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6715,"output_tokens":1418,"usd":0.020708},"stage2":{"model":"claude-opus-4-6","input_tokens":4663,"output_tokens":1597,"usd":0.09486},"total_usd":0.115568,"stage1_batch_id":"msgbatch_011kfMfE81o6CTcyhVUqumuW","stage2_batch_id":"msgbatch_01DTputS6WNkH745x1nVSoBo","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2018,\n      \"finding\": \"CFAP251 (WDR66) is a component of the calmodulin- and radial-spoke-associated complex, located adjacent to DNAH1 on the inner surface of the peripheral microtubule doublets of the axoneme, and loss of CFAP251 affects formation of the mitochondrial sheath during spermatozoon flagellum biogenesis.\",\n      \"method\": \"Immunofluorescence, transmission electron microscopy, whole-exome sequencing of loss-of-function mutations in human patients; Tetrahymena ortholog functional studies\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (IF, TEM, patient mutations) replicated across two independent studies in the same year\",\n      \"pmids\": [\"30122541\", \"30122540\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Homozygous deletion of the two penultimate exons of WDR66/CFAP251 causes MMAF; the Trypanosoma WDR66 ortholog (TbWDR66) localizes to the axoneme, and reproduction of the human deletion in TbWDR66 impaired flagellar movement, confirming the importance of the WDR66 C-terminal region for flagellar function.\",\n      \"method\": \"Whole-exome sequencing, SVA breakpoint analysis, Trypanosoma TbWDR66 genetic deletion and motility assay\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — genetic deletion in model organism with direct motility readout, confirmed in human patient cohort\",\n      \"pmids\": [\"30122540\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Biallelic loss-of-function mutations in CFAP251 result in absence of CFAP251 protein along the sperm flagellum, demonstrating that CFAP251 is required for flagellar development; a spermatozoa-specific isoform was identified that is likely generated during spermatozoon maturation.\",\n      \"method\": \"Whole-exome sequencing, immunofluorescence staining of patient spermatozoa, Western blot\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal human genetic and protein localization evidence with multiple patients\",\n      \"pmids\": [\"30122541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Biallelic loss-of-function mutations in CFAP251 identified in MMAF patients abolish CFAP251 protein expression in sperm flagella, supporting its role in flagellar function.\",\n      \"method\": \"Whole-exome sequencing, immunofluorescence staining of patient spermatozoa\",\n      \"journal\": \"Journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — single lab, patient-based evidence with IF confirmation\",\n      \"pmids\": [\"30310178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"A novel splicing mutation in CFAP251 (c.1192-3C>G) causes exon skipping, leading to significantly reduced CFAP251 protein expression in spermatozoa and MMAF phenotype.\",\n      \"method\": \"Splicing assay, Western blot, immunofluorescence in patient spermatozoa\",\n      \"journal\": \"Frontiers in genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct splicing mechanism demonstrated with protein expression validation\",\n      \"pmids\": [\"35087568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"CFAP251 interacts with TUBB4B, and its disruption leads to downregulation of TUBB4B, impairing spermiogenesis and ciliary function; CFAP251 also interacts with the mitochondrial protein SLC25A4, suggesting a role in regulating energy transport.\",\n      \"method\": \"Cfap251 knockout mouse model, co-immunoprecipitation/interaction assays, whole-exome sequencing, immunofluorescence\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse model replicating human phenotype plus interaction data with TUBB4B and SLC25A4, single study\",\n      \"pmids\": [\"41943837\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CFAP251 (WDR66) is an axonemal protein residing in the calmodulin- and radial-spoke-associated complex at the base of radial spoke 3 on the inner surface of peripheral microtubule doublets; it physically interacts with TUBB4B (required for spermiogenesis and ciliary function) and the mitochondrial carrier SLC25A4, and its loss-of-function—via homozygous deletion, frameshift, nonsense, or splice mutations—abolishes CFAP251 protein from the sperm flagellum, disrupts mitochondrial sheath formation and flagellar beating, and causes multiple morphological abnormalities of sperm flagella (MMAF) and male infertility.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"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].\",\n  \"teleology\": [\n    {\n      \"year\": 2018,\n      \"claim\": \"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.\",\n      \"evidence\": \"Whole-exome sequencing in independent MMAF patient cohorts with immunofluorescence, TEM, and Western blot validation; Tetrahymena ortholog studies\",\n      \"pmids\": [\"30122541\", \"30122540\", \"30310178\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct binding partners within the calmodulin- and radial-spoke-associated complex were not identified\",\n        \"Mechanism by which CFAP251 loss disrupts the mitochondrial sheath was unknown\",\n        \"No knockout animal model existed to confirm cell-autonomous function\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"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.\",\n      \"evidence\": \"Targeted deletion of TbWDR66 C-terminal exons in Trypanosoma brucei with flagellar motility assay\",\n      \"pmids\": [\"30122540\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for C-terminal domain function remains undefined\",\n        \"Whether the C-terminal region mediates protein–protein interactions within the radial spoke complex was untested\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"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.\",\n      \"evidence\": \"Minigene splicing assay, Western blot, and immunofluorescence in patient spermatozoa\",\n      \"pmids\": [\"35087568\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Threshold of CFAP251 protein required for normal flagellar function was not quantified\",\n        \"Whether heterozygous carriers have subclinical motility defects was not examined\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"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.\",\n      \"evidence\": \"Cfap251 KO mouse with co-immunoprecipitation, immunofluorescence, and whole-exome sequencing\",\n      \"pmids\": [\"41943837\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"TUBB4B and SLC25A4 interactions await reciprocal validation and structural mapping\",\n        \"Whether CFAP251–SLC25A4 interaction directly regulates ATP transport or is indirect remains untested\",\n        \"Role of CFAP251 in motile cilia outside the male germline (e.g., respiratory cilia) has not been characterized in the KO model\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"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.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No high-resolution structural data (cryo-ET or cryo-EM) placing CFAP251 within the 96-nm axonemal repeat\",\n        \"Systematic interactome of CFAP251 in ciliated cells has not been performed\",\n        \"Potential role in primary ciliary dyskinesia or respiratory ciliopathy phenotypes is unexplored\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [0, 1, 2, 3]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [0, 2, 5]}\n    ],\n    \"complexes\": [\n      \"Calmodulin- and radial-spoke-associated complex (CSC)\"\n    ],\n    \"partners\": [\n      \"TUBB4B\",\n      \"SLC25A4\",\n      \"DNAH1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}