{"gene":"CFAP410","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2015,"finding":"C21ORF2 (CFAP410) interacts with NEK1 kinase, and C21ORF2-depleted cells show impaired homologous recombination (but not non-homologous end joining) after ionizing radiation. Overexpression of NEK1 rescued the DNA repair defect in C21ORF2-knockdown cells, placing C21ORF2 in the same pathway as NEK1 for DNA damage repair.","method":"siRNA knockdown, colony survival assay, HR/NHEJ reporter assays, NEK1 overexpression rescue","journal":"Acta biochimica et biophysica Sinica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via rescue experiment and reporter assays, single lab, two orthogonal methods","pmids":["26290490"],"is_preprint":false},{"year":2015,"finding":"C21ORF2 (CFAP410) protein localizes to ciliary structures of photoreceptor cells, specifically the daughter basal body, the centriole adjacent to the basal body, and the connecting cilium, as determined by immunohistochemistry in human, pig, and mouse retinas.","method":"Immunohistochemistry on mammalian (human, pig, mouse) retinal sections","journal":"The British journal of ophthalmology","confidence":"Medium","confidence_rationale":"Tier 2 / Strong — direct localization experiment replicated across three species","pmids":["26294103"],"is_preprint":false},{"year":2016,"finding":"C21ORF2 (CFAP410) protein localizes to the connecting cilium of cone and rod photoreceptors, and is implicated in cartilage differentiation in chondrocytes based on functional data.","method":"Immunolocalization in retinal tissue; functional data in chondrocyte cells","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiment, single lab, two cell-type contexts","pmids":["26974433"],"is_preprint":false},{"year":2016,"finding":"Missense mutations in the leucine-rich repeat C-terminal (LRRCT) domain of C21ORF2 (CFAP410) reduce protein stability and alter cytoplasmic localization of mutant proteins compared to wild type in vitro, and C21ORF2 is required for ciliogenesis in retinal photoreceptors.","method":"In vitro expression assays, immunofluorescence, western blotting of mutant constructs in cell lines","journal":"Investigative ophthalmology & visual science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional mutagenesis with protein stability and localization readouts, single lab","pmids":["27548899"],"is_preprint":false},{"year":2020,"finding":"FBXO3 (substrate receptor of an SCF ubiquitin ligase complex) binds and ubiquitylates C21ORF2, targeting it for proteasomal degradation. NEK1-mediated phosphorylation of C21ORF2 attenuates its interaction with FBXO3, thereby stabilizing C21ORF2. The ALS-associated V58L mutant of C21ORF2 is hyperphosphorylated by NEK1, escapes FBXO3-mediated ubiquitylation, and accumulates together with NEK1. Expression of C21ORF2(V58L) in mouse ESC-derived motor neurons impaired neurite outgrowth.","method":"Co-immunoprecipitation, ubiquitylation assays, phosphorylation assays, motor neuron differentiation from mESCs, neurite outgrowth measurement","journal":"iScience","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reciprocal Co-IP, ubiquitylation assay, phosphorylation assay, and functional cellular readout in a single study with multiple orthogonal methods","pmids":["32891887"],"is_preprint":false},{"year":2023,"finding":"Endogenous NEK1 and C21ORF2 form a tight complex in human cells. A C-terminal interaction domain (CID) on NEK1 is necessary for association with C21ORF2, and pathogenic mutations in this region disrupt the complex. AlphaFold modelling predicts an extended binding interface between the leucine-rich repeat domain of C21ORF2 and the NEK1-CID. NEK1 mutations that inhibit kinase activity or weaken NEK1-C21ORF2 association severely compromise ciliogenesis. C21ORF2, like NEK1, is required for homologous recombination.","method":"Endogenous Co-immunoprecipitation, AlphaFold structural modelling, ciliogenesis assays, HR repair assays, pathogenic mutant analysis","journal":"Life science alliance","confidence":"High","confidence_rationale":"Tier 2 / Strong — endogenous reciprocal Co-IP, structural modelling, ciliogenesis and HR functional assays in same study with multiple orthogonal methods","pmids":["37188479"],"is_preprint":false},{"year":2023,"finding":"CFAP410 pathogenic variants (p.Tyr107His and p.Pro116Leu) reduce protein stability and alter the ubiquitination level of CFAP410, as measured by co-IP and western blotting in HEK293T cells, suggesting the ubiquitin-proteasome pathway mediates CFAP410 degradation.","method":"Co-immunoprecipitation, western blotting, immunofluorescence, cell cycle analysis in HEK293T cells","journal":"Frontiers in medicine","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and western blot with mutant constructs, single lab, two orthogonal methods","pmids":["37901396"],"is_preprint":false},{"year":2024,"finding":"The C-terminal domain (CTD) of CFAP410 forms a tetrameric helical bundle, as determined by crystal structures from Homo sapiens, Trypanosoma brucei, and Chlamydomonas reinhardtii. The tetrameric assembly is essential for correct basal body localization of CFAP410; the ALS/spondylometaphyseal dysplasia-associated L224P mutation disassembles the tetramer and disrupts basal body localization in T. brucei.","method":"X-ray crystallography (crystal structures from three organisms), functional localization assays with L224P mutant in T. brucei","journal":"Open biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure determination replicated across three organisms plus functional mutagenesis validating localization requirement","pmids":["39255848"],"is_preprint":false},{"year":2025,"finding":"C21ORF2 localizes to the basal body of the primary cilium in human motor neurons. ALS-associated mutations alter this basal body localization. Reduction of C21ORF2 levels causes fewer primary cilia and reduced ciliary length, leading to defective sonic hedgehog (SHH) signalling, reduced CRABP1 expression, and impaired neuromuscular junction formation. Overexpression of C21ORF2 in mutant motor neurons rescued ciliary frequency and length, CRABP1 expression, and NMJ formation.","method":"iPSC-derived motor neuron cultures, immunofluorescence, siRNA knockdown, SHH pathway reporter assays, compartmentalized co-culture NMJ assay, rescue by overexpression","journal":"Brain : a journal of neurology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (localization, KD, pathway assays, rescue) in patient-derived neurons and isogenic controls","pmids":["39703094"],"is_preprint":false},{"year":2024,"finding":"The ALS-associated C21ORF2-V58L variant causes increased apoptosis in mouse neurons, movement defects in zebrafish, and in iPSC-derived motor neurons: defects in DNA damage response, mitochondrial dysfunction, and changes in neuronal excitability. V58L induces post-transcriptional downregulation of NEK1 protein levels.","method":"iPSC-derived motor neurons (patient vs isogenic controls), zebrafish model, apoptosis assays, DNA damage response assays, mitochondrial assays, western blotting for NEK1","journal":"Acta neuropathologica communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods in patient-derived vs isogenic control neurons and in vivo zebrafish model, single lab","pmids":["39227882"],"is_preprint":false},{"year":2025,"finding":"Knock-in of ALS-associated Cfap410 variants in mouse ESCs results in impaired interaction with Nek1 and increased susceptibility to DNA damage in ESCs, neural progenitors, and differentiated neurons, without significant effects on primary cilia frequency.","method":"Gene editing (knock-in) in mESCs, neural differentiation, DNA damage assays, Co-immunoprecipitation for Nek1 interaction","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — endogenous knock-in model with Co-IP and functional DNA damage readouts, single lab","pmids":["40933646"],"is_preprint":false},{"year":2025,"finding":"CFAP410 adopts a bimodular architecture. The 1.0-Å resolution crystal structure of the N-terminal domain (NTD) of T. brucei CFAP410 reveals a leucine-rich repeat fold with a conserved surface patch. Disease-causing mutations in the NTD destabilize the structure and are predicted to disrupt interactions with binding partners.","method":"X-ray crystallography (1.0-Å resolution), structural analysis of disease-causing mutations","journal":"Frontiers in cell and developmental biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — high-resolution crystal structure with mutagenesis-based structural interpretation; single lab but rigorous structural method","pmids":["40018707"],"is_preprint":false},{"year":2026,"finding":"C21ORF2 promotes activation of the JAK2/STAT3 signaling pathway in prostate cancer cells, upregulating downstream targets c-MYC, Cyclin A1, Bcl-2, and Cleaved caspase-3. Co-immunoprecipitation demonstrated interaction between C21ORF2 and downstream targets; C21ORF2 showed negative correlation with KCTD5. In vivo xenograft models validated C21ORF2's role in tumor growth.","method":"Co-immunoprecipitation, western blotting, CCK-8 proliferation assay, Transwell assay, TUNEL staining, subcutaneous xenograft mouse model","journal":"BMC cancer","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, Co-IP without reciprocal validation, pathway activation inferred from western blot of downstream targets","pmids":["41612237"],"is_preprint":false}],"current_model":"CFAP410 (C21ORF2) is a basal body/ciliary protein whose C-terminal domain forms a tetrameric helical bundle required for basal body localization; it forms a tight complex with NEK1 kinase through a dedicated interaction domain, promotes homologous recombination-based DNA repair in the same pathway as NEK1, and is targeted for proteasomal degradation by the SCF-FBXO3 ubiquitin ligase—a process counteracted by NEK1-mediated phosphorylation of CFAP410; loss of CFAP410 function impairs primary ciliogenesis and downstream SHH signalling, leading to neuronal and photoreceptor dysfunction, while disease-associated mutations reduce protein stability, disrupt basal body localization, and impair NEK1 interaction."},"narrative":{"mechanistic_narrative":"CFAP410 (C21ORF2) is a basal body and connecting cilium protein that couples primary ciliogenesis to NEK1-dependent genome maintenance, and its loss of function disrupts both processes in neurons and photoreceptors [PMID:26294103, PMID:37188479, PMID:39703094]. It is built from two modules: an N-terminal leucine-rich repeat domain that engages binding partners and a C-terminal domain that assembles into a tetrameric helical bundle required for correct basal body localization, disruption of which (e.g. the L224P variant) disassembles the tetramer and mislocalizes the protein [PMID:39255848, PMID:40018707]. Through its leucine-rich repeat surface CFAP410 forms a tight complex with the kinase NEK1 via a dedicated C-terminal interaction domain on NEK1, and acts in the same pathway as NEK1 to promote homologous-recombination repair of DNA damage and to support ciliogenesis [PMID:26290490, PMID:37188479]. CFAP410 abundance is controlled by the ubiquitin–proteasome system: the SCF substrate receptor FBXO3 binds and ubiquitylates it for degradation, while NEK1-mediated phosphorylation attenuates the FBXO3 interaction and stabilizes the protein [PMID:32891887]. Reduced CFAP410 produces fewer and shorter primary cilia, defective sonic hedgehog signalling, and impaired neuromuscular junction formation, all reversible by re-expression [PMID:39703094]. Disease-associated missense and structural mutations destabilize the protein, alter its localization and ubiquitination, and weaken the NEK1 interaction, linking CFAP410 to ALS and spondylometaphyseal dysplasia phenotypes [PMID:27548899, PMID:37901396, PMID:39255848, PMID:40933646].","teleology":[{"year":2015,"claim":"Established that CFAP410 acts in DNA damage repair within the NEK1 pathway rather than as an isolated factor, defining its first functional partner.","evidence":"siRNA knockdown with HR/NHEJ reporter assays and NEK1 overexpression rescue in human cells","pmids":["26290490"],"confidence":"Medium","gaps":["Did not define the physical interaction interface","Mechanism by which CFAP410 promotes HR specifically was not resolved"]},{"year":2015,"claim":"Localized CFAP410 to specific ciliary substructures of photoreceptors, anchoring it to the basal body/connecting cilium compartment.","evidence":"Immunohistochemistry on human, pig, and mouse retinal sections","pmids":["26294103"],"confidence":"Medium","gaps":["Did not establish ciliary function mechanistically","No link to a molecular pathway at the cilium"]},{"year":2016,"claim":"Extended ciliary localization to cone and rod connecting cilia and tied CFAP410 to ciliogenesis and chondrocyte differentiation, broadening its tissue relevance.","evidence":"Immunolocalization in retina plus functional data in chondrocytes; mutant expression assays showing reduced stability and altered localization","pmids":["26974433","27548899"],"confidence":"Medium","gaps":["Did not identify the degradation machinery acting on mutants","Connection between cartilage and ciliary roles unresolved"]},{"year":2020,"claim":"Defined how CFAP410 levels are regulated, showing FBXO3 ubiquitylates it for degradation while NEK1 phosphorylation stabilizes it, and that an ALS mutant escapes this control.","evidence":"Reciprocal Co-IP, ubiquitylation and phosphorylation assays, and neurite outgrowth in mESC-derived motor neurons","pmids":["32891887"],"confidence":"High","gaps":["The SCF complex composition beyond FBXO3 not detailed","Phosphosites mediating FBXO3 escape not fully mapped"]},{"year":2023,"claim":"Characterized the endogenous NEK1–CFAP410 complex at structural resolution, mapping a C-terminal interaction domain on NEK1 and linking complex integrity to both ciliogenesis and HR.","evidence":"Endogenous reciprocal Co-IP, AlphaFold modelling, ciliogenesis and HR assays with pathogenic mutants","pmids":["37188479"],"confidence":"High","gaps":["Interface modelled by AlphaFold, not crystallographically resolved as a complex","How the same complex partitions between cilium and DNA repair unresolved"]},{"year":2023,"claim":"Confirmed that additional pathogenic CFAP410 variants destabilize the protein and alter its ubiquitination, generalizing the proteostatic mechanism of disease mutations.","evidence":"Co-IP, western blotting, immunofluorescence, and cell cycle analysis in HEK293T cells","pmids":["37901396"],"confidence":"Medium","gaps":["Did not test FBXO3/NEK1 dependence for these specific variants","Functional ciliary consequence not assayed"]},{"year":2024,"claim":"Solved the bimodular architecture, showing the CTD forms a tetrameric helical bundle essential for basal body localization and the NTD is an LRR fold whose mutations destabilize binding-partner interactions.","evidence":"X-ray crystallography across three organisms plus L224P localization assays in T. brucei; 1.0-Å NTD structure with disease-mutation analysis","pmids":["39255848","40018707"],"confidence":"High","gaps":["Identity of NTD surface-patch binding partners not experimentally confirmed","Structure of the full-length protein or partner complexes not determined"]},{"year":2024,"claim":"Demonstrated multi-organ pathogenicity of the V58L variant, linking it to DNA damage response defects, mitochondrial dysfunction, altered excitability, and reduced NEK1 protein.","evidence":"iPSC-derived motor neurons vs isogenic controls, zebrafish model, apoptosis/DNA damage/mitochondrial assays, NEK1 western blot","pmids":["39227882"],"confidence":"Medium","gaps":["Mechanism linking V58L to mitochondrial dysfunction unclear","How V58L downregulates NEK1 post-transcriptionally not defined"]},{"year":2025,"claim":"Connected CFAP410 loss to a ciliary signalling axis in motor neurons, showing reduced cilia impair SHH signalling, CRABP1 expression, and NMJ formation, all rescued by re-expression.","evidence":"iPSC-derived motor neurons, siRNA knockdown, SHH reporter assays, compartmentalized NMJ co-culture, overexpression rescue","pmids":["39703094"],"confidence":"High","gaps":["Whether SHH defect is upstream or parallel to NMJ phenotype not fully resolved","Relationship to the DNA-repair role in the same cells not integrated"]},{"year":2025,"claim":"Showed in an endogenous knock-in model that ALS variants impair the NEK1 interaction and increase DNA damage susceptibility without altering cilia frequency, dissociating the genome-maintenance from the ciliary phenotype.","evidence":"Knock-in mESCs, neural differentiation, DNA damage assays, Co-IP for Nek1 interaction","pmids":["40933646"],"confidence":"Medium","gaps":["Why cilia were unaffected here but affected in other models unresolved","Quantitative contribution of NEK1-binding loss to DNA damage not isolated"]},{"year":2026,"claim":"Proposed a context-specific oncogenic role via JAK2/STAT3 activation in prostate cancer, expanding CFAP410 function beyond cilia and DNA repair.","evidence":"Co-IP, western blotting, proliferation/migration assays, TUNEL, and xenograft model in prostate cancer cells","pmids":["41612237"],"confidence":"Low","gaps":["Co-IP without reciprocal validation; direct interaction unconfirmed","JAK2/STAT3 activation inferred only from downstream target western blots","No mechanistic link to the established ciliary/NEK1 functions"]},{"year":null,"claim":"How CFAP410 mechanistically integrates its basal body/ciliogenesis role with NEK1-dependent homologous recombination in the same cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No model explains the partition of the CFAP410–NEK1 complex between cilium and nucleus","NTD binding partners beyond NEK1 unidentified","Structure of the assembled CFAP410–NEK1 complex not experimentally determined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[7]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[1,2,8]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[1,7,8]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[0,5,10]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[5,8]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[8]}],"complexes":["CFAP410-NEK1 complex","SCF-FBXO3 ubiquitin ligase (substrate)"],"partners":["NEK1","FBXO3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O43822","full_name":"Cilia- and flagella-associated protein 410","aliases":["C21orf-HUMF09G8.5","Leucine-rich repeat-containing protein 76","YF5/A2"],"length_aa":256,"mass_kda":28.3,"function":"Plays a role in cilia formation and/or maintenance (By similarity). Plays a role in the regulation of cell morphology and cytoskeletal organization (PubMed:21834987). Involved in DNA damage repair (PubMed:26290490)","subcellular_location":"Mitochondrion; Cytoplasm, cytoskeleton, cilium basal body; Cell projection, cilium, photoreceptor outer segment; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/O43822/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CFAP410","classification":"Not Classified","n_dependent_lines":94,"n_total_lines":1208,"dependency_fraction":0.07781456953642384},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"VPS35","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CFAP410","total_profiled":1310},"omim":[{"mim_id":"617547","title":"RETINAL DYSTROPHY WITH OR WITHOUT MACULAR STAPHYLOMA; RDMS","url":"https://www.omim.org/entry/617547"},{"mim_id":"603191","title":"CILIA- AND FLAGELLA-ASSOCIATED PROTEIN 410; CFAP410","url":"https://www.omim.org/entry/603191"},{"mim_id":"602271","title":"SPONDYLOMETAPHYSEAL DYSPLASIA, AXIAL; SMDAX","url":"https://www.omim.org/entry/602271"},{"mim_id":"307030","title":"GLYCEROL KINASE DEFICIENCY; GKD","url":"https://www.omim.org/entry/307030"},{"mim_id":"300474","title":"GLYCEROL KINASE; GK","url":"https://www.omim.org/entry/300474"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Golgi apparatus","reliability":"Supported"},{"location":"Primary cilium transition zone","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"},{"location":"Basal body","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CFAP410"},"hgnc":{"alias_symbol":["YF5","A2","LRRC76"],"prev_symbol":["C21orf2"]},"alphafold":{"accession":"O43822","domains":[{"cath_id":"3.80.10.10","chopping":"6-138","consensus_level":"high","plddt":95.4369,"start":6,"end":138},{"cath_id":"1.20.5","chopping":"222-254","consensus_level":"high","plddt":83.4815,"start":222,"end":254}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O43822","model_url":"https://alphafold.ebi.ac.uk/files/AF-O43822-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O43822-F1-predicted_aligned_error_v6.png","plddt_mean":77.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CFAP410","jax_strain_url":"https://www.jax.org/strain/search?query=CFAP410"},"sequence":{"accession":"O43822","fasta_url":"https://rest.uniprot.org/uniprotkb/O43822.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O43822/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O43822"}},"corpus_meta":[{"pmid":"26290490","id":"PMC_26290490","title":"The NEK1 interactor, C21ORF2, is required for efficient DNA damage repair.","date":"2015","source":"Acta biochimica et biophysica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/26290490","citation_count":64,"is_preprint":false},{"pmid":"26294103","id":"PMC_26294103","title":"C21orf2 is mutated in recessive early-onset retinal dystrophy with macular staphyloma and encodes a protein that localises to the photoreceptor primary cilium.","date":"2015","source":"The British journal of ophthalmology","url":"https://pubmed.ncbi.nlm.nih.gov/26294103","citation_count":40,"is_preprint":false},{"pmid":"26974433","id":"PMC_26974433","title":"Axial Spondylometaphyseal Dysplasia Is Caused by C21orf2 Mutations.","date":"2016","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26974433","citation_count":38,"is_preprint":false},{"pmid":"23488799","id":"PMC_23488799","title":"Safety assessment and probiotic evaluation of Enterococcus faecium YF5 isolated from sourdough.","date":"2013","source":"Journal of food science","url":"https://pubmed.ncbi.nlm.nih.gov/23488799","citation_count":32,"is_preprint":false},{"pmid":"27548899","id":"PMC_27548899","title":"Identification of Novel Mutations in the LRR-Cap Domain of C21orf2 in Japanese Patients With Retinitis Pigmentosa and Cone-Rod Dystrophy.","date":"2016","source":"Investigative ophthalmology & visual science","url":"https://pubmed.ncbi.nlm.nih.gov/27548899","citation_count":29,"is_preprint":false},{"pmid":"32891887","id":"PMC_32891887","title":"An Amyotrophic Lateral Sclerosis-Associated Mutant of C21ORF2 Is Stabilized by NEK1-Mediated Hyperphosphorylation and the Inability to Bind FBXO3.","date":"2020","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/32891887","citation_count":26,"is_preprint":false},{"pmid":"9465297","id":"PMC_9465297","title":"Characterization of a novel gene, C21orf2, on human chromosome 21q22.3 and its exclusion as the APECED gene by mutation analysis.","date":"1998","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/9465297","citation_count":25,"is_preprint":false},{"pmid":"28422394","id":"PMC_28422394","title":"Homozygous variant in C21orf2 in a case of Jeune syndrome with severe thoracic involvement: Extending the phenotypic spectrum.","date":"2017","source":"American journal of medical genetics. 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Overexpression of NEK1 rescued the DNA repair defect in C21ORF2-knockdown cells, placing C21ORF2 in the same pathway as NEK1 for DNA damage repair.\",\n      \"method\": \"siRNA knockdown, colony survival assay, HR/NHEJ reporter assays, NEK1 overexpression rescue\",\n      \"journal\": \"Acta biochimica et biophysica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via rescue experiment and reporter assays, single lab, two orthogonal methods\",\n      \"pmids\": [\"26290490\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"C21ORF2 (CFAP410) protein localizes to ciliary structures of photoreceptor cells, specifically the daughter basal body, the centriole adjacent to the basal body, and the connecting cilium, as determined by immunohistochemistry in human, pig, and mouse retinas.\",\n      \"method\": \"Immunohistochemistry on mammalian (human, pig, mouse) retinal sections\",\n      \"journal\": \"The British journal of ophthalmology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization experiment replicated across three species\",\n      \"pmids\": [\"26294103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"C21ORF2 (CFAP410) protein localizes to the connecting cilium of cone and rod photoreceptors, and is implicated in cartilage differentiation in chondrocytes based on functional data.\",\n      \"method\": \"Immunolocalization in retinal tissue; functional data in chondrocyte cells\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiment, single lab, two cell-type contexts\",\n      \"pmids\": [\"26974433\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Missense mutations in the leucine-rich repeat C-terminal (LRRCT) domain of C21ORF2 (CFAP410) reduce protein stability and alter cytoplasmic localization of mutant proteins compared to wild type in vitro, and C21ORF2 is required for ciliogenesis in retinal photoreceptors.\",\n      \"method\": \"In vitro expression assays, immunofluorescence, western blotting of mutant constructs in cell lines\",\n      \"journal\": \"Investigative ophthalmology & visual science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional mutagenesis with protein stability and localization readouts, single lab\",\n      \"pmids\": [\"27548899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"FBXO3 (substrate receptor of an SCF ubiquitin ligase complex) binds and ubiquitylates C21ORF2, targeting it for proteasomal degradation. NEK1-mediated phosphorylation of C21ORF2 attenuates its interaction with FBXO3, thereby stabilizing C21ORF2. The ALS-associated V58L mutant of C21ORF2 is hyperphosphorylated by NEK1, escapes FBXO3-mediated ubiquitylation, and accumulates together with NEK1. Expression of C21ORF2(V58L) in mouse ESC-derived motor neurons impaired neurite outgrowth.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assays, phosphorylation assays, motor neuron differentiation from mESCs, neurite outgrowth measurement\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reciprocal Co-IP, ubiquitylation assay, phosphorylation assay, and functional cellular readout in a single study with multiple orthogonal methods\",\n      \"pmids\": [\"32891887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Endogenous NEK1 and C21ORF2 form a tight complex in human cells. A C-terminal interaction domain (CID) on NEK1 is necessary for association with C21ORF2, and pathogenic mutations in this region disrupt the complex. AlphaFold modelling predicts an extended binding interface between the leucine-rich repeat domain of C21ORF2 and the NEK1-CID. NEK1 mutations that inhibit kinase activity or weaken NEK1-C21ORF2 association severely compromise ciliogenesis. C21ORF2, like NEK1, is required for homologous recombination.\",\n      \"method\": \"Endogenous Co-immunoprecipitation, AlphaFold structural modelling, ciliogenesis assays, HR repair assays, pathogenic mutant analysis\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — endogenous reciprocal Co-IP, structural modelling, ciliogenesis and HR functional assays in same study with multiple orthogonal methods\",\n      \"pmids\": [\"37188479\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CFAP410 pathogenic variants (p.Tyr107His and p.Pro116Leu) reduce protein stability and alter the ubiquitination level of CFAP410, as measured by co-IP and western blotting in HEK293T cells, suggesting the ubiquitin-proteasome pathway mediates CFAP410 degradation.\",\n      \"method\": \"Co-immunoprecipitation, western blotting, immunofluorescence, cell cycle analysis in HEK293T cells\",\n      \"journal\": \"Frontiers in medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and western blot with mutant constructs, single lab, two orthogonal methods\",\n      \"pmids\": [\"37901396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The C-terminal domain (CTD) of CFAP410 forms a tetrameric helical bundle, as determined by crystal structures from Homo sapiens, Trypanosoma brucei, and Chlamydomonas reinhardtii. The tetrameric assembly is essential for correct basal body localization of CFAP410; the ALS/spondylometaphyseal dysplasia-associated L224P mutation disassembles the tetramer and disrupts basal body localization in T. brucei.\",\n      \"method\": \"X-ray crystallography (crystal structures from three organisms), functional localization assays with L224P mutant in T. brucei\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure determination replicated across three organisms plus functional mutagenesis validating localization requirement\",\n      \"pmids\": [\"39255848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"C21ORF2 localizes to the basal body of the primary cilium in human motor neurons. ALS-associated mutations alter this basal body localization. Reduction of C21ORF2 levels causes fewer primary cilia and reduced ciliary length, leading to defective sonic hedgehog (SHH) signalling, reduced CRABP1 expression, and impaired neuromuscular junction formation. Overexpression of C21ORF2 in mutant motor neurons rescued ciliary frequency and length, CRABP1 expression, and NMJ formation.\",\n      \"method\": \"iPSC-derived motor neuron cultures, immunofluorescence, siRNA knockdown, SHH pathway reporter assays, compartmentalized co-culture NMJ assay, rescue by overexpression\",\n      \"journal\": \"Brain : a journal of neurology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (localization, KD, pathway assays, rescue) in patient-derived neurons and isogenic controls\",\n      \"pmids\": [\"39703094\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The ALS-associated C21ORF2-V58L variant causes increased apoptosis in mouse neurons, movement defects in zebrafish, and in iPSC-derived motor neurons: defects in DNA damage response, mitochondrial dysfunction, and changes in neuronal excitability. V58L induces post-transcriptional downregulation of NEK1 protein levels.\",\n      \"method\": \"iPSC-derived motor neurons (patient vs isogenic controls), zebrafish model, apoptosis assays, DNA damage response assays, mitochondrial assays, western blotting for NEK1\",\n      \"journal\": \"Acta neuropathologica communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods in patient-derived vs isogenic control neurons and in vivo zebrafish model, single lab\",\n      \"pmids\": [\"39227882\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Knock-in of ALS-associated Cfap410 variants in mouse ESCs results in impaired interaction with Nek1 and increased susceptibility to DNA damage in ESCs, neural progenitors, and differentiated neurons, without significant effects on primary cilia frequency.\",\n      \"method\": \"Gene editing (knock-in) in mESCs, neural differentiation, DNA damage assays, Co-immunoprecipitation for Nek1 interaction\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — endogenous knock-in model with Co-IP and functional DNA damage readouts, single lab\",\n      \"pmids\": [\"40933646\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CFAP410 adopts a bimodular architecture. The 1.0-Å resolution crystal structure of the N-terminal domain (NTD) of T. brucei CFAP410 reveals a leucine-rich repeat fold with a conserved surface patch. Disease-causing mutations in the NTD destabilize the structure and are predicted to disrupt interactions with binding partners.\",\n      \"method\": \"X-ray crystallography (1.0-Å resolution), structural analysis of disease-causing mutations\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — high-resolution crystal structure with mutagenesis-based structural interpretation; single lab but rigorous structural method\",\n      \"pmids\": [\"40018707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"C21ORF2 promotes activation of the JAK2/STAT3 signaling pathway in prostate cancer cells, upregulating downstream targets c-MYC, Cyclin A1, Bcl-2, and Cleaved caspase-3. Co-immunoprecipitation demonstrated interaction between C21ORF2 and downstream targets; C21ORF2 showed negative correlation with KCTD5. In vivo xenograft models validated C21ORF2's role in tumor growth.\",\n      \"method\": \"Co-immunoprecipitation, western blotting, CCK-8 proliferation assay, Transwell assay, TUNEL staining, subcutaneous xenograft mouse model\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, Co-IP without reciprocal validation, pathway activation inferred from western blot of downstream targets\",\n      \"pmids\": [\"41612237\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CFAP410 (C21ORF2) is a basal body/ciliary protein whose C-terminal domain forms a tetrameric helical bundle required for basal body localization; it forms a tight complex with NEK1 kinase through a dedicated interaction domain, promotes homologous recombination-based DNA repair in the same pathway as NEK1, and is targeted for proteasomal degradation by the SCF-FBXO3 ubiquitin ligase—a process counteracted by NEK1-mediated phosphorylation of CFAP410; loss of CFAP410 function impairs primary ciliogenesis and downstream SHH signalling, leading to neuronal and photoreceptor dysfunction, while disease-associated mutations reduce protein stability, disrupt basal body localization, and impair NEK1 interaction.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CFAP410 (C21ORF2) is a basal body and connecting cilium protein that couples primary ciliogenesis to NEK1-dependent genome maintenance, and its loss of function disrupts both processes in neurons and photoreceptors [#1, #5, #8]. It is built from two modules: an N-terminal leucine-rich repeat domain that engages binding partners and a C-terminal domain that assembles into a tetrameric helical bundle required for correct basal body localization, disruption of which (e.g. the L224P variant) disassembles the tetramer and mislocalizes the protein [#7, #11]. Through its leucine-rich repeat surface CFAP410 forms a tight complex with the kinase NEK1 via a dedicated C-terminal interaction domain on NEK1, and acts in the same pathway as NEK1 to promote homologous-recombination repair of DNA damage and to support ciliogenesis [#0, #5]. CFAP410 abundance is controlled by the ubiquitin–proteasome system: the SCF substrate receptor FBXO3 binds and ubiquitylates it for degradation, while NEK1-mediated phosphorylation attenuates the FBXO3 interaction and stabilizes the protein [#4]. Reduced CFAP410 produces fewer and shorter primary cilia, defective sonic hedgehog signalling, and impaired neuromuscular junction formation, all reversible by re-expression [#8]. Disease-associated missense and structural mutations destabilize the protein, alter its localization and ubiquitination, and weaken the NEK1 interaction, linking CFAP410 to ALS and spondylometaphyseal dysplasia phenotypes [#3, #6, #7, #10].\",\n  \"teleology\": [\n    {\n      \"year\": 2015,\n      \"claim\": \"Established that CFAP410 acts in DNA damage repair within the NEK1 pathway rather than as an isolated factor, defining its first functional partner.\",\n      \"evidence\": \"siRNA knockdown with HR/NHEJ reporter assays and NEK1 overexpression rescue in human cells\",\n      \"pmids\": [\"26290490\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not define the physical interaction interface\", \"Mechanism by which CFAP410 promotes HR specifically was not resolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Localized CFAP410 to specific ciliary substructures of photoreceptors, anchoring it to the basal body/connecting cilium compartment.\",\n      \"evidence\": \"Immunohistochemistry on human, pig, and mouse retinal sections\",\n      \"pmids\": [\"26294103\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not establish ciliary function mechanistically\", \"No link to a molecular pathway at the cilium\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Extended ciliary localization to cone and rod connecting cilia and tied CFAP410 to ciliogenesis and chondrocyte differentiation, broadening its tissue relevance.\",\n      \"evidence\": \"Immunolocalization in retina plus functional data in chondrocytes; mutant expression assays showing reduced stability and altered localization\",\n      \"pmids\": [\"26974433\", \"27548899\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not identify the degradation machinery acting on mutants\", \"Connection between cartilage and ciliary roles unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined how CFAP410 levels are regulated, showing FBXO3 ubiquitylates it for degradation while NEK1 phosphorylation stabilizes it, and that an ALS mutant escapes this control.\",\n      \"evidence\": \"Reciprocal Co-IP, ubiquitylation and phosphorylation assays, and neurite outgrowth in mESC-derived motor neurons\",\n      \"pmids\": [\"32891887\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The SCF complex composition beyond FBXO3 not detailed\", \"Phosphosites mediating FBXO3 escape not fully mapped\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Characterized the endogenous NEK1–CFAP410 complex at structural resolution, mapping a C-terminal interaction domain on NEK1 and linking complex integrity to both ciliogenesis and HR.\",\n      \"evidence\": \"Endogenous reciprocal Co-IP, AlphaFold modelling, ciliogenesis and HR assays with pathogenic mutants\",\n      \"pmids\": [\"37188479\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Interface modelled by AlphaFold, not crystallographically resolved as a complex\", \"How the same complex partitions between cilium and DNA repair unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Confirmed that additional pathogenic CFAP410 variants destabilize the protein and alter its ubiquitination, generalizing the proteostatic mechanism of disease mutations.\",\n      \"evidence\": \"Co-IP, western blotting, immunofluorescence, and cell cycle analysis in HEK293T cells\",\n      \"pmids\": [\"37901396\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not test FBXO3/NEK1 dependence for these specific variants\", \"Functional ciliary consequence not assayed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Solved the bimodular architecture, showing the CTD forms a tetrameric helical bundle essential for basal body localization and the NTD is an LRR fold whose mutations destabilize binding-partner interactions.\",\n      \"evidence\": \"X-ray crystallography across three organisms plus L224P localization assays in T. brucei; 1.0-Å NTD structure with disease-mutation analysis\",\n      \"pmids\": [\"39255848\", \"40018707\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of NTD surface-patch binding partners not experimentally confirmed\", \"Structure of the full-length protein or partner complexes not determined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated multi-organ pathogenicity of the V58L variant, linking it to DNA damage response defects, mitochondrial dysfunction, altered excitability, and reduced NEK1 protein.\",\n      \"evidence\": \"iPSC-derived motor neurons vs isogenic controls, zebrafish model, apoptosis/DNA damage/mitochondrial assays, NEK1 western blot\",\n      \"pmids\": [\"39227882\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking V58L to mitochondrial dysfunction unclear\", \"How V58L downregulates NEK1 post-transcriptionally not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected CFAP410 loss to a ciliary signalling axis in motor neurons, showing reduced cilia impair SHH signalling, CRABP1 expression, and NMJ formation, all rescued by re-expression.\",\n      \"evidence\": \"iPSC-derived motor neurons, siRNA knockdown, SHH reporter assays, compartmentalized NMJ co-culture, overexpression rescue\",\n      \"pmids\": [\"39703094\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SHH defect is upstream or parallel to NMJ phenotype not fully resolved\", \"Relationship to the DNA-repair role in the same cells not integrated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showed in an endogenous knock-in model that ALS variants impair the NEK1 interaction and increase DNA damage susceptibility without altering cilia frequency, dissociating the genome-maintenance from the ciliary phenotype.\",\n      \"evidence\": \"Knock-in mESCs, neural differentiation, DNA damage assays, Co-IP for Nek1 interaction\",\n      \"pmids\": [\"40933646\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Why cilia were unaffected here but affected in other models unresolved\", \"Quantitative contribution of NEK1-binding loss to DNA damage not isolated\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Proposed a context-specific oncogenic role via JAK2/STAT3 activation in prostate cancer, expanding CFAP410 function beyond cilia and DNA repair.\",\n      \"evidence\": \"Co-IP, western blotting, proliferation/migration assays, TUNEL, and xenograft model in prostate cancer cells\",\n      \"pmids\": [\"41612237\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Co-IP without reciprocal validation; direct interaction unconfirmed\", \"JAK2/STAT3 activation inferred only from downstream target western blots\", \"No mechanistic link to the established ciliary/NEK1 functions\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CFAP410 mechanistically integrates its basal body/ciliogenesis role with NEK1-dependent homologous recombination in the same cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No model explains the partition of the CFAP410–NEK1 complex between cilium and nucleus\", \"NTD binding partners beyond NEK1 unidentified\", \"Structure of the assembled CFAP410–NEK1 complex not experimentally determined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [1, 2, 8]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [1, 7, 8]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [0, 5, 10]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [5, 8]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"complexes\": [\"CFAP410-NEK1 complex\", \"SCF-FBXO3 ubiquitin ligase (substrate)\"],\n    \"partners\": [\"NEK1\", \"FBXO3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}