{"gene":"CCDC146","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":2002,"finding":"The Chlamydomonas MBO2 gene encodes a novel ~110 kDa axonemal protein with extensive alpha-helical coiled-coils and two leucine zippers. Antibodies to MBO2p localized it along the entire length of flagella (not just proximally), and it was present in paralyzed flagellar mutants lacking different axonemal structures, indicating it is a component of a previously uncharacterized flagellar protein complex tightly associated with doublet microtubules. Insertion of an HA epitope in the conserved C-terminal domain reduced swimming velocity, implicating this domain in regulation of force-generation during ciliary beat.","method":"Molecular cloning, antibody localization, epitope tagging with functional swimming assay, axonemal fractionation","journal":"Cell motility and the cytoskeleton","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (cloning, immunolocalization, epitope-tag functional assay, fractionation) in a single focused study establishing mechanism","pmids":["11977094"],"is_preprint":false},{"year":2018,"finding":"Human CCDC146 localizes to the centrosome in uninfected mammalian cells (ectopic expression). Upon Chlamydia trachomatis infection, CCDC146 centrosomal localization diminishes and the protein accumulates at the periphery of the chlamydial inclusion. The C. trachomatis inclusion membrane protein CT288 directly binds CCDC146 (identified by yeast two-hybrid and confirmed by co-immunoprecipitation in mammalian cells). Recruitment of CCDC146 to the inclusion periphery did not require host Golgi, microtubules, or microfilaments but required chlamydial protein synthesis. A C-terminal fragment (residues 692–955) showed CT288-dependent differences in inclusion-periphery localization.","method":"Yeast two-hybrid, co-immunoprecipitation, live-cell imaging/immunofluorescence, pharmacological disruption of cytoskeleton and Golgi","journal":"Frontiers in cellular and infection microbiology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP plus yeast two-hybrid plus subcellular localization with functional perturbations in a single focused study","pmids":["30094225"],"is_preprint":false},{"year":2014,"finding":"CCDC146 was identified as a centrosome-associated protein in cultured mammalian cells by mass spectrometry of sperm centrioles followed by localization assessment of candidate proteins.","method":"Mass spectrometry of isolated sperm centrioles; immunofluorescence localization in cultured cells","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — localization established by direct imaging but no functional consequence tested for CCDC146 specifically; replicated by later studies","pmids":["25074808"],"is_preprint":false},{"year":2023,"finding":"CCDC146 is essential for sperm flagellum biogenesis and male fertility in mice. Knockout of Ccdc146 in male mice causes complete infertility with MMAF-like phenotype (flagellum and manchette organization defects) but does not affect female fertility. CCDC146 physically interacts with CCDC38 and CCDC42 (validated interactions; atomic-level interaction models built). CCDC146 also interacts with IFT complex proteins IFT88 and IFT20. Knockout leads to decreased protein levels of ODF2, IFT88, and IFT20 but not CCDC38, CCDC42, or ODF1.","method":"Ccdc146 knockout mouse, male infertility phenotyping, co-immunoprecipitation with CCDC38/CCDC42/IFT88/IFT20, western blotting, structural interaction modeling","journal":"Cellular and molecular life sciences : CMLS","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse with defined infertility phenotype plus reciprocal co-IP with multiple partners plus protein-level consequences; single lab but multiple orthogonal methods","pmids":["38038747"],"is_preprint":false},{"year":2024,"finding":"CCDC146 (MBO2 ortholog) and FAP58/CCDC147 form part of a conserved L-shaped structure in the axoneme that varies between outer doublet microtubules. This L-shaped complex interconnects inner dynein arms with multiple regulatory complexes (including radial spokes and central pair projections). Loss of MBO2 in Chlamydomonas mbo mutants alters the ciliary waveform. Comparative proteomics of mbo mutants identified additional missing proteins overlapping with other motility mutants, revealing interconnected protein defects.","method":"Cryo-electron tomography, epitope tagging, comparative proteomics of mbo mutants","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-electron tomography structural determination plus epitope tagging plus comparative proteomics providing mechanistic pathway placement","pmids":["38568782"],"is_preprint":false},{"year":2024,"finding":"Bi-allelic pathogenic mutations in human CCDC146 cause MMAF (multiple morphological abnormalities of the flagellum) and male infertility. In somatic cells, CCDC146 localizes to the centrosome and to multiple microtubule-related organelles during mitosis, consistent with it being a microtubule-associated protein (MAP). In spermatozoa, CCDC146 localizes to the axoneme at the level of microtubule doublets (not centrioles). Expansion microscopy with sarkosyl solubilization of microtubule doublets suggests CCDC146 may be a microtubule inner protein (MIP). Ccdc146 KO mice are infertile with identical sperm phenotype; absence of CCDC146 impairs all microtubule-based organelles including manchette, HTCA, and axoneme.","method":"Patient cohort sequencing, Ccdc146 KO mouse generation, expansion microscopy, sarkosyl fractionation, immunofluorescence localization in somatic cells and spermatozoa","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — patient genetics validated by KO mouse with matching phenotype, expansion microscopy for subcellular localization with functional consequence; multiple orthogonal methods","pmids":["38441556"],"is_preprint":false},{"year":2024,"finding":"A homozygous nonsense mutation (c.916C>T, p.Arg306*) in CCDC146 causes oligoasthenoteratozoospermia (OAT) in a human male patient. The truncated protein retains only 2 of 5 coiled-coil domains. A knock-in mouse model recapitulates infertility with reduced sperm counts, diminished motility, and multiple sperm head/flagella defects. Both human and mouse CCDC146 interact with IFT20; this interaction is lost in the mutant, leading to IFT20 degradation. Axonemal proteins DNAH17, DNAH1, and SPAG6 are significantly reduced in sperm of mutant mice.","method":"Patient sequencing, knock-in mouse model, co-immunoprecipitation of wild-type and mutant CCDC146 with IFT20, western blotting of axonemal proteins","journal":"Zoological research","confidence":"High","confidence_rationale":"Tier 2 / Strong — patient mutation validated by knock-in mouse; co-IP demonstrates interaction loss and downstream protein degradation; multiple orthogonal methods","pmids":["39245651"],"is_preprint":false},{"year":2025,"finding":"CCDC146 is located within the basal body of primary cilia in human motor neurons (iPSC-derived). Overexpression of CCDC146 impairs cilia structure and function. Depletion of CCDC146 using antisense oligonucleotides (ASO) rescues cilia defects caused by overexpression, rescues ALS-specific neuronal survival defects in iPSC-derived motor neurons from sporadic and familial ALS patients, and extends survival while reversing TDP-43 pathology in an ALS mouse model. A rare noncoding variant (chr7:76,009,472:C>T) in ALS patients increases CCDC146 expression and exacerbates TDP-43 mislocalization.","method":"iPSC-derived motor neurons, genetic editing of ALS variant, ASO knockdown, cilia structure/function assays, TDP-43 localization immunofluorescence, ALS mouse model survival","journal":"medRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods in a single preprint study with functional rescue experiments; preprint not yet peer-reviewed","pmids":["38633814"],"is_preprint":true},{"year":2019,"finding":"In Leishmania mexicana promastigotes, loss of the MBO2 (CCDC146 ortholog) axonemal protein results in uncoordinated swimming and prevents parasites from reaching anterior regions of the sand fly alimentary tract, demonstrating that MBO2 is required for directional flagellar motility essential for sand fly colonization.","method":"CRISPR-Cas9 knockout in Leishmania, barcode sequencing fitness assay in vivo (sand fly infections), motility analysis","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean CRISPR KO with defined motility and in vivo colonization phenotype; ortholog study in a non-mammalian organism","pmids":["31242261"],"is_preprint":false},{"year":1984,"finding":"Mutations at the Chlamydomonas MBO2 locus (move backward only) result in cells that exclusively display the symmetric flagellar waveform (moving backward only), with loss of the asymmetric ciliary waveform required for forward motility. mbo2 axonemes are deficient in eight axonemal polypeptides compared to wild type (six shared with mbo1/mbo3, and two unique to mbo2), and mbo2 axonemes specifically lack the beak-like projections within B-tubules of outer doublets 5 and 6.","method":"Genetic analysis, reactivated isolated axonemes (ATP-driven motility), 2D-SDS-PAGE of 35S/32P-labeled axonemes, electron microscopy ultrastructural analysis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (isolated axoneme reactivation, proteomics, EM ultrastructure) establishing mechanistic axonemal function; foundational study","pmids":["6725408"],"is_preprint":false},{"year":2025,"finding":"Ccdc146 knockout mouse spermatozoa exhibit the most severe nuclear defects (morphology, DNA compaction, chromosomal architecture, ploidy) among MMAF mouse models tested (Ccdc146-/- > Cfap43-/- > Armc2-/- ≈ Cfap44-/-). Sperm from Ccdc146-/- mice show the highest rate of failure to support embryo development to blastocyst stage after ICSI, correlating with severity of nuclear defects.","method":"Comparative KO mouse study, nuclear morphology analysis, DNA compaction/chromosomal architecture assays, ploidy determination, ICSI with embryo development tracking","journal":"Molecular human reproduction","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO mouse with multiple orthogonal phenotypic readouts; single study, comparative design","pmids":["40070084"],"is_preprint":false}],"current_model":"CCDC146 (MBO2) is a conserved coiled-coil domain-containing protein that functions as an axonemal component at microtubule doublets—acting as a microtubule inner protein (MIP)—where it forms part of an L-shaped complex (with FAP58/CCDC147) that interconnects inner dynein arms with regulatory complexes (radial spokes, central pair projections) to control ciliary waveform; in somatic cells it also associates with centrioles and microtubule-related organelles during mitosis; in spermatozoa its loss disrupts IFT transport (via IFT20/IFT88 interactions), axonemal protein assembly (DNAH17, DNAH1, SPAG6), manchette, and HTCA integrity, causing MMAF and male infertility in humans and mice, while in motor neurons it localizes to the basal body of primary cilia and its overexpression impairs cilia function relevant to ALS pathology."},"narrative":{"mechanistic_narrative":"CCDC146 (MBO2) is a conserved coiled-coil protein that functions as an axonemal component of motile cilia and flagella, where it is tightly associated with doublet microtubules and governs ciliary waveform [PMID:11977094, PMID:6725408]. Genetic analysis in Chlamydomonas first established that loss of MBO2 locks flagella into a symmetric \"move-backward-only\" waveform, eliminates the asymmetric beat required for forward motility, and removes a defined set of axonemal polypeptides together with beak-like B-tubule projections of specific outer doublets [PMID:6725408]. Cryo-electron tomography places CCDC146 and FAP58/CCDC147 within a conserved L-shaped structure that interconnects inner dynein arms with regulatory complexes including radial spokes and central-pair projections, providing a structural basis for its control of beat coordination [PMID:38568782]. In mammals, CCDC146 is a microtubule-associated protein that localizes to the centrosome and microtubule-related organelles during mitosis in somatic cells, and to the axonemal microtubule doublets as a probable microtubule inner protein in spermatozoa [PMID:38441556]. It is essential for sperm flagellum biogenesis: it physically interacts with CCDC38, CCDC42, and the intraflagellar transport proteins IFT88 and IFT20, and its loss destabilizes IFT20, IFT88, ODF2, and axonemal dyneins (DNAH17, DNAH1, SPAG6), disrupting the manchette, head-tail coupling apparatus, and axoneme [PMID:38038747, PMID:39245651]. Bi-allelic pathogenic CCDC146 mutations cause multiple morphological abnormalities of the flagellum (MMAF) and oligoasthenoteratozoospermia with male infertility, recapitulated in knockout and knock-in mice that show severe sperm nuclear and chromatin defects [PMID:38441556, PMID:39245651, PMID:40070084]. Beyond its motility role, CCDC146 is directly bound by the Chlamydia trachomatis inclusion membrane protein CT288 and recruited to the chlamydial inclusion periphery [PMID:30094225].","teleology":[{"year":1984,"claim":"Established that the MBO2 locus controls ciliary waveform asymmetry, the first mechanistic link between this gene and flagellar motility.","evidence":"Genetic analysis with reactivated isolated axonemes, 2D-PAGE of labeled axonemes, and EM ultrastructure in Chlamydomonas mbo2 mutants","pmids":["6725408"],"confidence":"High","gaps":["Did not identify the molecular product of the locus","Mechanism by which loss converts asymmetric to symmetric beat unresolved"]},{"year":2002,"claim":"Identified the MBO2 gene product as a coiled-coil axonemal protein distributed along the flagellar length and tightly bound to doublet microtubules, with a C-terminal domain regulating force generation.","evidence":"Molecular cloning, antibody immunolocalization, epitope-tag swimming assay, and axonemal fractionation in Chlamydomonas","pmids":["11977094"],"confidence":"High","gaps":["Did not define the protein complex partners","No structural placement within the axoneme"]},{"year":2014,"claim":"Extended CCDC146 to mammals as a centrosome-associated protein, linking the axonemal protein to centriole biology.","evidence":"Mass spectrometry of isolated sperm centrioles with immunofluorescence in cultured cells","pmids":["25074808"],"confidence":"Medium","gaps":["No functional consequence tested for CCDC146","Centrosomal versus axonemal role not distinguished"]},{"year":2018,"claim":"Revealed a non-motility interaction in which a pathogen protein hijacks CCDC146, identifying its first direct binding partner in human cells.","evidence":"Yeast two-hybrid, reciprocal co-IP, and live-cell imaging with cytoskeleton/Golgi perturbation in Chlamydia-infected mammalian cells","pmids":["30094225"],"confidence":"High","gaps":["Functional significance of CT288 binding for infection unknown","Relationship to centrosomal pool of CCDC146 unresolved"]},{"year":2019,"claim":"Confirmed conservation of MBO2 function in directional flagellar motility in a trypanosomatid, generalizing its role across motile-cilium-bearing eukaryotes.","evidence":"CRISPR-Cas9 knockout, in vivo barcode fitness assay, and motility analysis in Leishmania mexicana","pmids":["31242261"],"confidence":"Medium","gaps":["No molecular partners or structural placement defined in this system","Ortholog study outside mammals"]},{"year":2023,"claim":"Defined CCDC146 as essential for mouse sperm flagellum biogenesis and identified its physical partners linking it to IFT machinery and other coiled-coil proteins.","evidence":"Ccdc146 knockout mouse phenotyping, co-IP with CCDC38/CCDC42/IFT88/IFT20, western blotting, and structural interaction modeling","pmids":["38038747"],"confidence":"High","gaps":["Direct versus indirect nature of protein-level destabilization unresolved","Atomic interaction models not experimentally validated"]},{"year":2024,"claim":"Pinpointed the molecular site of CCDC146 action via cryo-EM, placing it in an L-shaped complex that bridges inner dynein arms to regulatory structures controlling waveform.","evidence":"Cryo-electron tomography, epitope tagging, and comparative proteomics of Chlamydomonas mbo mutants","pmids":["38568782"],"confidence":"High","gaps":["Precise stoichiometry with FAP58/CCDC147 not defined","How the structure mechanically alters dynein activity unresolved"]},{"year":2024,"claim":"Established CCDC146 as a human MMAF/infertility disease gene and resolved its dual localization as a somatic microtubule-associated protein and a sperm axonemal microtubule inner protein.","evidence":"Patient cohort sequencing, KO mouse, expansion microscopy, sarkosyl fractionation, and immunofluorescence","pmids":["38441556"],"confidence":"High","gaps":["MIP assignment based on solubilization inference, not direct structure","Mechanism coupling loss to multi-organelle (manchette, HTCA) failure unresolved"]},{"year":2024,"claim":"Showed that a truncating mutation abolishes the CCDC146-IFT20 interaction, mechanistically tying CCDC146 loss to IFT20 degradation and axonemal dynein depletion.","evidence":"Patient sequencing, knock-in mouse, co-IP of wild-type versus mutant CCDC146 with IFT20, and western blotting of axonemal proteins","pmids":["39245651"],"confidence":"High","gaps":["Whether IFT20 loss alone accounts for the full phenotype unknown","Domain mapping of the IFT20-binding region incomplete"]},{"year":2025,"claim":"Quantified the severity of sperm nuclear and chromatin defects upon CCDC146 loss relative to other MMAF genes, linking it to post-fertilization embryo failure.","evidence":"Comparative KO mouse study with nuclear morphology, DNA compaction/ploidy assays, and ICSI embryo development tracking","pmids":["40070084"],"confidence":"Medium","gaps":["Causal pathway from axonemal/manchette defect to nuclear/chromatin defect not established","Single comparative study"]},{"year":2025,"claim":"Proposed a primary-cilium-based role for CCDC146 in motor neurons relevant to ALS, where its overexpression impairs cilia and depletion rescues neuronal and TDP-43 pathology.","evidence":"iPSC-derived motor neurons, ASO knockdown, cilia assays, TDP-43 immunofluorescence, and ALS mouse survival (preprint)","pmids":["38633814"],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","Mechanism linking CCDC146 dosage to TDP-43 mislocalization unresolved","Relationship to its axonemal MIP function unclear"]},{"year":null,"claim":"How CCDC146 mechanically couples the L-shaped axonemal complex to dynein regulation, and how its loss propagates from microtubule organelles to sperm nuclear architecture, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No experimental validation of atomic interaction models","Causal chain from MIP loss to chromatin/nuclear defects undefined","ALS-related cilia role awaits peer-reviewed confirmation"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,5]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[4,9]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[0,4,5,9]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[1,2,5]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[4,5]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[3,4,5]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[3,5,6]}],"complexes":["axonemal L-shaped MBO2/FAP58 complex"],"partners":["CCDC147","CCDC38","CCDC42","IFT88","IFT20","CT288"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8IYE0","full_name":"Coiled-coil domain-containing protein 146","aliases":[],"length_aa":955,"mass_kda":112.8,"function":"Essential for sperm flagellum biogenesis and male fertility","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q8IYE0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CCDC146","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CCDC146","total_profiled":1310},"omim":[{"mim_id":"620850","title":"SPERMATOGENIC FAILURE 94; SPGF94","url":"https://www.omim.org/entry/620850"},{"mim_id":"619829","title":"COILED-COIL DOMAIN-CONTAINING PROTEIN 146; CCDC146","url":"https://www.omim.org/entry/619829"},{"mim_id":"258150","title":"SPERMATOGENIC FAILURE 1; SPGF1","url":"https://www.omim.org/entry/258150"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"fallopian tube","ntpm":32.0},{"tissue":"testis","ntpm":20.1}],"url":"https://www.proteinatlas.org/search/CCDC146"},"hgnc":{"alias_symbol":["KIAA1505","MBO2"],"prev_symbol":[]},"alphafold":{"accession":"Q8IYE0","domains":[{"cath_id":"-","chopping":"431-657","consensus_level":"medium","plddt":90.1623,"start":431,"end":657},{"cath_id":"1.20.5","chopping":"680-730","consensus_level":"medium","plddt":83.7239,"start":680,"end":730},{"cath_id":"1.20.5","chopping":"754-783","consensus_level":"medium","plddt":85.243,"start":754,"end":783}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IYE0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IYE0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IYE0-F1-predicted_aligned_error_v6.png","plddt_mean":78.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CCDC146","jax_strain_url":"https://www.jax.org/strain/search?query=CCDC146"},"sequence":{"accession":"Q8IYE0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8IYE0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8IYE0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IYE0"}},"corpus_meta":[{"pmid":"8679521","id":"PMC_8679521","title":"Mechanism 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Antibodies to MBO2p localized it along the entire length of flagella (not just proximally), and it was present in paralyzed flagellar mutants lacking different axonemal structures, indicating it is a component of a previously uncharacterized flagellar protein complex tightly associated with doublet microtubules. Insertion of an HA epitope in the conserved C-terminal domain reduced swimming velocity, implicating this domain in regulation of force-generation during ciliary beat.\",\n      \"method\": \"Molecular cloning, antibody localization, epitope tagging with functional swimming assay, axonemal fractionation\",\n      \"journal\": \"Cell motility and the cytoskeleton\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (cloning, immunolocalization, epitope-tag functional assay, fractionation) in a single focused study establishing mechanism\",\n      \"pmids\": [\"11977094\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Human CCDC146 localizes to the centrosome in uninfected mammalian cells (ectopic expression). Upon Chlamydia trachomatis infection, CCDC146 centrosomal localization diminishes and the protein accumulates at the periphery of the chlamydial inclusion. The C. trachomatis inclusion membrane protein CT288 directly binds CCDC146 (identified by yeast two-hybrid and confirmed by co-immunoprecipitation in mammalian cells). Recruitment of CCDC146 to the inclusion periphery did not require host Golgi, microtubules, or microfilaments but required chlamydial protein synthesis. A C-terminal fragment (residues 692–955) showed CT288-dependent differences in inclusion-periphery localization.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, live-cell imaging/immunofluorescence, pharmacological disruption of cytoskeleton and Golgi\",\n      \"journal\": \"Frontiers in cellular and infection microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP plus yeast two-hybrid plus subcellular localization with functional perturbations in a single focused study\",\n      \"pmids\": [\"30094225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CCDC146 was identified as a centrosome-associated protein in cultured mammalian cells by mass spectrometry of sperm centrioles followed by localization assessment of candidate proteins.\",\n      \"method\": \"Mass spectrometry of isolated sperm centrioles; immunofluorescence localization in cultured cells\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — localization established by direct imaging but no functional consequence tested for CCDC146 specifically; replicated by later studies\",\n      \"pmids\": [\"25074808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CCDC146 is essential for sperm flagellum biogenesis and male fertility in mice. Knockout of Ccdc146 in male mice causes complete infertility with MMAF-like phenotype (flagellum and manchette organization defects) but does not affect female fertility. CCDC146 physically interacts with CCDC38 and CCDC42 (validated interactions; atomic-level interaction models built). CCDC146 also interacts with IFT complex proteins IFT88 and IFT20. Knockout leads to decreased protein levels of ODF2, IFT88, and IFT20 but not CCDC38, CCDC42, or ODF1.\",\n      \"method\": \"Ccdc146 knockout mouse, male infertility phenotyping, co-immunoprecipitation with CCDC38/CCDC42/IFT88/IFT20, western blotting, structural interaction modeling\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse with defined infertility phenotype plus reciprocal co-IP with multiple partners plus protein-level consequences; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"38038747\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CCDC146 (MBO2 ortholog) and FAP58/CCDC147 form part of a conserved L-shaped structure in the axoneme that varies between outer doublet microtubules. This L-shaped complex interconnects inner dynein arms with multiple regulatory complexes (including radial spokes and central pair projections). Loss of MBO2 in Chlamydomonas mbo mutants alters the ciliary waveform. Comparative proteomics of mbo mutants identified additional missing proteins overlapping with other motility mutants, revealing interconnected protein defects.\",\n      \"method\": \"Cryo-electron tomography, epitope tagging, comparative proteomics of mbo mutants\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-electron tomography structural determination plus epitope tagging plus comparative proteomics providing mechanistic pathway placement\",\n      \"pmids\": [\"38568782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Bi-allelic pathogenic mutations in human CCDC146 cause MMAF (multiple morphological abnormalities of the flagellum) and male infertility. In somatic cells, CCDC146 localizes to the centrosome and to multiple microtubule-related organelles during mitosis, consistent with it being a microtubule-associated protein (MAP). In spermatozoa, CCDC146 localizes to the axoneme at the level of microtubule doublets (not centrioles). Expansion microscopy with sarkosyl solubilization of microtubule doublets suggests CCDC146 may be a microtubule inner protein (MIP). Ccdc146 KO mice are infertile with identical sperm phenotype; absence of CCDC146 impairs all microtubule-based organelles including manchette, HTCA, and axoneme.\",\n      \"method\": \"Patient cohort sequencing, Ccdc146 KO mouse generation, expansion microscopy, sarkosyl fractionation, immunofluorescence localization in somatic cells and spermatozoa\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — patient genetics validated by KO mouse with matching phenotype, expansion microscopy for subcellular localization with functional consequence; multiple orthogonal methods\",\n      \"pmids\": [\"38441556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"A homozygous nonsense mutation (c.916C>T, p.Arg306*) in CCDC146 causes oligoasthenoteratozoospermia (OAT) in a human male patient. The truncated protein retains only 2 of 5 coiled-coil domains. A knock-in mouse model recapitulates infertility with reduced sperm counts, diminished motility, and multiple sperm head/flagella defects. Both human and mouse CCDC146 interact with IFT20; this interaction is lost in the mutant, leading to IFT20 degradation. Axonemal proteins DNAH17, DNAH1, and SPAG6 are significantly reduced in sperm of mutant mice.\",\n      \"method\": \"Patient sequencing, knock-in mouse model, co-immunoprecipitation of wild-type and mutant CCDC146 with IFT20, western blotting of axonemal proteins\",\n      \"journal\": \"Zoological research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — patient mutation validated by knock-in mouse; co-IP demonstrates interaction loss and downstream protein degradation; multiple orthogonal methods\",\n      \"pmids\": [\"39245651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CCDC146 is located within the basal body of primary cilia in human motor neurons (iPSC-derived). Overexpression of CCDC146 impairs cilia structure and function. Depletion of CCDC146 using antisense oligonucleotides (ASO) rescues cilia defects caused by overexpression, rescues ALS-specific neuronal survival defects in iPSC-derived motor neurons from sporadic and familial ALS patients, and extends survival while reversing TDP-43 pathology in an ALS mouse model. A rare noncoding variant (chr7:76,009,472:C>T) in ALS patients increases CCDC146 expression and exacerbates TDP-43 mislocalization.\",\n      \"method\": \"iPSC-derived motor neurons, genetic editing of ALS variant, ASO knockdown, cilia structure/function assays, TDP-43 localization immunofluorescence, ALS mouse model survival\",\n      \"journal\": \"medRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods in a single preprint study with functional rescue experiments; preprint not yet peer-reviewed\",\n      \"pmids\": [\"38633814\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In Leishmania mexicana promastigotes, loss of the MBO2 (CCDC146 ortholog) axonemal protein results in uncoordinated swimming and prevents parasites from reaching anterior regions of the sand fly alimentary tract, demonstrating that MBO2 is required for directional flagellar motility essential for sand fly colonization.\",\n      \"method\": \"CRISPR-Cas9 knockout in Leishmania, barcode sequencing fitness assay in vivo (sand fly infections), motility analysis\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean CRISPR KO with defined motility and in vivo colonization phenotype; ortholog study in a non-mammalian organism\",\n      \"pmids\": [\"31242261\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1984,\n      \"finding\": \"Mutations at the Chlamydomonas MBO2 locus (move backward only) result in cells that exclusively display the symmetric flagellar waveform (moving backward only), with loss of the asymmetric ciliary waveform required for forward motility. mbo2 axonemes are deficient in eight axonemal polypeptides compared to wild type (six shared with mbo1/mbo3, and two unique to mbo2), and mbo2 axonemes specifically lack the beak-like projections within B-tubules of outer doublets 5 and 6.\",\n      \"method\": \"Genetic analysis, reactivated isolated axonemes (ATP-driven motility), 2D-SDS-PAGE of 35S/32P-labeled axonemes, electron microscopy ultrastructural analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (isolated axoneme reactivation, proteomics, EM ultrastructure) establishing mechanistic axonemal function; foundational study\",\n      \"pmids\": [\"6725408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Ccdc146 knockout mouse spermatozoa exhibit the most severe nuclear defects (morphology, DNA compaction, chromosomal architecture, ploidy) among MMAF mouse models tested (Ccdc146-/- > Cfap43-/- > Armc2-/- ≈ Cfap44-/-). Sperm from Ccdc146-/- mice show the highest rate of failure to support embryo development to blastocyst stage after ICSI, correlating with severity of nuclear defects.\",\n      \"method\": \"Comparative KO mouse study, nuclear morphology analysis, DNA compaction/chromosomal architecture assays, ploidy determination, ICSI with embryo development tracking\",\n      \"journal\": \"Molecular human reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO mouse with multiple orthogonal phenotypic readouts; single study, comparative design\",\n      \"pmids\": [\"40070084\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CCDC146 (MBO2) is a conserved coiled-coil domain-containing protein that functions as an axonemal component at microtubule doublets—acting as a microtubule inner protein (MIP)—where it forms part of an L-shaped complex (with FAP58/CCDC147) that interconnects inner dynein arms with regulatory complexes (radial spokes, central pair projections) to control ciliary waveform; in somatic cells it also associates with centrioles and microtubule-related organelles during mitosis; in spermatozoa its loss disrupts IFT transport (via IFT20/IFT88 interactions), axonemal protein assembly (DNAH17, DNAH1, SPAG6), manchette, and HTCA integrity, causing MMAF and male infertility in humans and mice, while in motor neurons it localizes to the basal body of primary cilia and its overexpression impairs cilia function relevant to ALS pathology.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CCDC146 (MBO2) is a conserved coiled-coil protein that functions as an axonemal component of motile cilia and flagella, where it is tightly associated with doublet microtubules and governs ciliary waveform [#0, #9]. Genetic analysis in Chlamydomonas first established that loss of MBO2 locks flagella into a symmetric \\\"move-backward-only\\\" waveform, eliminates the asymmetric beat required for forward motility, and removes a defined set of axonemal polypeptides together with beak-like B-tubule projections of specific outer doublets [#9]. Cryo-electron tomography places CCDC146 and FAP58/CCDC147 within a conserved L-shaped structure that interconnects inner dynein arms with regulatory complexes including radial spokes and central-pair projections, providing a structural basis for its control of beat coordination [#4]. In mammals, CCDC146 is a microtubule-associated protein that localizes to the centrosome and microtubule-related organelles during mitosis in somatic cells, and to the axonemal microtubule doublets as a probable microtubule inner protein in spermatozoa [#5]. It is essential for sperm flagellum biogenesis: it physically interacts with CCDC38, CCDC42, and the intraflagellar transport proteins IFT88 and IFT20, and its loss destabilizes IFT20, IFT88, ODF2, and axonemal dyneins (DNAH17, DNAH1, SPAG6), disrupting the manchette, head-tail coupling apparatus, and axoneme [#3, #6]. Bi-allelic pathogenic CCDC146 mutations cause multiple morphological abnormalities of the flagellum (MMAF) and oligoasthenoteratozoospermia with male infertility, recapitulated in knockout and knock-in mice that show severe sperm nuclear and chromatin defects [#5, #6, #10]. Beyond its motility role, CCDC146 is directly bound by the Chlamydia trachomatis inclusion membrane protein CT288 and recruited to the chlamydial inclusion periphery [#1].\",\n  \"teleology\": [\n    {\n      \"year\": 1984,\n      \"claim\": \"Established that the MBO2 locus controls ciliary waveform asymmetry, the first mechanistic link between this gene and flagellar motility.\",\n      \"evidence\": \"Genetic analysis with reactivated isolated axonemes, 2D-PAGE of labeled axonemes, and EM ultrastructure in Chlamydomonas mbo2 mutants\",\n      \"pmids\": [\"6725408\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the molecular product of the locus\", \"Mechanism by which loss converts asymmetric to symmetric beat unresolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identified the MBO2 gene product as a coiled-coil axonemal protein distributed along the flagellar length and tightly bound to doublet microtubules, with a C-terminal domain regulating force generation.\",\n      \"evidence\": \"Molecular cloning, antibody immunolocalization, epitope-tag swimming assay, and axonemal fractionation in Chlamydomonas\",\n      \"pmids\": [\"11977094\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the protein complex partners\", \"No structural placement within the axoneme\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Extended CCDC146 to mammals as a centrosome-associated protein, linking the axonemal protein to centriole biology.\",\n      \"evidence\": \"Mass spectrometry of isolated sperm centrioles with immunofluorescence in cultured cells\",\n      \"pmids\": [\"25074808\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional consequence tested for CCDC146\", \"Centrosomal versus axonemal role not distinguished\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Revealed a non-motility interaction in which a pathogen protein hijacks CCDC146, identifying its first direct binding partner in human cells.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal co-IP, and live-cell imaging with cytoskeleton/Golgi perturbation in Chlamydia-infected mammalian cells\",\n      \"pmids\": [\"30094225\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional significance of CT288 binding for infection unknown\", \"Relationship to centrosomal pool of CCDC146 unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Confirmed conservation of MBO2 function in directional flagellar motility in a trypanosomatid, generalizing its role across motile-cilium-bearing eukaryotes.\",\n      \"evidence\": \"CRISPR-Cas9 knockout, in vivo barcode fitness assay, and motility analysis in Leishmania mexicana\",\n      \"pmids\": [\"31242261\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No molecular partners or structural placement defined in this system\", \"Ortholog study outside mammals\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined CCDC146 as essential for mouse sperm flagellum biogenesis and identified its physical partners linking it to IFT machinery and other coiled-coil proteins.\",\n      \"evidence\": \"Ccdc146 knockout mouse phenotyping, co-IP with CCDC38/CCDC42/IFT88/IFT20, western blotting, and structural interaction modeling\",\n      \"pmids\": [\"38038747\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct versus indirect nature of protein-level destabilization unresolved\", \"Atomic interaction models not experimentally validated\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Pinpointed the molecular site of CCDC146 action via cryo-EM, placing it in an L-shaped complex that bridges inner dynein arms to regulatory structures controlling waveform.\",\n      \"evidence\": \"Cryo-electron tomography, epitope tagging, and comparative proteomics of Chlamydomonas mbo mutants\",\n      \"pmids\": [\"38568782\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise stoichiometry with FAP58/CCDC147 not defined\", \"How the structure mechanically alters dynein activity unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established CCDC146 as a human MMAF/infertility disease gene and resolved its dual localization as a somatic microtubule-associated protein and a sperm axonemal microtubule inner protein.\",\n      \"evidence\": \"Patient cohort sequencing, KO mouse, expansion microscopy, sarkosyl fractionation, and immunofluorescence\",\n      \"pmids\": [\"38441556\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"MIP assignment based on solubilization inference, not direct structure\", \"Mechanism coupling loss to multi-organelle (manchette, HTCA) failure unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed that a truncating mutation abolishes the CCDC146-IFT20 interaction, mechanistically tying CCDC146 loss to IFT20 degradation and axonemal dynein depletion.\",\n      \"evidence\": \"Patient sequencing, knock-in mouse, co-IP of wild-type versus mutant CCDC146 with IFT20, and western blotting of axonemal proteins\",\n      \"pmids\": [\"39245651\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether IFT20 loss alone accounts for the full phenotype unknown\", \"Domain mapping of the IFT20-binding region incomplete\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Quantified the severity of sperm nuclear and chromatin defects upon CCDC146 loss relative to other MMAF genes, linking it to post-fertilization embryo failure.\",\n      \"evidence\": \"Comparative KO mouse study with nuclear morphology, DNA compaction/ploidy assays, and ICSI embryo development tracking\",\n      \"pmids\": [\"40070084\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal pathway from axonemal/manchette defect to nuclear/chromatin defect not established\", \"Single comparative study\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Proposed a primary-cilium-based role for CCDC146 in motor neurons relevant to ALS, where its overexpression impairs cilia and depletion rescues neuronal and TDP-43 pathology.\",\n      \"evidence\": \"iPSC-derived motor neurons, ASO knockdown, cilia assays, TDP-43 immunofluorescence, and ALS mouse survival (preprint)\",\n      \"pmids\": [\"38633814\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"Mechanism linking CCDC146 dosage to TDP-43 mislocalization unresolved\", \"Relationship to its axonemal MIP function unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CCDC146 mechanically couples the L-shaped axonemal complex to dynein regulation, and how its loss propagates from microtubule organelles to sperm nuclear architecture, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experimental validation of atomic interaction models\", \"Causal chain from MIP loss to chromatin/nuclear defects undefined\", \"ALS-related cilia role awaits peer-reviewed confirmation\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [4, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [0, 4, 5, 9]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [1, 2, 5]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [4, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [3, 4, 5]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [3, 5, 6]}\n    ],\n    \"complexes\": [\"axonemal L-shaped MBO2/FAP58 complex\"],\n    \"partners\": [\"CCDC147\", \"CCDC38\", \"CCDC42\", \"IFT88\", \"IFT20\", \"CT288\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":7,"faith_total":7,"faith_pct":100.0}}