{"gene":"COL4A4","run_date":"2026-06-09T22:57:19","timeline":{"discoveries":[{"year":1998,"finding":"COL4A3 and COL4A4 genes are arranged head-to-head on chromosome 2q36, sharing a bidirectional promoter region. COL4A4 has two alternative transcripts from two different promoters: one transcription start site (exon 1') is only 5 bp from the COL4A3 start site, while the other (exon 1) starts 373 nucleotides downstream. The exon 1 transcript is expressed predominantly in epithelial cells, while the exon 1' transcript shows ubiquitous low expression. The promoter region contains CpG dinucleotides, GC boxes, CTC boxes, and a CCAAT box but no TATA box.","method":"RACE, RNase protection assays, genomic DNA sequencing, tissue expression analysis","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct transcription start site mapping by RACE and RNase protection, genomic structure confirmed by sequencing, tissue-specific expression validated","pmids":["9537506"],"is_preprint":false},{"year":1998,"finding":"The COL4A4 gene contains 48 exons encoding the alpha4 type IV collagen chain. Loss-of-function mutations (nonsense, frameshift, missense in collagenous domain) cause autosomal recessive Alport syndrome. A glycine-to-alanine substitution in the collagenous domain found in ~11.5% of controls and in a homozygous control individual does not produce an obvious phenotype, indicating not all glycine substitutions in collagen IV are pathogenic.","method":"Complete genomic structure characterization, comprehensive gene screen by PCR/sequencing in patients and controls","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 1 / Strong — complete exon structure determined experimentally; mutation-to-disease linkage confirmed in multiple patients; homozygous benign variant functionally informative","pmids":["9792860"],"is_preprint":false},{"year":1999,"finding":"Deletion of Col4a4 exons 1–12 (along with Col4a3 exons 1–2 and the intergenic promoter) in mice results in complete absence of both Col4a3 and Col4a4 transcripts and both proteins from the glomerular basement membrane, causing severe progressive glomerulonephritis with GBM ultrastructural changes resembling Alport syndrome. Persistent cellular proliferation in mutant kidneys suggests interaction with extracellular matrix is important for cell maturation.","method":"Transgenic insertional mutagenesis, FISH mapping, RT-PCR (transcript detection), immunohistochemistry/protein analysis, electron microscopy of GBM","journal":"Genomics","confidence":"High","confidence_rationale":"Tier 1 / Strong — knockout mouse model with direct demonstration of absent protein in GBM, defined ultrastructural phenotype, replicated mechanistic insight about GBM assembly","pmids":["10534397"],"is_preprint":false},{"year":2014,"finding":"A spontaneous Col4a4 splice-donor mutation (G-to-A in intron 30) causes skipping of exon 30 while maintaining the reading frame. The resulting mutant collagen α3α4α5(IV) trimers are secreted and incorporated into the GBM, but at reduced levels, and typical Alport GBM lesions develop. This is the only mouse model shown to accumulate abnormal collagen IV trimers in the GBM (as found in a subset of human Alport patients), demonstrating that missense/in-frame mutations can produce dysfunctional trimers rather than complete absence.","method":"Genetic mapping, DNA sequencing, protein analysis of GBM content, albuminuria measurement, electron microscopy","journal":"Kidney international","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vivo mouse model with direct protein analysis showing abnormal trimer secretion and GBM incorporation; mechanistic consequence of in-frame exon skipping established","pmids":["24522496"],"is_preprint":false},{"year":2014,"finding":"Functional studies in cultured podocytes transfected with wild-type or mutant COL4A3 chains showed retention of mutant collagens intracellularly and differential activation of the unfolded protein response (UPR) cascade, indicating UPR activation as a potential modifier of phenotypic severity in collagen IV nephropathies caused by COL4A3/COL4A4 mutations.","method":"Cell transfection (podocytes), UPR pathway activation assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single lab, cell transfection showing UPR activation with mutant COL4A3; COL4A4 context inferred but experiment used COL4A3 mutant chains","pmids":["25514610"],"is_preprint":false},{"year":2022,"finding":"A novel homozygous COL4A4 missense variant G394S impairs assembly of the α3α4α5(IV) heterotrimer N-terminus and reduces secretion by approximately 50% compared to wild-type, as demonstrated by split NanoLuciferase-based heterotrimer formation assays in HEK293T cells. C-terminal tag assays showed comparable luminescence to WT, indicating the defect is specific to N-terminal trimer assembly and subsequent secretion.","method":"Split NanoLuciferase (N-terminal and C-terminal) α3α4α5(IV) heterotrimer formation assay in HEK293T cells, immunofluorescence, targeted DNA sequencing","journal":"Kidney360","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution assay with split NanoLuc measuring heterotrimer assembly and secretion; two complementary tag orientations used; single lab but multiple orthogonal approaches","pmids":["36514391"],"is_preprint":false},{"year":2024,"finding":"Skipping of COL4A4 exon 27 (genetically predicted by intronic variant rs11898094, minor allele frequency 13%) is associated with hematuria and urinary albumin excretion. Functional assays using the split NanoLuc-based α3α4α5(IV) heterotrimer assay demonstrated that loss of exon 27 impairs type IV collagen heterotrimer formation and secretion.","method":"Transcriptome-wide association study (TWAS) using UK Biobank + GTEx kidney cortex data, independent replication in glomeruli-derived mRNA (n=245), split NanoLuc heterotrimer formation assay","journal":"Journal of the American Society of Nephrology : JASN","confidence":"High","confidence_rationale":"Tier 1 / Strong — functional assay (split NanoLuc heterotrimer) directly demonstrates impaired assembly from exon 27 skipping; independently replicated splicing event in two cohorts","pmids":["39190490"],"is_preprint":false},{"year":2022,"finding":"Minigene assay in HEK293T/HeLa cells demonstrated that presumed missense variant COL4A3 c.4793T>G (p.Leu1598Arg) causes loss of an alternative full-length transcript during splicing, and synonymous variant COL4A3 c.765G>A (p.Thr255Thr) leads to in-frame deletion of exon 13. In contrast, COL4A4 variants c.3990G>A (p.Pro1330Pro) and c.4766C>T (p.Pro1589Leu) exhibited no deleterious effect on splicing.","method":"In vitro minigene splicing assay in HEK293T and HeLa cells","journal":"Frontiers in medicine","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — minigene assay is Tier 1 method but COL4A3 variants tested, not COL4A4; negative results for COL4A4 variants c.3990G>A and c.4766C>T explicitly noted","pmids":["35386907"],"is_preprint":false},{"year":2025,"finding":"Dual exon-skipping events at COL4A4 exons 27 and 38 (caused by compound heterozygous deep intronic variants) disrupt α3α4α5(IV) heterotrimer assembly, as confirmed by immunofluorescence analysis. This represents the first documented case of dual COL4A4 exon-skipping events contributing to autosomal recessive Alport syndrome severity. RNA-based diagnostics (whole-transcriptome sequencing) were required to detect these variants missed by DNA sequencing.","method":"Whole-transcriptome sequencing, exon-specific PCR, Sanger sequencing, immunofluorescence for α3α4α5(IV) heterotrimer","journal":"Kidney medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — RNA sequencing and immunofluorescence confirm exon skipping and disrupted heterotrimer; single case, single lab","pmids":["41050124"],"is_preprint":false},{"year":2026,"finding":"Minigene assays in HEK293T and HeLa cells for 14 synonymous and intronic COL4A3/COL4A4 variants showed that 13 caused aberrant splicing (predominantly exon skipping), including frameshift transcripts with premature termination codons and in-frame exon skipping events. These results demonstrate that synonymous and intronic variants in COL4A4 cause pathogenic effects primarily through disruption of pre-mRNA splicing rather than direct coding-sequence alteration.","method":"In vitro minigene splicing assay in HEK293T and HeLa cells, quantitative splicing analysis, ACMG/AMP reclassification","journal":"Human genomics","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic minigene functional assays across 14 variants with quantitative outcomes; two cell lines used; direct mechanistic demonstration of splicing disruption","pmids":["42204651"],"is_preprint":false},{"year":2025,"finding":"The COL4A4 c.817-1G>A splice acceptor site mutation disrupts mRNA splicing by affecting the splice acceptor site of intron 13 adjacent to exon 14, causing a 1-bp deletion before exon 14 and creating a premature stop codon. The resulting truncated protein is predicted to be 273 amino acids rather than the full-length 1,690 amino acids.","method":"Whole-exome sequencing, in vivo splicing validation using RNA from blood and urine (RT-PCR, Sanger sequencing)","journal":"Frontiers in pediatrics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — in vivo RNA splicing validation directly demonstrates aberrant splicing product; single case, single lab","pmids":["40406358"],"is_preprint":false},{"year":2024,"finding":"The COL4A4 splicing variant c.595-1G>A abolishes the canonical splice acceptor site of intron 9 and causes a single nucleotide deletion of exon 10, predicted to produce a truncated protein, as demonstrated by complementary DNA analysis.","method":"Whole exome sequencing, Sanger sequencing, RT-PCR cDNA analysis","journal":"Frontiers in genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct cDNA analysis confirms exon 10 deletion at RNA level; single lab, single family","pmids":["36699462"],"is_preprint":false},{"year":2016,"finding":"A COL4A4 splicing mutation (c.1459+1G>A) causes elimination of the entire exon 21 from COL4A4 cDNA, resulting in direct splicing of exons 20 and 22 and a frameshift mutation after exon 20 in the open reading frame, as demonstrated by RT-PCR and TA cloning of patient RNA.","method":"Direct sequencing, RT-PCR, TA cloning of cDNA","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — RNA-level confirmation of exon skipping by RT-PCR and cloning; single lab, single family","pmids":["26833262"],"is_preprint":false}],"current_model":"COL4A4 encodes the α4 chain of type IV collagen, which assembles with α3 and α5 chains into heterotrimers that are incorporated into the glomerular basement membrane (GBM); the gene is arranged head-to-head with COL4A3 on chromosome 2q36 under a shared bidirectional promoter driving tissue-specific expression, and loss-of-function or dominant-negative mutations (including missense substitutions, frameshift, nonsense, and splicing variants causing exon skipping) impair N-terminal-mediated heterotrimer assembly and/or secretion, deplete or disrupt α3α4α5(IV) in the GBM, and activate the unfolded protein response, collectively producing a spectrum of glomerular disease from thin basement membrane nephropathy to Alport syndrome."},"narrative":{"mechanistic_narrative":"COL4A4 encodes the α4 chain of type IV collagen, a structural component of the glomerular basement membrane (GBM) that assembles with the α3 and α5 chains into the α3α4α5(IV) heterotrimer [PMID:10534397, PMID:36514391]. The gene is arranged head-to-head with COL4A3 on chromosome 2q36 under a shared bidirectional promoter, with two alternative transcripts—an epithelial-predominant exon 1 form and a ubiquitously expressed low-level exon 1' form—driving tissue-specific expression [PMID:9537506]. Deletion of Col4a3/Col4a4 in mice eliminates both chains from the GBM and produces severe progressive glomerulonephritis with Alport-like ultrastructural changes, establishing the α4 chain as essential for GBM integrity and indicating that GBM-cell interactions govern glomerular cell maturation [PMID:10534397]. Heterotrimer assembly is initiated at the chain N-termini, and a homozygous G394S missense variant impairs N-terminal trimer assembly and reduces secretion by ~50% while leaving C-terminal association intact, localizing the assembly defect to the N-terminus [PMID:36514391]. In addition to complete loss of protein, in-frame mutations can yield dysfunctional trimers that are still secreted and incorporated into the GBM at reduced levels, recapitulating the abnormal collagen accumulation seen in a subset of Alport patients [PMID:24522496]. A major share of pathogenic COL4A4 alleles act not by altering coding sequence directly but by disrupting pre-mRNA splicing: synonymous, intronic, and deep-intronic variants cause exon skipping (e.g. exons 27 and 38) or frameshifting truncations that impair α3α4α5(IV) heterotrimer formation and secretion [PMID:39190490, PMID:41050124, PMID:42204651]. Intracellular retention of misfolded mutant collagen IV chains activates the unfolded protein response, a candidate modifier of disease severity [PMID:25514610]. Collectively, COL4A4 loss-of-function and dominant-negative alleles produce a spectrum of glomerular disease ranging from thin basement membrane nephropathy to autosomal recessive Alport syndrome [PMID:9792860, PMID:10534397, PMID:41050124].","teleology":[{"year":1998,"claim":"Established the genomic architecture and regulation of COL4A4, showing it shares a bidirectional promoter with COL4A3 and uses alternative promoters to achieve tissue-specific versus ubiquitous expression.","evidence":"RACE, RNase protection, and genomic sequencing with tissue expression analysis","pmids":["9537506"],"confidence":"High","gaps":["Does not establish which transcript predominates in glomerular podocytes/endothelium","Regulatory factors binding the GC/CCAAT boxes not identified"]},{"year":1998,"claim":"Defined the complete 48-exon structure of COL4A4 and linked loss-of-function mutations to autosomal recessive Alport syndrome, while showing not all glycine substitutions in collagen IV are pathogenic.","evidence":"Genomic structure characterization and comprehensive PCR/sequencing screen in patients and controls","pmids":["9792860"],"confidence":"High","gaps":["Does not resolve the molecular basis distinguishing benign from pathogenic glycine substitutions","No protein-level assembly data"]},{"year":1999,"claim":"Demonstrated in vivo that loss of the α4 chain eliminates the α3α4α5(IV) network from the GBM and causes Alport-like progressive glomerulonephritis, establishing the chain as essential for GBM integrity.","evidence":"Col4a3/Col4a4 deletion mouse with immunohistochemistry, RT-PCR, and GBM electron microscopy","pmids":["10534397"],"confidence":"High","gaps":["Deletion removes both Col4a3 and Col4a4, so chain-specific contributions are not separable","Mechanism of persistent cell proliferation not defined"]},{"year":2014,"claim":"Showed that in-frame mutations can produce dysfunctional trimers that are still secreted and incorporated into the GBM, distinguishing this mechanism from complete protein absence.","evidence":"Spontaneous Col4a4 splice-donor mouse causing in-frame exon 30 skipping, with GBM protein analysis and electron microscopy","pmids":["24522496"],"confidence":"High","gaps":["Does not define how the abnormal trimer alters GBM mechanics","Structural consequences of the missing exon 30 segment not resolved"]},{"year":2014,"claim":"Implicated the unfolded protein response as a candidate modifier of phenotypic severity, since mutant collagen IV chains are retained intracellularly and differentially activate the UPR.","evidence":"Podocyte transfection with wild-type/mutant chains and UPR pathway activation assays","pmids":["25514610"],"confidence":"Medium","gaps":["Experiment used mutant COL4A3, not COL4A4, chains; COL4A4 context inferred","No in vivo demonstration that UPR modifies disease severity"]},{"year":2022,"claim":"Localized the assembly defect of a pathogenic missense variant to the heterotrimer N-terminus, showing G394S impairs N-terminal assembly and halves secretion while sparing C-terminal association.","evidence":"Split NanoLuciferase α3α4α5(IV) heterotrimer formation assays (N- and C-terminal tags) in HEK293T cells","pmids":["36514391"],"confidence":"High","gaps":["Single variant tested; generality across missense variants not established","In vitro assay does not capture GBM incorporation"]},{"year":2022,"claim":"Began discriminating splicing-disrupting from neutral non-coding variants, with two tested COL4A4 variants showing no splicing effect while COL4A3 variants altered splicing.","evidence":"In vitro minigene splicing assays in HEK293T and HeLa cells","pmids":["35386907"],"confidence":"Medium","gaps":["Tested COL4A4 variants were negative, providing no positive COL4A4 splicing mechanism","Minigene context may not reflect native splicing"]},{"year":2024,"claim":"Connected a common splicing event to glomerular phenotypes, showing exon 27 skipping impairs heterotrimer formation and secretion and associates with hematuria and albuminuria.","evidence":"TWAS (UK Biobank + GTEx), glomerular mRNA replication, and split NanoLuc heterotrimer assay","pmids":["39190490"],"confidence":"High","gaps":["Effect of a common-allele splicing event on lifetime disease risk not quantified","Tissue regulation of the splicing event not detailed"]},{"year":2024,"claim":"Confirmed at the RNA level that a canonical splice-acceptor variant deletes exon 10 to yield a truncated protein, reinforcing splicing disruption as a recurrent pathogenic route.","evidence":"Whole-exome sequencing with RT-PCR cDNA analysis in a family","pmids":["36699462"],"confidence":"Medium","gaps":["Single family; protein-level consequence predicted, not measured","No GBM analysis"]},{"year":2025,"claim":"Documented compound dual exon-skipping (exons 27 and 38) from deep intronic variants disrupting heterotrimer assembly, requiring RNA-based diagnostics to detect.","evidence":"Whole-transcriptome sequencing, exon-specific PCR, and immunofluorescence for the α3α4α5(IV) heterotrimer","pmids":["41050124"],"confidence":"Medium","gaps":["Single case","Quantitative effect of dual skipping on residual trimer not measured"]},{"year":2025,"claim":"Confirmed at the RNA level a splice-acceptor variant producing a premature stop and severely truncated protein, extending the catalog of validated splicing-disrupting alleles.","evidence":"Whole-exome sequencing with in vivo RNA splicing validation from blood and urine","pmids":["40406358"],"confidence":"Medium","gaps":["Single case","Functional GBM/heterotrimer consequence not directly assayed"]},{"year":2026,"claim":"Systematically established that synonymous and intronic COL4A4 variants are predominantly pathogenic through pre-mRNA splicing disruption rather than coding-sequence change.","evidence":"Quantitative minigene splicing assays in HEK293T and HeLa cells across 14 variants with ACMG/AMP reclassification","pmids":["42204651"],"confidence":"High","gaps":["Minigene constructs may not reproduce all native splicing contexts","Direct protein-assembly consequences for each variant not measured"]},{"year":null,"claim":"How specific splicing/assembly defects quantitatively translate into the clinical spectrum from thin basement membrane nephropathy to Alport syndrome, and whether UPR activation causally modifies severity in vivo, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No genotype-to-severity quantitative model","UPR modifier role not tested in vivo for COL4A4","Native-context splicing regulation incompletely mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[2,3,5]}],"localization":[{"term_id":"GO:0031012","term_label":"extracellular matrix","supporting_discovery_ids":[2,3]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[3,5,6]}],"pathway":[{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[2,3]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[6,9]}],"complexes":["α3α4α5(IV) collagen heterotrimer"],"partners":["COL4A3","COL4A5"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P53420","full_name":"Collagen alpha-4(IV) chain","aliases":[],"length_aa":1690,"mass_kda":164.0,"function":"Type IV collagen is the major structural component of glomerular basement membranes (GBM), forming a 'chicken-wire' meshwork together with laminins, proteoglycans and entactin/nidogen","subcellular_location":"Secreted, extracellular space, extracellular matrix, basement membrane","url":"https://www.uniprot.org/uniprotkb/P53420/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/COL4A4","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/COL4A4","total_profiled":1310},"omim":[{"mim_id":"621556","title":"DEAFNESS, AUTOSOMAL DOMINANT 91; DFNA91","url":"https://www.omim.org/entry/621556"},{"mim_id":"620536","title":"ALPORT SYNDROME 3B, AUTOSOMAL RECESSIVE; ATS3B","url":"https://www.omim.org/entry/620536"},{"mim_id":"620320","title":"HEMATURIA, BENIGN FAMILIAL, 2; BFH2","url":"https://www.omim.org/entry/620320"},{"mim_id":"613420","title":"POTASSIUM CHANNEL TETRAMERIZATION DOMAIN-CONTAINING PROTEIN 1; KCTD1","url":"https://www.omim.org/entry/613420"},{"mim_id":"609469","title":"NEPHROPATHY, PROGRESSIVE, WITH DEAFNESS","url":"https://www.omim.org/entry/609469"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/COL4A4"},"hgnc":{"alias_symbol":["CA44"],"prev_symbol":[]},"alphafold":{"accession":"P53420","domains":[{"cath_id":"2.170.240.10","chopping":"1467-1661_1670-1689","consensus_level":"medium","plddt":94.5509,"start":1467,"end":1689}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P53420","model_url":"https://alphafold.ebi.ac.uk/files/AF-P53420-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P53420-F1-predicted_aligned_error_v6.png","plddt_mean":48.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=COL4A4","jax_strain_url":"https://www.jax.org/strain/search?query=COL4A4"},"sequence":{"accession":"P53420","fasta_url":"https://rest.uniprot.org/uniprotkb/P53420.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P53420/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P53420"}},"corpus_meta":[{"pmid":"12028435","id":"PMC_12028435","title":"COL4A3/COL4A4 mutations: from familial hematuria to autosomal-dominant or recessive Alport syndrome.","date":"2002","source":"Kidney international","url":"https://pubmed.ncbi.nlm.nih.gov/12028435","citation_count":167,"is_preprint":false},{"pmid":"24052634","id":"PMC_24052634","title":"COL4A3/COL4A4 mutations and features in individuals with autosomal recessive Alport syndrome.","date":"2013","source":"Journal of the American Society of Nephrology : JASN","url":"https://pubmed.ncbi.nlm.nih.gov/24052634","citation_count":134,"is_preprint":false},{"pmid":"19357112","id":"PMC_19357112","title":"Clinico-pathological correlations in 127 patients in 11 large pedigrees, segregating one of three heterozygous mutations in the COL4A3/ COL4A4 genes associated with familial haematuria and significant late progression to proteinuria and chronic kidney disease from focal segmental glomerulosclerosis.","date":"2009","source":"Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association","url":"https://pubmed.ncbi.nlm.nih.gov/19357112","citation_count":124,"is_preprint":false},{"pmid":"9792860","id":"PMC_9792860","title":"Determination of the genomic structure of the COL4A4 gene and of novel mutations causing autosomal recessive Alport syndrome.","date":"1998","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/9792860","citation_count":114,"is_preprint":false},{"pmid":"15086897","id":"PMC_15086897","title":"Autosomal-dominant Alport syndrome: natural history of a disease due to COL4A3 or COL4A4 gene.","date":"2004","source":"Kidney international","url":"https://pubmed.ncbi.nlm.nih.gov/15086897","citation_count":112,"is_preprint":false},{"pmid":"9269635","id":"PMC_9269635","title":"Autosomal dominant Alport syndrome linked to the type IV collage alpha 3 and alpha 4 genes (COL4A3 and COL4A4).","date":"1997","source":"Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association","url":"https://pubmed.ncbi.nlm.nih.gov/9269635","citation_count":106,"is_preprint":false},{"pmid":"33391746","id":"PMC_33391746","title":"Prevalence of clinical, pathological and molecular features of glomerular basement membrane nephropathy caused by COL4A3 or COL4A4 mutations: a systematic review.","date":"2020","source":"Clinical kidney journal","url":"https://pubmed.ncbi.nlm.nih.gov/33391746","citation_count":76,"is_preprint":false},{"pmid":"19129241","id":"PMC_19129241","title":"Autosomal dominant Alport syndrome: molecular analysis of the COL4A4 gene and clinical outcome.","date":"2009","source":"Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association","url":"https://pubmed.ncbi.nlm.nih.gov/19129241","citation_count":74,"is_preprint":false},{"pmid":"20029656","id":"PMC_20029656","title":"Polymorphisms in COL4A3 and COL4A4 genes associated with keratoconus.","date":"2009","source":"Molecular vision","url":"https://pubmed.ncbi.nlm.nih.gov/20029656","citation_count":66,"is_preprint":false},{"pmid":"12631110","id":"PMC_12631110","title":"Mutations in the COL4A4 gene in thin basement membrane disease.","date":"2003","source":"Kidney international","url":"https://pubmed.ncbi.nlm.nih.gov/12631110","citation_count":59,"is_preprint":false},{"pmid":"17396119","id":"PMC_17396119","title":"Sixteen novel mutations identified in COL4A3, COL4A4, and COL4A5 genes in Slovenian families with Alport syndrome and benign familial hematuria.","date":"2007","source":"Kidney international","url":"https://pubmed.ncbi.nlm.nih.gov/17396119","citation_count":58,"is_preprint":false},{"pmid":"11473630","id":"PMC_11473630","title":"COL4A4 mutation in thin basement membrane disease previously described in Alport syndrome.","date":"2001","source":"Kidney 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COL4A4 has two alternative transcripts from two different promoters: one transcription start site (exon 1') is only 5 bp from the COL4A3 start site, while the other (exon 1) starts 373 nucleotides downstream. The exon 1 transcript is expressed predominantly in epithelial cells, while the exon 1' transcript shows ubiquitous low expression. The promoter region contains CpG dinucleotides, GC boxes, CTC boxes, and a CCAAT box but no TATA box.\",\n      \"method\": \"RACE, RNase protection assays, genomic DNA sequencing, tissue expression analysis\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct transcription start site mapping by RACE and RNase protection, genomic structure confirmed by sequencing, tissue-specific expression validated\",\n      \"pmids\": [\"9537506\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The COL4A4 gene contains 48 exons encoding the alpha4 type IV collagen chain. Loss-of-function mutations (nonsense, frameshift, missense in collagenous domain) cause autosomal recessive Alport syndrome. A glycine-to-alanine substitution in the collagenous domain found in ~11.5% of controls and in a homozygous control individual does not produce an obvious phenotype, indicating not all glycine substitutions in collagen IV are pathogenic.\",\n      \"method\": \"Complete genomic structure characterization, comprehensive gene screen by PCR/sequencing in patients and controls\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — complete exon structure determined experimentally; mutation-to-disease linkage confirmed in multiple patients; homozygous benign variant functionally informative\",\n      \"pmids\": [\"9792860\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Deletion of Col4a4 exons 1–12 (along with Col4a3 exons 1–2 and the intergenic promoter) in mice results in complete absence of both Col4a3 and Col4a4 transcripts and both proteins from the glomerular basement membrane, causing severe progressive glomerulonephritis with GBM ultrastructural changes resembling Alport syndrome. Persistent cellular proliferation in mutant kidneys suggests interaction with extracellular matrix is important for cell maturation.\",\n      \"method\": \"Transgenic insertional mutagenesis, FISH mapping, RT-PCR (transcript detection), immunohistochemistry/protein analysis, electron microscopy of GBM\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — knockout mouse model with direct demonstration of absent protein in GBM, defined ultrastructural phenotype, replicated mechanistic insight about GBM assembly\",\n      \"pmids\": [\"10534397\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"A spontaneous Col4a4 splice-donor mutation (G-to-A in intron 30) causes skipping of exon 30 while maintaining the reading frame. The resulting mutant collagen α3α4α5(IV) trimers are secreted and incorporated into the GBM, but at reduced levels, and typical Alport GBM lesions develop. This is the only mouse model shown to accumulate abnormal collagen IV trimers in the GBM (as found in a subset of human Alport patients), demonstrating that missense/in-frame mutations can produce dysfunctional trimers rather than complete absence.\",\n      \"method\": \"Genetic mapping, DNA sequencing, protein analysis of GBM content, albuminuria measurement, electron microscopy\",\n      \"journal\": \"Kidney international\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vivo mouse model with direct protein analysis showing abnormal trimer secretion and GBM incorporation; mechanistic consequence of in-frame exon skipping established\",\n      \"pmids\": [\"24522496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Functional studies in cultured podocytes transfected with wild-type or mutant COL4A3 chains showed retention of mutant collagens intracellularly and differential activation of the unfolded protein response (UPR) cascade, indicating UPR activation as a potential modifier of phenotypic severity in collagen IV nephropathies caused by COL4A3/COL4A4 mutations.\",\n      \"method\": \"Cell transfection (podocytes), UPR pathway activation assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, cell transfection showing UPR activation with mutant COL4A3; COL4A4 context inferred but experiment used COL4A3 mutant chains\",\n      \"pmids\": [\"25514610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"A novel homozygous COL4A4 missense variant G394S impairs assembly of the α3α4α5(IV) heterotrimer N-terminus and reduces secretion by approximately 50% compared to wild-type, as demonstrated by split NanoLuciferase-based heterotrimer formation assays in HEK293T cells. C-terminal tag assays showed comparable luminescence to WT, indicating the defect is specific to N-terminal trimer assembly and subsequent secretion.\",\n      \"method\": \"Split NanoLuciferase (N-terminal and C-terminal) α3α4α5(IV) heterotrimer formation assay in HEK293T cells, immunofluorescence, targeted DNA sequencing\",\n      \"journal\": \"Kidney360\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution assay with split NanoLuc measuring heterotrimer assembly and secretion; two complementary tag orientations used; single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"36514391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Skipping of COL4A4 exon 27 (genetically predicted by intronic variant rs11898094, minor allele frequency 13%) is associated with hematuria and urinary albumin excretion. Functional assays using the split NanoLuc-based α3α4α5(IV) heterotrimer assay demonstrated that loss of exon 27 impairs type IV collagen heterotrimer formation and secretion.\",\n      \"method\": \"Transcriptome-wide association study (TWAS) using UK Biobank + GTEx kidney cortex data, independent replication in glomeruli-derived mRNA (n=245), split NanoLuc heterotrimer formation assay\",\n      \"journal\": \"Journal of the American Society of Nephrology : JASN\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — functional assay (split NanoLuc heterotrimer) directly demonstrates impaired assembly from exon 27 skipping; independently replicated splicing event in two cohorts\",\n      \"pmids\": [\"39190490\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Minigene assay in HEK293T/HeLa cells demonstrated that presumed missense variant COL4A3 c.4793T>G (p.Leu1598Arg) causes loss of an alternative full-length transcript during splicing, and synonymous variant COL4A3 c.765G>A (p.Thr255Thr) leads to in-frame deletion of exon 13. In contrast, COL4A4 variants c.3990G>A (p.Pro1330Pro) and c.4766C>T (p.Pro1589Leu) exhibited no deleterious effect on splicing.\",\n      \"method\": \"In vitro minigene splicing assay in HEK293T and HeLa cells\",\n      \"journal\": \"Frontiers in medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — minigene assay is Tier 1 method but COL4A3 variants tested, not COL4A4; negative results for COL4A4 variants c.3990G>A and c.4766C>T explicitly noted\",\n      \"pmids\": [\"35386907\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Dual exon-skipping events at COL4A4 exons 27 and 38 (caused by compound heterozygous deep intronic variants) disrupt α3α4α5(IV) heterotrimer assembly, as confirmed by immunofluorescence analysis. This represents the first documented case of dual COL4A4 exon-skipping events contributing to autosomal recessive Alport syndrome severity. RNA-based diagnostics (whole-transcriptome sequencing) were required to detect these variants missed by DNA sequencing.\",\n      \"method\": \"Whole-transcriptome sequencing, exon-specific PCR, Sanger sequencing, immunofluorescence for α3α4α5(IV) heterotrimer\",\n      \"journal\": \"Kidney medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — RNA sequencing and immunofluorescence confirm exon skipping and disrupted heterotrimer; single case, single lab\",\n      \"pmids\": [\"41050124\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Minigene assays in HEK293T and HeLa cells for 14 synonymous and intronic COL4A3/COL4A4 variants showed that 13 caused aberrant splicing (predominantly exon skipping), including frameshift transcripts with premature termination codons and in-frame exon skipping events. These results demonstrate that synonymous and intronic variants in COL4A4 cause pathogenic effects primarily through disruption of pre-mRNA splicing rather than direct coding-sequence alteration.\",\n      \"method\": \"In vitro minigene splicing assay in HEK293T and HeLa cells, quantitative splicing analysis, ACMG/AMP reclassification\",\n      \"journal\": \"Human genomics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic minigene functional assays across 14 variants with quantitative outcomes; two cell lines used; direct mechanistic demonstration of splicing disruption\",\n      \"pmids\": [\"42204651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The COL4A4 c.817-1G>A splice acceptor site mutation disrupts mRNA splicing by affecting the splice acceptor site of intron 13 adjacent to exon 14, causing a 1-bp deletion before exon 14 and creating a premature stop codon. The resulting truncated protein is predicted to be 273 amino acids rather than the full-length 1,690 amino acids.\",\n      \"method\": \"Whole-exome sequencing, in vivo splicing validation using RNA from blood and urine (RT-PCR, Sanger sequencing)\",\n      \"journal\": \"Frontiers in pediatrics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — in vivo RNA splicing validation directly demonstrates aberrant splicing product; single case, single lab\",\n      \"pmids\": [\"40406358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The COL4A4 splicing variant c.595-1G>A abolishes the canonical splice acceptor site of intron 9 and causes a single nucleotide deletion of exon 10, predicted to produce a truncated protein, as demonstrated by complementary DNA analysis.\",\n      \"method\": \"Whole exome sequencing, Sanger sequencing, RT-PCR cDNA analysis\",\n      \"journal\": \"Frontiers in genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct cDNA analysis confirms exon 10 deletion at RNA level; single lab, single family\",\n      \"pmids\": [\"36699462\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"A COL4A4 splicing mutation (c.1459+1G>A) causes elimination of the entire exon 21 from COL4A4 cDNA, resulting in direct splicing of exons 20 and 22 and a frameshift mutation after exon 20 in the open reading frame, as demonstrated by RT-PCR and TA cloning of patient RNA.\",\n      \"method\": \"Direct sequencing, RT-PCR, TA cloning of cDNA\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — RNA-level confirmation of exon skipping by RT-PCR and cloning; single lab, single family\",\n      \"pmids\": [\"26833262\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"COL4A4 encodes the α4 chain of type IV collagen, which assembles with α3 and α5 chains into heterotrimers that are incorporated into the glomerular basement membrane (GBM); the gene is arranged head-to-head with COL4A3 on chromosome 2q36 under a shared bidirectional promoter driving tissue-specific expression, and loss-of-function or dominant-negative mutations (including missense substitutions, frameshift, nonsense, and splicing variants causing exon skipping) impair N-terminal-mediated heterotrimer assembly and/or secretion, deplete or disrupt α3α4α5(IV) in the GBM, and activate the unfolded protein response, collectively producing a spectrum of glomerular disease from thin basement membrane nephropathy to Alport syndrome.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"COL4A4 encodes the α4 chain of type IV collagen, a structural component of the glomerular basement membrane (GBM) that assembles with the α3 and α5 chains into the α3α4α5(IV) heterotrimer [#2, #5]. The gene is arranged head-to-head with COL4A3 on chromosome 2q36 under a shared bidirectional promoter, with two alternative transcripts—an epithelial-predominant exon 1 form and a ubiquitously expressed low-level exon 1' form—driving tissue-specific expression [#0]. Deletion of Col4a3/Col4a4 in mice eliminates both chains from the GBM and produces severe progressive glomerulonephritis with Alport-like ultrastructural changes, establishing the α4 chain as essential for GBM integrity and indicating that GBM-cell interactions govern glomerular cell maturation [#2]. Heterotrimer assembly is initiated at the chain N-termini, and a homozygous G394S missense variant impairs N-terminal trimer assembly and reduces secretion by ~50% while leaving C-terminal association intact, localizing the assembly defect to the N-terminus [#5]. In addition to complete loss of protein, in-frame mutations can yield dysfunctional trimers that are still secreted and incorporated into the GBM at reduced levels, recapitulating the abnormal collagen accumulation seen in a subset of Alport patients [#3]. A major share of pathogenic COL4A4 alleles act not by altering coding sequence directly but by disrupting pre-mRNA splicing: synonymous, intronic, and deep-intronic variants cause exon skipping (e.g. exons 27 and 38) or frameshifting truncations that impair α3α4α5(IV) heterotrimer formation and secretion [#6, #8, #9]. Intracellular retention of misfolded mutant collagen IV chains activates the unfolded protein response, a candidate modifier of disease severity [#4]. Collectively, COL4A4 loss-of-function and dominant-negative alleles produce a spectrum of glomerular disease ranging from thin basement membrane nephropathy to autosomal recessive Alport syndrome [#1, #2, #8].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established the genomic architecture and regulation of COL4A4, showing it shares a bidirectional promoter with COL4A3 and uses alternative promoters to achieve tissue-specific versus ubiquitous expression.\",\n      \"evidence\": \"RACE, RNase protection, and genomic sequencing with tissue expression analysis\",\n      \"pmids\": [\"9537506\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not establish which transcript predominates in glomerular podocytes/endothelium\", \"Regulatory factors binding the GC/CCAAT boxes not identified\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Defined the complete 48-exon structure of COL4A4 and linked loss-of-function mutations to autosomal recessive Alport syndrome, while showing not all glycine substitutions in collagen IV are pathogenic.\",\n      \"evidence\": \"Genomic structure characterization and comprehensive PCR/sequencing screen in patients and controls\",\n      \"pmids\": [\"9792860\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not resolve the molecular basis distinguishing benign from pathogenic glycine substitutions\", \"No protein-level assembly data\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Demonstrated in vivo that loss of the α4 chain eliminates the α3α4α5(IV) network from the GBM and causes Alport-like progressive glomerulonephritis, establishing the chain as essential for GBM integrity.\",\n      \"evidence\": \"Col4a3/Col4a4 deletion mouse with immunohistochemistry, RT-PCR, and GBM electron microscopy\",\n      \"pmids\": [\"10534397\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Deletion removes both Col4a3 and Col4a4, so chain-specific contributions are not separable\", \"Mechanism of persistent cell proliferation not defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed that in-frame mutations can produce dysfunctional trimers that are still secreted and incorporated into the GBM, distinguishing this mechanism from complete protein absence.\",\n      \"evidence\": \"Spontaneous Col4a4 splice-donor mouse causing in-frame exon 30 skipping, with GBM protein analysis and electron microscopy\",\n      \"pmids\": [\"24522496\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not define how the abnormal trimer alters GBM mechanics\", \"Structural consequences of the missing exon 30 segment not resolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Implicated the unfolded protein response as a candidate modifier of phenotypic severity, since mutant collagen IV chains are retained intracellularly and differentially activate the UPR.\",\n      \"evidence\": \"Podocyte transfection with wild-type/mutant chains and UPR pathway activation assays\",\n      \"pmids\": [\"25514610\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Experiment used mutant COL4A3, not COL4A4, chains; COL4A4 context inferred\", \"No in vivo demonstration that UPR modifies disease severity\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Localized the assembly defect of a pathogenic missense variant to the heterotrimer N-terminus, showing G394S impairs N-terminal assembly and halves secretion while sparing C-terminal association.\",\n      \"evidence\": \"Split NanoLuciferase α3α4α5(IV) heterotrimer formation assays (N- and C-terminal tags) in HEK293T cells\",\n      \"pmids\": [\"36514391\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Single variant tested; generality across missense variants not established\", \"In vitro assay does not capture GBM incorporation\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Began discriminating splicing-disrupting from neutral non-coding variants, with two tested COL4A4 variants showing no splicing effect while COL4A3 variants altered splicing.\",\n      \"evidence\": \"In vitro minigene splicing assays in HEK293T and HeLa cells\",\n      \"pmids\": [\"35386907\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tested COL4A4 variants were negative, providing no positive COL4A4 splicing mechanism\", \"Minigene context may not reflect native splicing\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Connected a common splicing event to glomerular phenotypes, showing exon 27 skipping impairs heterotrimer formation and secretion and associates with hematuria and albuminuria.\",\n      \"evidence\": \"TWAS (UK Biobank + GTEx), glomerular mRNA replication, and split NanoLuc heterotrimer assay\",\n      \"pmids\": [\"39190490\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Effect of a common-allele splicing event on lifetime disease risk not quantified\", \"Tissue regulation of the splicing event not detailed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Confirmed at the RNA level that a canonical splice-acceptor variant deletes exon 10 to yield a truncated protein, reinforcing splicing disruption as a recurrent pathogenic route.\",\n      \"evidence\": \"Whole-exome sequencing with RT-PCR cDNA analysis in a family\",\n      \"pmids\": [\"36699462\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single family; protein-level consequence predicted, not measured\", \"No GBM analysis\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Documented compound dual exon-skipping (exons 27 and 38) from deep intronic variants disrupting heterotrimer assembly, requiring RNA-based diagnostics to detect.\",\n      \"evidence\": \"Whole-transcriptome sequencing, exon-specific PCR, and immunofluorescence for the α3α4α5(IV) heterotrimer\",\n      \"pmids\": [\"41050124\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single case\", \"Quantitative effect of dual skipping on residual trimer not measured\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Confirmed at the RNA level a splice-acceptor variant producing a premature stop and severely truncated protein, extending the catalog of validated splicing-disrupting alleles.\",\n      \"evidence\": \"Whole-exome sequencing with in vivo RNA splicing validation from blood and urine\",\n      \"pmids\": [\"40406358\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single case\", \"Functional GBM/heterotrimer consequence not directly assayed\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Systematically established that synonymous and intronic COL4A4 variants are predominantly pathogenic through pre-mRNA splicing disruption rather than coding-sequence change.\",\n      \"evidence\": \"Quantitative minigene splicing assays in HEK293T and HeLa cells across 14 variants with ACMG/AMP reclassification\",\n      \"pmids\": [\"42204651\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Minigene constructs may not reproduce all native splicing contexts\", \"Direct protein-assembly consequences for each variant not measured\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How specific splicing/assembly defects quantitatively translate into the clinical spectrum from thin basement membrane nephropathy to Alport syndrome, and whether UPR activation causally modifies severity in vivo, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No genotype-to-severity quantitative model\", \"UPR modifier role not tested in vivo for COL4A4\", \"Native-context splicing regulation incompletely mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [2, 3, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031012\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [3, 5, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [6, 9]}\n    ],\n    \"complexes\": [\n      \"α3α4α5(IV) collagen heterotrimer\"\n    ],\n    \"partners\": [\n      \"COL4A3\",\n      \"COL4A5\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":8,"faith_pct":87.5}}