{"gene":"MAD1L1","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2001,"finding":"HsMAD1 (MAD1L1) localizes to kinetochores during prometaphase and is absent from kinetochores at metaphase/anaphase; it physically associates with HsMAD2 but not with p55CDC; both HsMAD1 and HsMAD2 co-localize with nuclear pore complexes throughout interphase, confirmed by co-labeling with nuclear pore antibodies and co-purification with enriched nuclear envelope fractions.","method":"Immunofluorescence co-labeling, subcellular fractionation/co-purification, co-immunoprecipitation","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus independent fractionation and co-labeling, replicated across multiple cell lines","pmids":["11181178"],"is_preprint":false},{"year":2001,"finding":"A truncated dominant-negative mutant form of MAD1L1 (found in a lymphoma sample) is less inhibitory than wild-type MAD1L1 at decreasing cell proliferation; co-expression experiments confirmed dominant-negative activity; the mutant impaired the mitotic checkpoint, demonstrated by decreased mitotic indices in HOS cells expressing mutant MAD1L1 after nocodazole treatment.","method":"Transfection of wild-type vs. mutant MAD1L1 constructs, co-expression experiments, mitotic index assay with nocodazole","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional loss-of-checkpoint phenotype with dominant-negative construct in multiple cell lines, single lab","pmids":["11423979"],"is_preprint":false},{"year":2002,"finding":"Two leucine zipper domains in hsMAD1 (amino acids 501–522 and 557–571) are required for binding to hsMAD2; a coding SNP at codon 558 (Arg→His) within the second leucine zipper reduces MAD1–MAD2 binding affinity and impairs mitotic arrest; loss-of-heterozygosity at the hsMAD1 558 locus was documented in a human breast cancer.","method":"Deletion/point-mutation mapping of leucine zipper domains, Co-IP binding assays, mitotic arrest assay, LOH analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — domain mutagenesis with functional validation (binding + checkpoint assay), single lab with multiple orthogonal methods","pmids":["12042300"],"is_preprint":false},{"year":2002,"finding":"The hsMAD1 promoter is GC-rich and TATA-box-less; a core region spanning −73 to −31 is essential for transcription; the promoter is active predominantly in G1 (not induced by microtubule inhibitors), responds to mitogenic stimuli, and is preferentially activated by a gain-of-function p53 mutant.","method":"Promoter deletion/mutational analysis, reporter assays, cell-cycle synchronization experiments","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — systematic mutational analysis with reporter assays, single lab","pmids":["11980658"],"is_preprint":false},{"year":2010,"finding":"Cells expressing the MAD1L1 Arg558His variant display impaired spindle assembly checkpoint function, shown by lower 4N-DNA content and lower mitotic index following nocodazole treatment.","method":"Flow cytometry (4N-DNA content), mitotic index assay with nocodazole","journal":"Journal of medical genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal functional assays, single lab, consistent with prior mutagenesis data","pmids":["20516147"],"is_preprint":false},{"year":2017,"finding":"A RARS–MAD1L1 fusion protein interacts with AIMP2, activating the FUBP1/c-Myc pathway; silencing FUBP1 or inhibiting c-Myc abrogated the cancer stem cell-like characteristics induced by RARS–MAD1L1.","method":"Co-immunoprecipitation, chromatin immunoprecipitation assay, MTT/colony/sphere formation assays, in vivo xenograft assay","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ChIP plus functional rescue experiments, single lab","pmids":["29133573"],"is_preprint":false},{"year":2022,"finding":"Biallelic germline loss-of-function mutations in MAD1L1 result in absence of full-length MAD1 protein, deficient spindle assembly checkpoint (SAC) response, and ~30–40% aneuploid blood cells; single-cell RNA analysis revealed mitochondrial stress and systemic inflammation (enhanced interferon and NFκB signaling) in both aneuploid and euploid cells, and specific clonal expansions of γδ T cells (chromosome 18 gains) and intermediate B cells (chromosome 12 gains) with leukemic transcriptomic signatures.","method":"Germline mutation functional studies, SAC response assays, single-cell RNA sequencing, protein expression analysis","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (protein assay, SAC assay, scRNA-seq), physiological human patient context","pmids":["36322655"],"is_preprint":false},{"year":2023,"finding":"MAD1L1 physically interacts with CHPF (chondroitin polymerizing factor) in glioma cells, as demonstrated by immunoprecipitation, co-immunoprecipitation, GST pulldown, and LC-MS/MS.","method":"Co-immunoprecipitation, GST pulldown, LC-MS/MS","journal":"Aging","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — three orthogonal binding assays (Co-IP, GST pulldown, MS), single lab","pmids":["37851364"],"is_preprint":false}],"current_model":"MAD1L1 encodes a mitotic spindle assembly checkpoint (SAC) protein that localizes to kinetochores in prometaphase and to nuclear pore complexes in interphase; it directly binds MAD2 through two leucine zipper domains (residues 501–522 and 557–571), and this interaction is required for MAD2 kinetochore recruitment and enforcement of mitotic arrest; a common Arg558His polymorphism within the second leucine zipper reduces MAD1–MAD2 binding and attenuates SAC function; biallelic loss of MAD1L1 causes profound aneuploidy with systemic inflammation via NFκB/interferon signaling; the MAD1L1 promoter is regulated by mitogenic signals and gain-of-function p53 mutants; and MAD1L1 also participates in non-mitotic protein–protein interactions (e.g., with CHPF in glioma) whose functional significance is under investigation."},"narrative":{"mechanistic_narrative":"MAD1L1 encodes a core mitotic spindle assembly checkpoint (SAC) protein that enforces accurate chromosome segregation by recruiting MAD2 to unattached kinetochores [PMID:11181178, PMID:12042300]. It localizes dynamically through the cell cycle, decorating kinetochores specifically in prometaphase and departing by metaphase/anaphase, while in interphase it co-localizes with nuclear pore complexes [PMID:11181178]. MAD1L1 physically associates with MAD2 through two leucine zipper domains (residues 501–522 and 557–571), and this interaction is essential for checkpoint enforcement; a coding polymorphism at codon 558 (Arg→His) within the second leucine zipper reduces MAD1–MAD2 binding affinity and impairs nocodazole-induced mitotic arrest [PMID:12042300, PMID:20516147]. Loss of MAD1L1 function compromises the checkpoint and drives chromosomal instability: a truncated dominant-negative mutant from lymphoma weakens the mitotic checkpoint [PMID:11423979], and biallelic germline loss-of-function abolishes full-length MAD1, producing a deficient SAC, profound aneuploidy in blood cells, mitochondrial stress, and systemic interferon and NFκB inflammatory signaling with clonal expansions bearing leukemic signatures [PMID:36322655]. The MAD1L1 promoter is GC-rich and TATA-less, active in G1, responsive to mitogenic stimuli, and preferentially activated by a gain-of-function p53 mutant [PMID:11980658]. Beyond mitosis, MAD1L1 engages non-checkpoint protein interactions, including binding to CHPF in glioma cells, whose functional role is not characterized in the available corpus [PMID:37851364].","teleology":[{"year":2001,"claim":"Establishing where MAD1L1 acts and what it partners with anchored it as a kinetochore-localized checkpoint component physically coupled to MAD2.","evidence":"Immunofluorescence co-labeling, subcellular fractionation, and reciprocal co-immunoprecipitation across cell lines","pmids":["11181178"],"confidence":"High","gaps":["Did not map the MAD1–MAD2 interaction interface","Functional consequence of nuclear pore localization in interphase unresolved"]},{"year":2001,"claim":"Demonstrating that a lymphoma-derived truncated MAD1L1 acts as a dominant-negative and weakens the checkpoint linked MAD1L1 loss of function to tumorigenic chromosome instability.","evidence":"Transfection of wild-type vs. mutant constructs with nocodazole mitotic index assays in HOS cells","pmids":["11423979"],"confidence":"Medium","gaps":["Single dominant-negative allele; not a clean loss-of-function genetic test","Did not quantify aneuploidy directly"]},{"year":2002,"claim":"Mapping two leucine zipper domains required for MAD2 binding and showing the codon-558 variant weakens it connected a defined protein region to checkpoint strength and a cancer-associated allele.","evidence":"Deletion/point-mutation mapping, Co-IP binding assays, mitotic arrest assay, and LOH analysis in breast cancer","pmids":["12042300"],"confidence":"High","gaps":["No structural model of the bound interface","Population-level penetrance of the 558 variant not addressed"]},{"year":2002,"claim":"Defining the MAD1L1 promoter architecture and its cell-cycle and p53-mutant responsiveness explained how checkpoint protein levels are coupled to proliferative signaling.","evidence":"Promoter deletion/mutational analysis with reporter assays and cell-cycle synchronization","pmids":["11980658"],"confidence":"Medium","gaps":["Specific transcription factors binding the core region not identified","Mechanism of gain-of-function p53 activation unresolved"]},{"year":2010,"claim":"Independent functional testing of the Arg558His variant confirmed it impairs checkpoint responses, corroborating the earlier binding-based prediction in a clinical-genetic context.","evidence":"Flow cytometry for 4N-DNA content and nocodazole mitotic index assays","pmids":["20516147"],"confidence":"Medium","gaps":["Did not test endogenous heterozygous vs homozygous variant carriers","No direct aneuploidy measurement"]},{"year":2017,"claim":"Characterizing a RARS–MAD1L1 fusion oncoprotein that activates FUBP1/c-Myc revealed MAD1L1 sequence in a gain-of-function rearrangement distinct from its checkpoint role.","evidence":"Co-IP, ChIP, in vitro proliferation/sphere assays, and xenografts with FUBP1 silencing and c-Myc inhibition","pmids":["29133573"],"confidence":"Medium","gaps":["Contribution of the MAD1L1 portion to fusion activity not dissected","Relevance to wild-type MAD1L1 function unclear"]},{"year":2022,"claim":"Identifying biallelic germline loss-of-function patients tied complete MAD1L1 deficiency to SAC failure, aneuploidy, and a systemic inflammatory/leukemic phenotype, establishing physiological consequences in humans.","evidence":"Germline mutation functional studies, SAC assays, protein expression, and single-cell RNA sequencing of patient cells","pmids":["36322655"],"confidence":"High","gaps":["Mechanism linking aneuploidy to interferon/NFκB activation not fully resolved","Why specific chromosome gains undergo clonal expansion unknown"]},{"year":2023,"claim":"Detecting a MAD1L1–CHPF physical interaction in glioma raised a candidate non-mitotic partnership for MAD1L1.","evidence":"Co-IP, GST pulldown, and LC-MS/MS in glioma cells","pmids":["37851364"],"confidence":"Medium","gaps":["Functional consequence of the MAD1L1–CHPF interaction not established","Interaction not validated outside glioma context"]},{"year":null,"claim":"How MAD1L1-dependent aneuploidy is mechanistically translated into NFκB/interferon inflammatory signaling and selective clonal expansion remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No defined signaling intermediary between mis-segregation and innate immune activation","No structural basis for the MAD1–MAD2 kinetochore recruitment complex in the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,2]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[3]}],"localization":[{"term_id":"GO:0005635","term_label":"nuclear envelope","supporting_discovery_ids":[0]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,1,2,6]}],"complexes":["spindle assembly checkpoint (MAD1–MAD2)","kinetochore","nuclear pore complex"],"partners":["MAD2","CHPF"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y6D9","full_name":"Mitotic spindle assembly checkpoint protein MAD1","aliases":["Mitotic arrest deficient 1-like protein 1","MAD1-like protein 1","Mitotic checkpoint MAD1 protein homolog","HsMAD1","hMAD1","Tax-binding protein 181"],"length_aa":718,"mass_kda":83.1,"function":"Component of the spindle-assembly checkpoint that prevents the onset of anaphase until all chromosomes are properly aligned at the metaphase plate (PubMed:10049595, PubMed:20133940, PubMed:29162720). Forms a heterotetrameric complex with the closed conformation form of MAD2L1 (C-MAD2) at unattached kinetochores during prometaphase, recruits an open conformation of MAD2L1 (O-MAD2) and promotes the conversion of O-MAD2 to C-MAD2, which ensures mitotic checkpoint signaling (PubMed:29162720) Sequesters MAD2L1 in the cytoplasm preventing its function as an activator of the mitotic spindle assembly checkpoint (SAC) resulting in SAC impairment and chromosomal instability in hepatocellular carcinomas","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9Y6D9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MAD1L1","classification":"Not Classified","n_dependent_lines":54,"n_total_lines":1208,"dependency_fraction":0.04470198675496689},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MAD1L1","total_profiled":1310},"omim":[{"mim_id":"620189","title":"MOSAIC VARIEGATED ANEUPLOIDY SYNDROME 7 WITH INFLAMMATION AND TUMOR PREDISPOSITION; MVA7","url":"https://www.omim.org/entry/620189"},{"mim_id":"618136","title":"MAD2L1-BINDING PROTEIN; MAD2L1BP","url":"https://www.omim.org/entry/618136"},{"mim_id":"615890","title":"DYNEIN, CYTOPLASMIC 1, LIGHT INTERMEDIATE CHAIN 1; DYNC1LI1","url":"https://www.omim.org/entry/615890"},{"mim_id":"613713","title":"PCI DOMAIN-CONTAINING PROTEIN 2; PCID2","url":"https://www.omim.org/entry/613713"},{"mim_id":"610266","title":"TAO KINASE 1; TAOK1","url":"https://www.omim.org/entry/610266"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nuclear membrane","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MAD1L1"},"hgnc":{"alias_symbol":["HsMAD1","TXBP181","MAD1","PIG9","TP53I9"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y6D9","domains":[{"cath_id":"-","chopping":"334-491","consensus_level":"medium","plddt":81.7897,"start":334,"end":491},{"cath_id":"3.30.457.60","chopping":"639-718","consensus_level":"high","plddt":81.658,"start":639,"end":718},{"cath_id":"1.20.5","chopping":"59-242","consensus_level":"medium","plddt":93.0331,"start":59,"end":242}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y6D9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y6D9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y6D9-F1-predicted_aligned_error_v6.png","plddt_mean":81.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MAD1L1","jax_strain_url":"https://www.jax.org/strain/search?query=MAD1L1"},"sequence":{"accession":"Q9Y6D9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y6D9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y6D9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y6D9"}},"corpus_meta":[{"pmid":"11181178","id":"PMC_11181178","title":"Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase.","date":"2001","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/11181178","citation_count":162,"is_preprint":false},{"pmid":"11423979","id":"PMC_11423979","title":"Mutations in the mitotic check point gene, MAD1L1, in human cancers.","date":"2001","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/11423979","citation_count":89,"is_preprint":false},{"pmid":"29133573","id":"PMC_29133573","title":"The RARS-MAD1L1 Fusion Gene Induces Cancer Stem Cell-like Properties and Therapeutic Resistance in Nasopharyngeal Carcinoma.","date":"2017","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/29133573","citation_count":52,"is_preprint":false},{"pmid":"16130125","id":"PMC_16130125","title":"Gain of a region on 7p22.3, containing MAD1L1, is the most frequent event in small-cell lung cancer cell lines.","date":"2006","source":"Genes, chromosomes & cancer","url":"https://pubmed.ncbi.nlm.nih.gov/16130125","citation_count":48,"is_preprint":false},{"pmid":"20516147","id":"PMC_20516147","title":"Functional evaluation of missense variations in the human MAD1L1 and MAD2L1 genes and their impact on susceptibility to lung cancer.","date":"2010","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/20516147","citation_count":42,"is_preprint":false},{"pmid":"11980658","id":"PMC_11980658","title":"Expression of mitotic spindle checkpoint protein hsMAD1 correlates with cellular proliferation and is activated by a gain-of-function p53 mutant.","date":"2002","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/11980658","citation_count":39,"is_preprint":false},{"pmid":"12042300","id":"PMC_12042300","title":"Characterization of regions in hsMAD1 needed for binding hsMAD2. A polymorphic change in an hsMAD1 leucine zipper affects MAD1-MAD2 interaction and spindle checkpoint function.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12042300","citation_count":39,"is_preprint":false},{"pmid":"27503294","id":"PMC_27503294","title":"Epigenetic Variability across Human Populations: A Focus on DNA Methylation Profiles of the KRTCAP3, MAD1L1 and BRSK2 Genes.","date":"2016","source":"Genome biology and evolution","url":"https://pubmed.ncbi.nlm.nih.gov/27503294","citation_count":30,"is_preprint":false},{"pmid":"30531795","id":"PMC_30531795","title":"Replicated associations of FADS1, MAD1L1, and a rare variant at 10q26.13 with bipolar disorder in Chinese population.","date":"2018","source":"Translational psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/30531795","citation_count":25,"is_preprint":false},{"pmid":"26183163","id":"PMC_26183163","title":"MAD1L1 Arg558His and MAD2L1 Leu84Met interaction with smoking increase the risk of colorectal cancer.","date":"2015","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/26183163","citation_count":21,"is_preprint":false},{"pmid":"36600305","id":"PMC_36600305","title":"Methylation in MAD1L1 is associated with the severity of suicide attempt and phenotypes of depression.","date":"2023","source":"Clinical epigenetics","url":"https://pubmed.ncbi.nlm.nih.gov/36600305","citation_count":19,"is_preprint":false},{"pmid":"27184339","id":"PMC_27184339","title":"Investigating the Impact of a Genome-Wide Supported Bipolar Risk Variant of MAD1L1 on the Human Reward System.","date":"2016","source":"Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/27184339","citation_count":18,"is_preprint":false},{"pmid":"35987687","id":"PMC_35987687","title":"Schizophrenia-associated differential DNA methylation in brain is distributed across the genome and annotated to MAD1L1, a locus at which DNA methylation and transcription phenotypes share genetic variation with schizophrenia risk.","date":"2022","source":"Translational psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/35987687","citation_count":17,"is_preprint":false},{"pmid":"36322655","id":"PMC_36322655","title":"Biallelic germline mutations in MAD1L1 induce a syndrome of aneuploidy with high tumor susceptibility.","date":"2022","source":"Science advances","url":"https://pubmed.ncbi.nlm.nih.gov/36322655","citation_count":11,"is_preprint":false},{"pmid":"26528791","id":"PMC_26528791","title":"Genetic association of GWAS-supported MAD1L1 gene polymorphism rs12666575 with schizophrenia susceptibility in a Chinese population.","date":"2015","source":"Neuroscience letters","url":"https://pubmed.ncbi.nlm.nih.gov/26528791","citation_count":11,"is_preprint":false},{"pmid":"34481484","id":"PMC_34481484","title":"MAD1L1 and TSNARE gene polymorphisms are associated with schizophrenia susceptibility in the Han Chinese population.","date":"2021","source":"BMC medical genomics","url":"https://pubmed.ncbi.nlm.nih.gov/34481484","citation_count":8,"is_preprint":false},{"pmid":"37851364","id":"PMC_37851364","title":"The HNF4A-CHPF pathway promotes proliferation and invasion through interactions with MAD1L1 in glioma.","date":"2023","source":"Aging","url":"https://pubmed.ncbi.nlm.nih.gov/37851364","citation_count":6,"is_preprint":false},{"pmid":"37007941","id":"PMC_37007941","title":"Maternal genetic polymorphisms in the major mitotic checkpoint genes MAD1L1 and MAD2L1 associated with the risk of survival in abnormal chromosomal fetuses.","date":"2023","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/37007941","citation_count":4,"is_preprint":false},{"pmid":"39079086","id":"PMC_39079086","title":"DNA methylation near MAD1L1, KDM2B, and SOCS3 mediates the effect of socioeconomic status on elevated body mass index in African American 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biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/42003906","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13895,"output_tokens":2009,"usd":0.03591,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9029,"output_tokens":2814,"usd":0.057748,"stage2_stop_reason":"end_turn"},"total_usd":0.093658,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"HsMAD1 (MAD1L1) localizes to kinetochores during prometaphase and is absent from kinetochores at metaphase/anaphase; it physically associates with HsMAD2 but not with p55CDC; both HsMAD1 and HsMAD2 co-localize with nuclear pore complexes throughout interphase, confirmed by co-labeling with nuclear pore antibodies and co-purification with enriched nuclear envelope fractions.\",\n      \"method\": \"Immunofluorescence co-labeling, subcellular fractionation/co-purification, co-immunoprecipitation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus independent fractionation and co-labeling, replicated across multiple cell lines\",\n      \"pmids\": [\"11181178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"A truncated dominant-negative mutant form of MAD1L1 (found in a lymphoma sample) is less inhibitory than wild-type MAD1L1 at decreasing cell proliferation; co-expression experiments confirmed dominant-negative activity; the mutant impaired the mitotic checkpoint, demonstrated by decreased mitotic indices in HOS cells expressing mutant MAD1L1 after nocodazole treatment.\",\n      \"method\": \"Transfection of wild-type vs. mutant MAD1L1 constructs, co-expression experiments, mitotic index assay with nocodazole\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional loss-of-checkpoint phenotype with dominant-negative construct in multiple cell lines, single lab\",\n      \"pmids\": [\"11423979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Two leucine zipper domains in hsMAD1 (amino acids 501–522 and 557–571) are required for binding to hsMAD2; a coding SNP at codon 558 (Arg→His) within the second leucine zipper reduces MAD1–MAD2 binding affinity and impairs mitotic arrest; loss-of-heterozygosity at the hsMAD1 558 locus was documented in a human breast cancer.\",\n      \"method\": \"Deletion/point-mutation mapping of leucine zipper domains, Co-IP binding assays, mitotic arrest assay, LOH analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — domain mutagenesis with functional validation (binding + checkpoint assay), single lab with multiple orthogonal methods\",\n      \"pmids\": [\"12042300\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The hsMAD1 promoter is GC-rich and TATA-box-less; a core region spanning −73 to −31 is essential for transcription; the promoter is active predominantly in G1 (not induced by microtubule inhibitors), responds to mitogenic stimuli, and is preferentially activated by a gain-of-function p53 mutant.\",\n      \"method\": \"Promoter deletion/mutational analysis, reporter assays, cell-cycle synchronization experiments\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic mutational analysis with reporter assays, single lab\",\n      \"pmids\": [\"11980658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Cells expressing the MAD1L1 Arg558His variant display impaired spindle assembly checkpoint function, shown by lower 4N-DNA content and lower mitotic index following nocodazole treatment.\",\n      \"method\": \"Flow cytometry (4N-DNA content), mitotic index assay with nocodazole\",\n      \"journal\": \"Journal of medical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal functional assays, single lab, consistent with prior mutagenesis data\",\n      \"pmids\": [\"20516147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A RARS–MAD1L1 fusion protein interacts with AIMP2, activating the FUBP1/c-Myc pathway; silencing FUBP1 or inhibiting c-Myc abrogated the cancer stem cell-like characteristics induced by RARS–MAD1L1.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation assay, MTT/colony/sphere formation assays, in vivo xenograft assay\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ChIP plus functional rescue experiments, single lab\",\n      \"pmids\": [\"29133573\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Biallelic germline loss-of-function mutations in MAD1L1 result in absence of full-length MAD1 protein, deficient spindle assembly checkpoint (SAC) response, and ~30–40% aneuploid blood cells; single-cell RNA analysis revealed mitochondrial stress and systemic inflammation (enhanced interferon and NFκB signaling) in both aneuploid and euploid cells, and specific clonal expansions of γδ T cells (chromosome 18 gains) and intermediate B cells (chromosome 12 gains) with leukemic transcriptomic signatures.\",\n      \"method\": \"Germline mutation functional studies, SAC response assays, single-cell RNA sequencing, protein expression analysis\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (protein assay, SAC assay, scRNA-seq), physiological human patient context\",\n      \"pmids\": [\"36322655\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MAD1L1 physically interacts with CHPF (chondroitin polymerizing factor) in glioma cells, as demonstrated by immunoprecipitation, co-immunoprecipitation, GST pulldown, and LC-MS/MS.\",\n      \"method\": \"Co-immunoprecipitation, GST pulldown, LC-MS/MS\",\n      \"journal\": \"Aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — three orthogonal binding assays (Co-IP, GST pulldown, MS), single lab\",\n      \"pmids\": [\"37851364\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MAD1L1 encodes a mitotic spindle assembly checkpoint (SAC) protein that localizes to kinetochores in prometaphase and to nuclear pore complexes in interphase; it directly binds MAD2 through two leucine zipper domains (residues 501–522 and 557–571), and this interaction is required for MAD2 kinetochore recruitment and enforcement of mitotic arrest; a common Arg558His polymorphism within the second leucine zipper reduces MAD1–MAD2 binding and attenuates SAC function; biallelic loss of MAD1L1 causes profound aneuploidy with systemic inflammation via NFκB/interferon signaling; the MAD1L1 promoter is regulated by mitogenic signals and gain-of-function p53 mutants; and MAD1L1 also participates in non-mitotic protein–protein interactions (e.g., with CHPF in glioma) whose functional significance is under investigation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MAD1L1 encodes a core mitotic spindle assembly checkpoint (SAC) protein that enforces accurate chromosome segregation by recruiting MAD2 to unattached kinetochores [#0, #2]. It localizes dynamically through the cell cycle, decorating kinetochores specifically in prometaphase and departing by metaphase/anaphase, while in interphase it co-localizes with nuclear pore complexes [#0]. MAD1L1 physically associates with MAD2 through two leucine zipper domains (residues 501–522 and 557–571), and this interaction is essential for checkpoint enforcement; a coding polymorphism at codon 558 (Arg→His) within the second leucine zipper reduces MAD1–MAD2 binding affinity and impairs nocodazole-induced mitotic arrest [#2, #4]. Loss of MAD1L1 function compromises the checkpoint and drives chromosomal instability: a truncated dominant-negative mutant from lymphoma weakens the mitotic checkpoint [#1], and biallelic germline loss-of-function abolishes full-length MAD1, producing a deficient SAC, profound aneuploidy in blood cells, mitochondrial stress, and systemic interferon and NF\\u03baB inflammatory signaling with clonal expansions bearing leukemic signatures [#6]. The MAD1L1 promoter is GC-rich and TATA-less, active in G1, responsive to mitogenic stimuli, and preferentially activated by a gain-of-function p53 mutant [#3]. Beyond mitosis, MAD1L1 engages non-checkpoint protein interactions, including binding to CHPF in glioma cells, whose functional role is not characterized in the available corpus [#7].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Establishing where MAD1L1 acts and what it partners with anchored it as a kinetochore-localized checkpoint component physically coupled to MAD2.\",\n      \"evidence\": \"Immunofluorescence co-labeling, subcellular fractionation, and reciprocal co-immunoprecipitation across cell lines\",\n      \"pmids\": [\"11181178\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not map the MAD1–MAD2 interaction interface\", \"Functional consequence of nuclear pore localization in interphase unresolved\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrating that a lymphoma-derived truncated MAD1L1 acts as a dominant-negative and weakens the checkpoint linked MAD1L1 loss of function to tumorigenic chromosome instability.\",\n      \"evidence\": \"Transfection of wild-type vs. mutant constructs with nocodazole mitotic index assays in HOS cells\",\n      \"pmids\": [\"11423979\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single dominant-negative allele; not a clean loss-of-function genetic test\", \"Did not quantify aneuploidy directly\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Mapping two leucine zipper domains required for MAD2 binding and showing the codon-558 variant weakens it connected a defined protein region to checkpoint strength and a cancer-associated allele.\",\n      \"evidence\": \"Deletion/point-mutation mapping, Co-IP binding assays, mitotic arrest assay, and LOH analysis in breast cancer\",\n      \"pmids\": [\"12042300\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural model of the bound interface\", \"Population-level penetrance of the 558 variant not addressed\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defining the MAD1L1 promoter architecture and its cell-cycle and p53-mutant responsiveness explained how checkpoint protein levels are coupled to proliferative signaling.\",\n      \"evidence\": \"Promoter deletion/mutational analysis with reporter assays and cell-cycle synchronization\",\n      \"pmids\": [\"11980658\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific transcription factors binding the core region not identified\", \"Mechanism of gain-of-function p53 activation unresolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Independent functional testing of the Arg558His variant confirmed it impairs checkpoint responses, corroborating the earlier binding-based prediction in a clinical-genetic context.\",\n      \"evidence\": \"Flow cytometry for 4N-DNA content and nocodazole mitotic index assays\",\n      \"pmids\": [\"20516147\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not test endogenous heterozygous vs homozygous variant carriers\", \"No direct aneuploidy measurement\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Characterizing a RARS–MAD1L1 fusion oncoprotein that activates FUBP1/c-Myc revealed MAD1L1 sequence in a gain-of-function rearrangement distinct from its checkpoint role.\",\n      \"evidence\": \"Co-IP, ChIP, in vitro proliferation/sphere assays, and xenografts with FUBP1 silencing and c-Myc inhibition\",\n      \"pmids\": [\"29133573\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Contribution of the MAD1L1 portion to fusion activity not dissected\", \"Relevance to wild-type MAD1L1 function unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identifying biallelic germline loss-of-function patients tied complete MAD1L1 deficiency to SAC failure, aneuploidy, and a systemic inflammatory/leukemic phenotype, establishing physiological consequences in humans.\",\n      \"evidence\": \"Germline mutation functional studies, SAC assays, protein expression, and single-cell RNA sequencing of patient cells\",\n      \"pmids\": [\"36322655\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking aneuploidy to interferon/NF\\u03baB activation not fully resolved\", \"Why specific chromosome gains undergo clonal expansion unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Detecting a MAD1L1–CHPF physical interaction in glioma raised a candidate non-mitotic partnership for MAD1L1.\",\n      \"evidence\": \"Co-IP, GST pulldown, and LC-MS/MS in glioma cells\",\n      \"pmids\": [\"37851364\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of the MAD1L1–CHPF interaction not established\", \"Interaction not validated outside glioma context\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How MAD1L1-dependent aneuploidy is mechanistically translated into NF\\u03baB/interferon inflammatory signaling and selective clonal expansion remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No defined signaling intermediary between mis-segregation and innate immune activation\", \"No structural basis for the MAD1–MAD2 kinetochore recruitment complex in the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005635\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 1, 2, 6]}\n    ],\n    \"complexes\": [\"spindle assembly checkpoint (MAD1–MAD2)\", \"kinetochore\", \"nuclear pore complex\"],\n    \"partners\": [\"MAD2\", \"CHPF\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}