{"gene":"RAPSN","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2021,"finding":"Rapsyn undergoes liquid-liquid phase separation (LLPS) to form liquid-like condensates that recruit acetylcholine receptors (AChRs), cytoskeletal proteins, and signaling proteins for postsynaptic differentiation at the neuromuscular junction. LLPS requires multivalent binding of tetratricopeptide repeat (TPR) domains and is enhanced by MuSK signaling. CMS-associated mutations compromise Rapsyn LLPS and co-condensation with interaction partners, and mice carrying a CMS-associated LLPS-deficient mutation show impaired NMJ formation.","method":"In vitro LLPS assays, co-condensation experiments, mutagenesis of TPR domains, MuSK signaling modulation, transgenic mouse model with LLPS-deficient CMS mutation","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods including in vitro reconstitution, mutagenesis, signaling modulation, and in vivo mouse genetic model in a single rigorous study","pmids":["34033754"],"is_preprint":false},{"year":2003,"finding":"Two E-box mutations in the RAPSN promoter region (-27C→G and -38A→G) reduce transcriptional activity of the RAPSN promoter. The -27C→G mutation abolishes transcription from the mutant allele and reduces binding of myogenic transcription factors (MyoD/myogenin), while -38A→G alters binding affinity for nuclear extract components and attenuates reporter expression in myotubes. The major transcriptional start site was mapped to 172 nucleotides upstream of the translational start.","method":"Electrophoretic mobility shift assay (EMSA), luciferase reporter assay in C2C12 myotubes and HEK cells transfected with MyoD/myogenin, haplotype analysis, transcript analysis from patient muscle","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal functional methods (EMSA, reporter assay, patient allele transcription analysis) in a single study with clear mechanistic outcome","pmids":["12651869"],"is_preprint":false},{"year":2006,"finding":"RAPSN missense mutations R164C and L283P diminish co-clustering of AChR with rapsyn as shown by co-transfection of wild-type AChR subunits with mutant RAPSN constructs. A splice mutation (IVS1-15C>A) generates a novel acceptor splice site causing retention of 13 nucleotides of intron 1, leading to a frameshift transcript.","method":"RAPSN minigene transfection and RNA analysis for splice mutation; co-transfection of AChR subunits with mutant RAPSN constructs and co-clustering assay for missense mutations","journal":"Neurology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional cell-based co-clustering assay and minigene splicing analysis, single lab, two orthogonal methods","pmids":["16931511"],"is_preprint":false},{"year":1994,"finding":"The mouse Rapsn gene spans 12 kb, consists of 8 exons, and the exon/intron organization is consistent with structural domains predicted from amino acid sequence conservation. The gene was mapped to the central region of mouse chromosome 2.","method":"Genomic cloning, RNase protection assay, sequence analysis of intron/exon boundaries, genetic mapping","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct genomic characterization with multiple methods (cloning, RNase protection, sequencing), single lab","pmids":["7698761"],"is_preprint":false},{"year":1996,"finding":"Human rapsyn cDNA encodes a 412-amino-acid protein with predicted molecular mass of 46,330 Da, showing 96% sequence identity with mouse rapsyn. The human RAPSN gene locus was mapped to chromosome 11p11.2-p11.1.","method":"cDNA cloning and sequencing, PCR from somatic cell hybrids and radiation hybrids for chromosomal mapping","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct cloning, sequencing, and chromosomal mapping; single lab with multiple orthogonal methods","pmids":["8812503"],"is_preprint":false},{"year":2008,"finding":"A homozygous RAPSN frameshift mutation (c.1177-1178delAA) was identified in a family with lethal fetal akinesia, demonstrating that severe (complete) loss of rapsyn function causes lethal fetal akinesia, while incomplete loss of function causes congenital myasthenia, establishing a genotype-severity relationship.","method":"Mutation analysis (direct sequencing), functional studies in patient samples, familial segregation analysis","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct sequencing with functional studies in patient tissue, single lab, but functional studies not fully described in abstract","pmids":["18179903"],"is_preprint":false},{"year":2012,"finding":"A novel mutation (p.224insT) in the TPR6 domain of RAPSN was identified in CMS patients, providing evidence that the TPR6 domain is important for rapsyn self-association and co-clustering with AChR at the postsynaptic membrane.","method":"Genetic sequencing; functional implication inferred from domain localization of mutation in compound heterozygosity with known E-box promoter mutation","journal":"Journal of the neurological sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single mutation identification in clinical patients; functional role of TPR6 inferred without direct in vitro or cellular assay described in abstract","pmids":["22326364"],"is_preprint":false}],"current_model":"Rapsyn is a 43-kDa postsynaptic peripheral membrane protein essential for clustering nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction; it undergoes liquid-liquid phase separation via multivalent TPR domain interactions to form condensates that recruit AChRs, cytoskeletal, and signaling proteins, a process enhanced by MuSK signaling and disrupted by CMS-associated mutations; its transcription is driven by E-box elements in the promoter that are bound by myogenic factors such as MyoD and myogenin, and loss-of-function mutations ranging from promoter, splice, missense, to frameshift cause a spectrum from congenital myasthenic syndrome to lethal fetal akinesia depending on severity of functional impairment."},"narrative":{"mechanistic_narrative":"RAPSN encodes rapsyn, a postsynaptic scaffolding protein essential for clustering nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction [PMID:34033754]. Rapsyn drives postsynaptic differentiation by undergoing liquid-liquid phase separation through multivalent interactions of its tetratricopeptide repeat (TPR) domains, forming liquid-like condensates that recruit AChRs along with cytoskeletal and signaling proteins; this condensation is enhanced by MuSK signaling [PMID:34033754]. Self-association and co-clustering depend on intact TPR architecture, and a mutation in the TPR6 domain disrupts these functions in CMS patients [PMID:34033754, PMID:22326364]. RAPSN transcription in muscle is governed by E-box elements in its promoter that bind the myogenic transcription factors MyoD and myogenin; promoter mutations reduce transcriptional output [PMID:12651869]. Loss-of-function mutations span the regulatory and coding sequence — promoter E-box, splice-site, missense, and frameshift alleles — and produce a severity spectrum in which incomplete loss of function causes congenital myasthenic syndrome while complete loss causes lethal fetal akinesia [PMID:12651869, PMID:16931511, PMID:18179903]. Missense mutations such as R164C and L283P specifically impair co-clustering of AChR with rapsyn [PMID:16931511].","teleology":[{"year":1994,"claim":"Establishing the genomic structure of the gene was the first step in defining the protein's domain organization and enabling later mutation analysis.","evidence":"Genomic cloning, RNase protection, and genetic mapping of the mouse Rapsn gene","pmids":["7698761"],"confidence":"Medium","gaps":["Did not address protein function or AChR clustering","Mouse gene only; human locus not yet defined"]},{"year":1996,"claim":"Cloning the human cDNA and mapping the locus provided the reagents and chromosomal position needed to link the gene to human disease.","evidence":"cDNA cloning/sequencing and radiation hybrid mapping placing RAPSN at 11p11.2-p11.1","pmids":["8812503"],"confidence":"Medium","gaps":["No functional assay of the human protein","No disease association established yet"]},{"year":2003,"claim":"It was unknown how RAPSN transcription is controlled; this work showed E-box promoter elements bound by myogenic factors drive expression and that promoter mutations cause disease.","evidence":"EMSA, luciferase reporter assays in C2C12 myotubes/HEK with MyoD/myogenin, and patient allele transcription analysis","pmids":["12651869"],"confidence":"High","gaps":["Did not quantify how reduced transcription maps to clinical severity","Other promoter/enhancer elements not excluded"]},{"year":2006,"claim":"To connect coding mutations to molecular dysfunction, missense and splice mutations were shown to impair AChR co-clustering or generate frameshift transcripts.","evidence":"Minigene splicing analysis and AChR-rapsyn co-clustering assays of mutant constructs","pmids":["16931511"],"confidence":"Medium","gaps":["Single-lab cellular assay","Mechanism of clustering loss at protein level not defined"]},{"year":2008,"claim":"The relationship between mutation severity and phenotype was unresolved; a frameshift allele established that complete loss of function causes lethal fetal akinesia versus milder congenital myasthenia for partial loss.","evidence":"Direct sequencing, familial segregation, and patient-sample functional studies in a fetal akinesia family","pmids":["18179903"],"confidence":"Medium","gaps":["Functional studies incompletely described","Quantitative genotype-phenotype boundary not defined"]},{"year":2012,"claim":"A TPR6-domain mutation in CMS patients implicated this domain in rapsyn self-association and AChR co-clustering.","evidence":"Genetic sequencing with functional role inferred from domain localization in compound heterozygosity","pmids":["22326364"],"confidence":"Low","gaps":["No direct in vitro or cellular assay of TPR6 function performed","Compound heterozygosity confounds attribution to single allele"]},{"year":2021,"claim":"The physical mechanism of AChR clustering was unresolved; this work showed rapsyn forms phase-separated condensates via multivalent TPR interactions that recruit AChRs and that CMS mutations disrupt this LLPS in vitro and in mice.","evidence":"In vitro LLPS and co-condensation assays, TPR mutagenesis, MuSK signaling modulation, and a transgenic mouse carrying an LLPS-deficient CMS mutation","pmids":["34033754"],"confidence":"High","gaps":["Stoichiometry and composition of in vivo condensates not fully defined","Mechanistic link between MuSK signaling and condensate enhancement incompletely mapped"]},{"year":null,"claim":"How rapsyn condensate composition and dynamics are regulated across development and how the full spectrum of CMS alleles quantitatively maps to LLPS defects remain open.","evidence":"","pmids":[],"confidence":"High","gaps":["No structural model of TPR-mediated multivalency reported in the corpus","Quantitative coupling of LLPS impairment to clinical severity not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,2]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[0]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0]}],"complexes":[],"partners":["CHRNA1","MUSK"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13702","full_name":"43 kDa receptor-associated protein of the synapse","aliases":["43 kDa postsynaptic protein","Acetylcholine receptor-associated 43 kDa protein","RING finger protein 205"],"length_aa":412,"mass_kda":46.3,"function":"Postsynaptic protein required for clustering of nicotinic acetylcholine receptors (nAChRs) at the neuromuscular junction. It may link the receptor to the underlying postsynaptic cytoskeleton, possibly by direct association with actin or spectrin","subcellular_location":"Cell membrane; Postsynaptic cell membrane; Cytoplasm, cytoskeleton","url":"https://www.uniprot.org/uniprotkb/Q13702/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RAPSN","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/RAPSN","total_profiled":1310},"omim":[{"mim_id":"618388","title":"FETAL AKINESIA DEFORMATION SEQUENCE 2; FADS2","url":"https://www.omim.org/entry/618388"},{"mim_id":"616326","title":"MYASTHENIC SYNDROME, CONGENITAL, 11, ASSOCIATED WITH ACETYLCHOLINE RECEPTOR DEFICIENCY; CMS11","url":"https://www.omim.org/entry/616326"},{"mim_id":"616323","title":"MYASTHENIC SYNDROME, CONGENITAL, 3C, ASSOCIATED WITH ACETYLCHOLINE RECEPTOR DEFICIENCY; CMS3C","url":"https://www.omim.org/entry/616323"},{"mim_id":"604275","title":"CATENIN, DELTA-2; CTNND2","url":"https://www.omim.org/entry/604275"},{"mim_id":"602507","title":"RING FINGER PROTEIN 103; RNF103","url":"https://www.omim.org/entry/602507"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Centrosome","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"skeletal muscle","ntpm":83.3},{"tissue":"tongue","ntpm":31.0}],"url":"https://www.proteinatlas.org/search/RAPSN"},"hgnc":{"alias_symbol":["RNF205","CMS1D","CMS1E"],"prev_symbol":[]},"alphafold":{"accession":"Q13702","domains":[{"cath_id":"3.30.40","chopping":"362-412","consensus_level":"medium","plddt":83.7796,"start":362,"end":412}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13702","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13702-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13702-F1-predicted_aligned_error_v6.png","plddt_mean":93.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RAPSN","jax_strain_url":"https://www.jax.org/strain/search?query=RAPSN"},"sequence":{"accession":"Q13702","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13702.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13702/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13702"}},"corpus_meta":[{"pmid":"18179903","id":"PMC_18179903","title":"Mutation analysis of CHRNA1, CHRNB1, CHRND, and RAPSN genes in multiple pterygium syndrome/fetal akinesia patients.","date":"2008","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/18179903","citation_count":81,"is_preprint":false},{"pmid":"12651869","id":"PMC_12651869","title":"E-box mutations in the RAPSN promoter region in eight cases with congenital myasthenic syndrome.","date":"2003","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/12651869","citation_count":77,"is_preprint":false},{"pmid":"15036330","id":"PMC_15036330","title":"Novel truncating RAPSN mutations causing congenital myasthenic syndrome responsive to 3,4-diaminopyridine.","date":"2004","source":"Neuromuscular disorders : NMD","url":"https://pubmed.ncbi.nlm.nih.gov/15036330","citation_count":37,"is_preprint":false},{"pmid":"26782015","id":"PMC_26782015","title":"Long-term follow-up in patients with congenital myasthenic syndrome due to RAPSN mutations.","date":"2015","source":"Neuromuscular disorders : NMD","url":"https://pubmed.ncbi.nlm.nih.gov/26782015","citation_count":31,"is_preprint":false},{"pmid":"27577081","id":"PMC_27577081","title":"DNA methylation array analysis identifies breast cancer associated RPTOR, MGRN1 and RAPSN hypomethylation in peripheral blood DNA.","date":"2016","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/27577081","citation_count":30,"is_preprint":false},{"pmid":"16931511","id":"PMC_16931511","title":"Impaired receptor clustering in congenital myasthenic syndrome with novel RAPSN mutations.","date":"2006","source":"Neurology","url":"https://pubmed.ncbi.nlm.nih.gov/16931511","citation_count":29,"is_preprint":false},{"pmid":"27966543","id":"PMC_27966543","title":"Limb girdle myasthenia with digenic RAPSN and a novel disease gene AK9 mutations.","date":"2016","source":"European journal of human genetics : EJHG","url":"https://pubmed.ncbi.nlm.nih.gov/27966543","citation_count":18,"is_preprint":false},{"pmid":"28495245","id":"PMC_28495245","title":"Massive parallel sequencing identifies RAPSN and PDHA1 mutations causing fetal akinesia deformation sequence.","date":"2017","source":"European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society","url":"https://pubmed.ncbi.nlm.nih.gov/28495245","citation_count":17,"is_preprint":false},{"pmid":"34033754","id":"PMC_34033754","title":"Membraneless condensates by Rapsn phase separation as a platform for neuromuscular junction formation.","date":"2021","source":"Neuron","url":"https://pubmed.ncbi.nlm.nih.gov/34033754","citation_count":16,"is_preprint":false},{"pmid":"33173339","id":"PMC_33173339","title":"The Association Between RAPSN Methylation in Peripheral Blood and Early Stage Lung Cancer Detected in Case-Control Cohort.","date":"2020","source":"Cancer management and research","url":"https://pubmed.ncbi.nlm.nih.gov/33173339","citation_count":16,"is_preprint":false},{"pmid":"8812503","id":"PMC_8812503","title":"Cloning of cDNA encoding human rapsyn and mapping of the RAPSN gene locus to chromosome 11p11.2-p11.1.","date":"1996","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/8812503","citation_count":16,"is_preprint":false},{"pmid":"30266223","id":"PMC_30266223","title":"Clinical variability of early-onset congenital myasthenic syndrome due to biallelic RAPSN mutations in Brazil.","date":"2018","source":"Neuromuscular disorders : NMD","url":"https://pubmed.ncbi.nlm.nih.gov/30266223","citation_count":14,"is_preprint":false},{"pmid":"20157724","id":"PMC_20157724","title":"Identification of previously unreported mutations in CHRNA1, CHRNE and RAPSN genes in three unrelated Italian patients with congenital myasthenic syndromes.","date":"2010","source":"Journal of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/20157724","citation_count":14,"is_preprint":false},{"pmid":"21305573","id":"PMC_21305573","title":"Investigation for RAPSN and DOK-7 mutations in a cohort of seronegative myasthenia gravis patients.","date":"2011","source":"Muscle & nerve","url":"https://pubmed.ncbi.nlm.nih.gov/21305573","citation_count":13,"is_preprint":false},{"pmid":"22326364","id":"PMC_22326364","title":"A novel mutation in the TPR6 domain of the RAPSN gene associated with congenital myasthenic syndrome.","date":"2012","source":"Journal of the neurological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/22326364","citation_count":10,"is_preprint":false},{"pmid":"20930056","id":"PMC_20930056","title":"Multiexon deletions account for 15% of congenital myasthenic syndromes with RAPSN mutations after negative DNA sequencing.","date":"2010","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/20930056","citation_count":9,"is_preprint":false},{"pmid":"7698761","id":"PMC_7698761","title":"Characterization and mapping of the Rapsn gene encoding the 43-kDa acetylcholine receptor-associated protein.","date":"1994","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/7698761","citation_count":8,"is_preprint":false},{"pmid":"33958711","id":"PMC_33958711","title":"The association between RAPSN methylation in peripheral blood and breast cancer in the Chinese population.","date":"2021","source":"Journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/33958711","citation_count":5,"is_preprint":false},{"pmid":"31037086","id":"PMC_31037086","title":"No Hot Spot Mutations CHRNE c.1327 delG, CHAT c.914T>C, and RAPSN c.264C>A in Iranian Patients with Congenital Myasthenic Syndrome.","date":"2019","source":"Iranian journal of child neurology","url":"https://pubmed.ncbi.nlm.nih.gov/31037086","citation_count":3,"is_preprint":false},{"pmid":"34139178","id":"PMC_34139178","title":"Clustering acetylcholine receptors in neuromuscular junction by phase-separated Rapsn condensates.","date":"2021","source":"Neuron","url":"https://pubmed.ncbi.nlm.nih.gov/34139178","citation_count":2,"is_preprint":false},{"pmid":"33465529","id":"PMC_33465529","title":"Generation and characterization of an induced pluripotent stem cell line SDQLCHi018-A from a congenital myasthenic syndrome patient carrying compound heterozygote mutations in RAPSN gene.","date":"2021","source":"Stem cell research","url":"https://pubmed.ncbi.nlm.nih.gov/33465529","citation_count":1,"is_preprint":false},{"pmid":"39589458","id":"PMC_39589458","title":"Clinical and genetic diversity in Iranian individuals with RAPSN-related congenital myasthenic syndrome.","date":"2024","source":"Neurogenetics","url":"https://pubmed.ncbi.nlm.nih.gov/39589458","citation_count":0,"is_preprint":false},{"pmid":"42237298","id":"PMC_42237298","title":"Late diagnosis of RAPSN mutation-associated congenital myasthenic syndrome with obstructive sleep apnea in a 5-year-old girl.","date":"2026","source":"BMC pediatrics","url":"https://pubmed.ncbi.nlm.nih.gov/42237298","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12082,"output_tokens":1879,"usd":0.032216,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8807,"output_tokens":2287,"usd":0.050605,"stage2_stop_reason":"end_turn"},"total_usd":0.082821,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2021,\n      \"finding\": \"Rapsyn undergoes liquid-liquid phase separation (LLPS) to form liquid-like condensates that recruit acetylcholine receptors (AChRs), cytoskeletal proteins, and signaling proteins for postsynaptic differentiation at the neuromuscular junction. LLPS requires multivalent binding of tetratricopeptide repeat (TPR) domains and is enhanced by MuSK signaling. CMS-associated mutations compromise Rapsyn LLPS and co-condensation with interaction partners, and mice carrying a CMS-associated LLPS-deficient mutation show impaired NMJ formation.\",\n      \"method\": \"In vitro LLPS assays, co-condensation experiments, mutagenesis of TPR domains, MuSK signaling modulation, transgenic mouse model with LLPS-deficient CMS mutation\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods including in vitro reconstitution, mutagenesis, signaling modulation, and in vivo mouse genetic model in a single rigorous study\",\n      \"pmids\": [\"34033754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Two E-box mutations in the RAPSN promoter region (-27C→G and -38A→G) reduce transcriptional activity of the RAPSN promoter. The -27C→G mutation abolishes transcription from the mutant allele and reduces binding of myogenic transcription factors (MyoD/myogenin), while -38A→G alters binding affinity for nuclear extract components and attenuates reporter expression in myotubes. The major transcriptional start site was mapped to 172 nucleotides upstream of the translational start.\",\n      \"method\": \"Electrophoretic mobility shift assay (EMSA), luciferase reporter assay in C2C12 myotubes and HEK cells transfected with MyoD/myogenin, haplotype analysis, transcript analysis from patient muscle\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal functional methods (EMSA, reporter assay, patient allele transcription analysis) in a single study with clear mechanistic outcome\",\n      \"pmids\": [\"12651869\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"RAPSN missense mutations R164C and L283P diminish co-clustering of AChR with rapsyn as shown by co-transfection of wild-type AChR subunits with mutant RAPSN constructs. A splice mutation (IVS1-15C>A) generates a novel acceptor splice site causing retention of 13 nucleotides of intron 1, leading to a frameshift transcript.\",\n      \"method\": \"RAPSN minigene transfection and RNA analysis for splice mutation; co-transfection of AChR subunits with mutant RAPSN constructs and co-clustering assay for missense mutations\",\n      \"journal\": \"Neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional cell-based co-clustering assay and minigene splicing analysis, single lab, two orthogonal methods\",\n      \"pmids\": [\"16931511\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"The mouse Rapsn gene spans 12 kb, consists of 8 exons, and the exon/intron organization is consistent with structural domains predicted from amino acid sequence conservation. The gene was mapped to the central region of mouse chromosome 2.\",\n      \"method\": \"Genomic cloning, RNase protection assay, sequence analysis of intron/exon boundaries, genetic mapping\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct genomic characterization with multiple methods (cloning, RNase protection, sequencing), single lab\",\n      \"pmids\": [\"7698761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Human rapsyn cDNA encodes a 412-amino-acid protein with predicted molecular mass of 46,330 Da, showing 96% sequence identity with mouse rapsyn. The human RAPSN gene locus was mapped to chromosome 11p11.2-p11.1.\",\n      \"method\": \"cDNA cloning and sequencing, PCR from somatic cell hybrids and radiation hybrids for chromosomal mapping\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct cloning, sequencing, and chromosomal mapping; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"8812503\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"A homozygous RAPSN frameshift mutation (c.1177-1178delAA) was identified in a family with lethal fetal akinesia, demonstrating that severe (complete) loss of rapsyn function causes lethal fetal akinesia, while incomplete loss of function causes congenital myasthenia, establishing a genotype-severity relationship.\",\n      \"method\": \"Mutation analysis (direct sequencing), functional studies in patient samples, familial segregation analysis\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct sequencing with functional studies in patient tissue, single lab, but functional studies not fully described in abstract\",\n      \"pmids\": [\"18179903\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"A novel mutation (p.224insT) in the TPR6 domain of RAPSN was identified in CMS patients, providing evidence that the TPR6 domain is important for rapsyn self-association and co-clustering with AChR at the postsynaptic membrane.\",\n      \"method\": \"Genetic sequencing; functional implication inferred from domain localization of mutation in compound heterozygosity with known E-box promoter mutation\",\n      \"journal\": \"Journal of the neurological sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single mutation identification in clinical patients; functional role of TPR6 inferred without direct in vitro or cellular assay described in abstract\",\n      \"pmids\": [\"22326364\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Rapsyn is a 43-kDa postsynaptic peripheral membrane protein essential for clustering nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction; it undergoes liquid-liquid phase separation via multivalent TPR domain interactions to form condensates that recruit AChRs, cytoskeletal, and signaling proteins, a process enhanced by MuSK signaling and disrupted by CMS-associated mutations; its transcription is driven by E-box elements in the promoter that are bound by myogenic factors such as MyoD and myogenin, and loss-of-function mutations ranging from promoter, splice, missense, to frameshift cause a spectrum from congenital myasthenic syndrome to lethal fetal akinesia depending on severity of functional impairment.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RAPSN encodes rapsyn, a postsynaptic scaffolding protein essential for clustering nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction [#0]. Rapsyn drives postsynaptic differentiation by undergoing liquid-liquid phase separation through multivalent interactions of its tetratricopeptide repeat (TPR) domains, forming liquid-like condensates that recruit AChRs along with cytoskeletal and signaling proteins; this condensation is enhanced by MuSK signaling [#0]. Self-association and co-clustering depend on intact TPR architecture, and a mutation in the TPR6 domain disrupts these functions in CMS patients [#0, #6]. RAPSN transcription in muscle is governed by E-box elements in its promoter that bind the myogenic transcription factors MyoD and myogenin; promoter mutations reduce transcriptional output [#1]. Loss-of-function mutations span the regulatory and coding sequence — promoter E-box, splice-site, missense, and frameshift alleles — and produce a severity spectrum in which incomplete loss of function causes congenital myasthenic syndrome while complete loss causes lethal fetal akinesia [#1, #2, #5]. Missense mutations such as R164C and L283P specifically impair co-clustering of AChR with rapsyn [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Establishing the genomic structure of the gene was the first step in defining the protein's domain organization and enabling later mutation analysis.\",\n      \"evidence\": \"Genomic cloning, RNase protection, and genetic mapping of the mouse Rapsn gene\",\n      \"pmids\": [\"7698761\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not address protein function or AChR clustering\", \"Mouse gene only; human locus not yet defined\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Cloning the human cDNA and mapping the locus provided the reagents and chromosomal position needed to link the gene to human disease.\",\n      \"evidence\": \"cDNA cloning/sequencing and radiation hybrid mapping placing RAPSN at 11p11.2-p11.1\",\n      \"pmids\": [\"8812503\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional assay of the human protein\", \"No disease association established yet\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"It was unknown how RAPSN transcription is controlled; this work showed E-box promoter elements bound by myogenic factors drive expression and that promoter mutations cause disease.\",\n      \"evidence\": \"EMSA, luciferase reporter assays in C2C12 myotubes/HEK with MyoD/myogenin, and patient allele transcription analysis\",\n      \"pmids\": [\"12651869\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not quantify how reduced transcription maps to clinical severity\", \"Other promoter/enhancer elements not excluded\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"To connect coding mutations to molecular dysfunction, missense and splice mutations were shown to impair AChR co-clustering or generate frameshift transcripts.\",\n      \"evidence\": \"Minigene splicing analysis and AChR-rapsyn co-clustering assays of mutant constructs\",\n      \"pmids\": [\"16931511\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab cellular assay\", \"Mechanism of clustering loss at protein level not defined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"The relationship between mutation severity and phenotype was unresolved; a frameshift allele established that complete loss of function causes lethal fetal akinesia versus milder congenital myasthenia for partial loss.\",\n      \"evidence\": \"Direct sequencing, familial segregation, and patient-sample functional studies in a fetal akinesia family\",\n      \"pmids\": [\"18179903\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional studies incompletely described\", \"Quantitative genotype-phenotype boundary not defined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"A TPR6-domain mutation in CMS patients implicated this domain in rapsyn self-association and AChR co-clustering.\",\n      \"evidence\": \"Genetic sequencing with functional role inferred from domain localization in compound heterozygosity\",\n      \"pmids\": [\"22326364\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct in vitro or cellular assay of TPR6 function performed\", \"Compound heterozygosity confounds attribution to single allele\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The physical mechanism of AChR clustering was unresolved; this work showed rapsyn forms phase-separated condensates via multivalent TPR interactions that recruit AChRs and that CMS mutations disrupt this LLPS in vitro and in mice.\",\n      \"evidence\": \"In vitro LLPS and co-condensation assays, TPR mutagenesis, MuSK signaling modulation, and a transgenic mouse carrying an LLPS-deficient CMS mutation\",\n      \"pmids\": [\"34033754\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and composition of in vivo condensates not fully defined\", \"Mechanistic link between MuSK signaling and condensate enhancement incompletely mapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How rapsyn condensate composition and dynamics are regulated across development and how the full spectrum of CMS alleles quantitatively maps to LLPS defects remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural model of TPR-mediated multivalency reported in the corpus\", \"Quantitative coupling of LLPS impairment to clinical severity not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CHRNA1\", \"MUSK\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}