{"gene":"RAPSN","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2021,"finding":"Rapsyn undergoes liquid-liquid phase separation (LLPS) via multivalent binding of tetratricopeptide repeat (TPR) domains, forming liquid-like condensates that recruit acetylcholine receptors (AChRs), cytoskeletal proteins, and signaling proteins to establish the postsynaptic compartment of the neuromuscular junction (NMJ). MuSK signaling increases Rapsyn LLPS. CMS-associated mutations impair LLPS and co-condensation with interaction partners, and NMJ formation is disrupted in mice carrying a LLPS-deficient CMS mutation.","method":"In vitro LLPS reconstitution, live-cell imaging, co-condensation assays, mutagenesis of TPR domains, mouse genetic model with CMS-associated LLPS-deficient mutation","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 1 — reconstitution in vitro combined with mutagenesis and in vivo mouse model validation","pmids":["34033754"],"is_preprint":false},{"year":2003,"finding":"Two E-box mutations in the RAPSN promoter region (-27C→G and -38A→G) impair transcriptional regulation of RAPSN in muscle cells. The -27C→G mutation abolishes allelic transcription entirely; -38A→G alters nuclear protein binding affinity. Both mutations reduce luciferase reporter expression in C2C12 myotubes and MyoD/myogenin-transfected HEK cells, predicting reduced rapsyn expression and endplate AChR deficiency.","method":"Electrophoretic mobility shift assay (EMSA), luciferase reporter assay, transcriptional start site mapping, transfection in C2C12 and HEK cells","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal functional assays (EMSA, reporter, mutagenesis) in a single study","pmids":["12651869"],"is_preprint":false},{"year":2006,"finding":"RAPSN missense mutations R164C and L283P diminish co-clustering of AChR with rapsyn, demonstrating that these residues are required for rapsyn-mediated AChR clustering at the postsynaptic membrane. A splice mutation (IVS1-15C>A) creates a novel acceptor splice site retaining 13 nucleotides of intron 1, causing a frameshift in the mature mRNA.","method":"Cotransfection of AChR subunits with mutant RAPSN constructs (in vitro clustering assay), RAPSN minigene transfection with RNA analysis for splice mutation","journal":"Neurology","confidence":"Medium","confidence_rationale":"Tier 2 — cell-based co-clustering assay with defined mutations, single lab","pmids":["16931511"],"is_preprint":false},{"year":1996,"finding":"Human rapsyn is encoded by a 412-amino-acid cDNA showing 96% sequence identity with mouse rapsyn, and the RAPSN gene locus maps to chromosome 11p11.2-p11.1.","method":"cDNA cloning, sequencing, somatic cell hybrid and radiation hybrid PCR mapping","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 — direct molecular cloning and chromosomal mapping","pmids":["8812503"],"is_preprint":false},{"year":1994,"finding":"The mouse Rapsn gene spans ~12 kb and consists of 8 exons; exon-intron organization is consistent with structural domains predicted from amino acid sequence conservation, and the locus maps 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 — genomic characterization with structural domain inference, single lab","pmids":["7698761"],"is_preprint":false},{"year":2008,"finding":"A homozygous RAPSN frameshift mutation (c.1177-1178delAA) causes complete loss of rapsyn function resulting in lethal fetal akinesia sequence, while incomplete loss of rapsyn function (as seen with milder mutations) causes congenital myasthenia, establishing a genotype-phenotype continuum based on degree of rapsyn loss.","method":"Mutation analysis with functional interpretation; comparison of clinical severity across alleles with different predicted functional impact","journal":"American journal of human genetics","confidence":"Low","confidence_rationale":"Tier 3 — genotype-phenotype correlation without direct biochemical reconstitution of each allele","pmids":["18179903"],"is_preprint":false},{"year":2004,"finding":"Rapsyn deficiency due to truncating/nonsense RAPSN mutations causes decreased AChRs per endplate and decreased amplitude of the miniature endplate potential, confirming rapsyn's essential role in clustering AChRs at the postsynaptic membrane in vivo.","method":"Intercostal muscle biopsy with AChR quantification and miniature endplate potential recording","journal":"Neuromuscular disorders : NMD","confidence":"Medium","confidence_rationale":"Tier 2 — direct electrophysiological and morphological measurement in patient biopsy tissue","pmids":["15036330"],"is_preprint":false},{"year":2012,"finding":"A novel mutation (p.224 insT) in the TPR6 domain of RAPSN is associated with CMS, providing evidence that the TPR6 domain contributes to rapsyn self-association and co-clustering with AChR at the postsynaptic membrane.","method":"Mutation identification and domain analysis (genetic/structural inference)","journal":"Journal of the neurological sciences","confidence":"Low","confidence_rationale":"Tier 3 — mutation identification with domain inference, no direct functional assay of TPR6 in this paper","pmids":["22326364"],"is_preprint":false}],"current_model":"Rapsyn is a postsynaptic peripheral membrane scaffold protein that clusters nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction by undergoing liquid-liquid phase separation through multivalent TPR-domain interactions, forming condensates that recruit AChRs, cytoskeletal, and signaling proteins; its expression is driven by E-box-dependent muscle-specific transcriptional regulation, and MuSK signaling enhances its LLPS activity, while loss-of-function mutations impair AChR clustering and NMJ assembly with severity proportional to the degree of rapsyn loss."},"narrative":{"teleology":[{"year":1994,"claim":"Determining the genomic architecture of Rapsn established that the gene comprises 8 exons spanning ~12 kb, with exon-intron boundaries aligning to predicted structural domains, providing a framework for interpreting future mutations.","evidence":"Genomic cloning, RNase protection, and genetic mapping in mouse","pmids":["7698761"],"confidence":"Medium","gaps":["No functional assays of individual domains performed","Human gene structure not yet characterized"]},{"year":1996,"claim":"Cloning the human RAPSN cDNA and mapping it to chromosome 11p11.2-p11.1 enabled subsequent mutation analysis in human neuromuscular disease.","evidence":"cDNA cloning, sequencing, somatic cell hybrid and radiation hybrid PCR mapping","pmids":["8812503"],"confidence":"Medium","gaps":["No functional characterization of human rapsyn protein performed"]},{"year":2003,"claim":"Identifying that E-box elements in the RAPSN promoter are required for muscle-specific transcription revealed how rapsyn expression is restricted to the postsynaptic compartment and how promoter mutations cause rapsyn deficiency.","evidence":"EMSA, luciferase reporter assays in C2C12 myotubes and MyoD/myogenin-transfected HEK cells","pmids":["12651869"],"confidence":"High","gaps":["In vivo consequences of promoter mutations on NMJ morphology not directly tested","Identity of specific transcription factors binding E-boxes not fully resolved"]},{"year":2004,"claim":"Electrophysiological demonstration that rapsyn-deficient patient endplates have decreased AChR density and reduced miniature endplate potential amplitude confirmed rapsyn's essential in vivo role in AChR clustering.","evidence":"Intercostal muscle biopsy with AChR quantification and miniature endplate potential recording from CMS patients","pmids":["15036330"],"confidence":"Medium","gaps":["Mechanism by which rapsyn loss leads to AChR dispersal not addressed at a molecular level","Small number of patient biopsies"]},{"year":2006,"claim":"Showing that missense mutations R164C and L283P diminish rapsyn–AChR co-clustering identified specific residues required for the scaffolding interaction, while a splice-site mutation revealed an additional mechanism of rapsyn loss.","evidence":"Co-transfection clustering assay with mutant RAPSN constructs; minigene splicing analysis","pmids":["16931511"],"confidence":"Medium","gaps":["Direct binding interface between rapsyn and AChR subunits not mapped","Structural basis of R164C and L283P defects not resolved"]},{"year":2008,"claim":"Establishing that complete rapsyn loss causes lethal fetal akinesia while partial loss causes CMS defined a genotype–phenotype continuum and confirmed rapsyn as indispensable for NMJ formation.","evidence":"Genotype–phenotype correlation across RAPSN alleles of graded severity","pmids":["18179903"],"confidence":"Low","gaps":["No direct biochemical reconstitution of each allele's residual activity","Mechanism of fetal akinesia beyond NMJ failure not explored"]},{"year":2021,"claim":"Reconstituting rapsyn LLPS in vitro demonstrated that multivalent TPR-domain interactions drive phase separation to form condensates that recruit AChRs and associated proteins, providing the biophysical mechanism underlying postsynaptic clustering.","evidence":"In vitro LLPS reconstitution, live-cell imaging, co-condensation assays, mutagenesis, and mouse genetic model with CMS-associated LLPS-deficient mutation","pmids":["34033754"],"confidence":"High","gaps":["How MuSK signaling biochemically modulates rapsyn LLPS (e.g., phosphorylation sites, kinetics) is not fully defined","Whether LLPS is the sole clustering mechanism or acts in concert with classical scaffolding interactions remains open"]},{"year":null,"claim":"The structural basis of the rapsyn–AChR interface, the precise post-translational modifications by which MuSK signaling potentiates rapsyn LLPS, and whether rapsyn condensates undergo regulated disassembly during synaptic remodeling remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of a rapsyn–AChR complex","Post-translational regulation of LLPS not mapped","Role of rapsyn in activity-dependent synaptic plasticity at the NMJ unexplored"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,2,6]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,2,6]}],"pathway":[{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[0,6]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[0,2]}],"complexes":[],"partners":["CHRNA1","MUSK"],"other_free_text":[]},"mechanistic_narrative":"Rapsyn (RAPSN) is a postsynaptic scaffold protein essential for clustering nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). It achieves this through liquid-liquid phase separation (LLPS) driven by multivalent tetratricopeptide repeat (TPR) domain interactions, forming condensates that recruit AChRs, cytoskeletal proteins, and signaling molecules, a process enhanced by MuSK signaling [PMID:34033754]. Muscle-specific transcription of RAPSN depends on E-box elements in its promoter, and mutations disrupting these elements abolish allelic transcription and reduce rapsyn expression [PMID:12651869]. Loss-of-function RAPSN mutations cause congenital myasthenic syndrome (CMS) with severity proportional to the degree of rapsyn loss, ranging from impaired AChR clustering and decreased miniature endplate potentials in partial loss to lethal fetal akinesia in complete loss [PMID:15036330, PMID:18179903]."},"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":12,"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":1,"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}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10836,"output_tokens":1877,"usd":0.030332},"stage2":{"model":"claude-opus-4-6","input_tokens":5160,"output_tokens":1757,"usd":0.104587},"total_usd":0.134919,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","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) via multivalent binding of tetratricopeptide repeat (TPR) domains, forming liquid-like condensates that recruit acetylcholine receptors (AChRs), cytoskeletal proteins, and signaling proteins to establish the postsynaptic compartment of the neuromuscular junction (NMJ). MuSK signaling increases Rapsyn LLPS. CMS-associated mutations impair LLPS and co-condensation with interaction partners, and NMJ formation is disrupted in mice carrying a LLPS-deficient CMS mutation.\",\n      \"method\": \"In vitro LLPS reconstitution, live-cell imaging, co-condensation assays, mutagenesis of TPR domains, mouse genetic model with CMS-associated LLPS-deficient mutation\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution in vitro combined with mutagenesis and in vivo mouse model validation\",\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) impair transcriptional regulation of RAPSN in muscle cells. The -27C→G mutation abolishes allelic transcription entirely; -38A→G alters nuclear protein binding affinity. Both mutations reduce luciferase reporter expression in C2C12 myotubes and MyoD/myogenin-transfected HEK cells, predicting reduced rapsyn expression and endplate AChR deficiency.\",\n      \"method\": \"Electrophoretic mobility shift assay (EMSA), luciferase reporter assay, transcriptional start site mapping, transfection in C2C12 and HEK cells\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal functional assays (EMSA, reporter, mutagenesis) in a single study\",\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, demonstrating that these residues are required for rapsyn-mediated AChR clustering at the postsynaptic membrane. A splice mutation (IVS1-15C>A) creates a novel acceptor splice site retaining 13 nucleotides of intron 1, causing a frameshift in the mature mRNA.\",\n      \"method\": \"Cotransfection of AChR subunits with mutant RAPSN constructs (in vitro clustering assay), RAPSN minigene transfection with RNA analysis for splice mutation\",\n      \"journal\": \"Neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cell-based co-clustering assay with defined mutations, single lab\",\n      \"pmids\": [\"16931511\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Human rapsyn is encoded by a 412-amino-acid cDNA showing 96% sequence identity with mouse rapsyn, and the RAPSN gene locus maps to chromosome 11p11.2-p11.1.\",\n      \"method\": \"cDNA cloning, sequencing, somatic cell hybrid and radiation hybrid PCR mapping\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct molecular cloning and chromosomal mapping\",\n      \"pmids\": [\"8812503\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"The mouse Rapsn gene spans ~12 kb and consists of 8 exons; exon-intron organization is consistent with structural domains predicted from amino acid sequence conservation, and the locus maps 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 — genomic characterization with structural domain inference, single lab\",\n      \"pmids\": [\"7698761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"A homozygous RAPSN frameshift mutation (c.1177-1178delAA) causes complete loss of rapsyn function resulting in lethal fetal akinesia sequence, while incomplete loss of rapsyn function (as seen with milder mutations) causes congenital myasthenia, establishing a genotype-phenotype continuum based on degree of rapsyn loss.\",\n      \"method\": \"Mutation analysis with functional interpretation; comparison of clinical severity across alleles with different predicted functional impact\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — genotype-phenotype correlation without direct biochemical reconstitution of each allele\",\n      \"pmids\": [\"18179903\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Rapsyn deficiency due to truncating/nonsense RAPSN mutations causes decreased AChRs per endplate and decreased amplitude of the miniature endplate potential, confirming rapsyn's essential role in clustering AChRs at the postsynaptic membrane in vivo.\",\n      \"method\": \"Intercostal muscle biopsy with AChR quantification and miniature endplate potential recording\",\n      \"journal\": \"Neuromuscular disorders : NMD\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct electrophysiological and morphological measurement in patient biopsy tissue\",\n      \"pmids\": [\"15036330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"A novel mutation (p.224 insT) in the TPR6 domain of RAPSN is associated with CMS, providing evidence that the TPR6 domain contributes to rapsyn self-association and co-clustering with AChR at the postsynaptic membrane.\",\n      \"method\": \"Mutation identification and domain analysis (genetic/structural inference)\",\n      \"journal\": \"Journal of the neurological sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — mutation identification with domain inference, no direct functional assay of TPR6 in this paper\",\n      \"pmids\": [\"22326364\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Rapsyn is a postsynaptic peripheral membrane scaffold protein that clusters nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction by undergoing liquid-liquid phase separation through multivalent TPR-domain interactions, forming condensates that recruit AChRs, cytoskeletal, and signaling proteins; its expression is driven by E-box-dependent muscle-specific transcriptional regulation, and MuSK signaling enhances its LLPS activity, while loss-of-function mutations impair AChR clustering and NMJ assembly with severity proportional to the degree of rapsyn loss.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"Rapsyn (RAPSN) is a postsynaptic scaffold protein essential for clustering nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). It achieves this through liquid-liquid phase separation (LLPS) driven by multivalent tetratricopeptide repeat (TPR) domain interactions, forming condensates that recruit AChRs, cytoskeletal proteins, and signaling molecules, a process enhanced by MuSK signaling [PMID:34033754]. Muscle-specific transcription of RAPSN depends on E-box elements in its promoter, and mutations disrupting these elements abolish allelic transcription and reduce rapsyn expression [PMID:12651869]. Loss-of-function RAPSN mutations cause congenital myasthenic syndrome (CMS) with severity proportional to the degree of rapsyn loss, ranging from impaired AChR clustering and decreased miniature endplate potentials in partial loss to lethal fetal akinesia in complete loss [PMID:15036330, PMID:18179903].\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Determining the genomic architecture of Rapsn established that the gene comprises 8 exons spanning ~12 kb, with exon-intron boundaries aligning to predicted structural domains, providing a framework for interpreting future mutations.\",\n      \"evidence\": \"Genomic cloning, RNase protection, and genetic mapping in mouse\",\n      \"pmids\": [\"7698761\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional assays of individual domains performed\", \"Human gene structure not yet characterized\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Cloning the human RAPSN cDNA and mapping it to chromosome 11p11.2-p11.1 enabled subsequent mutation analysis in human neuromuscular disease.\",\n      \"evidence\": \"cDNA cloning, sequencing, somatic cell hybrid and radiation hybrid PCR mapping\",\n      \"pmids\": [\"8812503\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional characterization of human rapsyn protein performed\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identifying that E-box elements in the RAPSN promoter are required for muscle-specific transcription revealed how rapsyn expression is restricted to the postsynaptic compartment and how promoter mutations cause rapsyn deficiency.\",\n      \"evidence\": \"EMSA, luciferase reporter assays in C2C12 myotubes and MyoD/myogenin-transfected HEK cells\",\n      \"pmids\": [\"12651869\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo consequences of promoter mutations on NMJ morphology not directly tested\", \"Identity of specific transcription factors binding E-boxes not fully resolved\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Electrophysiological demonstration that rapsyn-deficient patient endplates have decreased AChR density and reduced miniature endplate potential amplitude confirmed rapsyn's essential in vivo role in AChR clustering.\",\n      \"evidence\": \"Intercostal muscle biopsy with AChR quantification and miniature endplate potential recording from CMS patients\",\n      \"pmids\": [\"15036330\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which rapsyn loss leads to AChR dispersal not addressed at a molecular level\", \"Small number of patient biopsies\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Showing that missense mutations R164C and L283P diminish rapsyn–AChR co-clustering identified specific residues required for the scaffolding interaction, while a splice-site mutation revealed an additional mechanism of rapsyn loss.\",\n      \"evidence\": \"Co-transfection clustering assay with mutant RAPSN constructs; minigene splicing analysis\",\n      \"pmids\": [\"16931511\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding interface between rapsyn and AChR subunits not mapped\", \"Structural basis of R164C and L283P defects not resolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Establishing that complete rapsyn loss causes lethal fetal akinesia while partial loss causes CMS defined a genotype–phenotype continuum and confirmed rapsyn as indispensable for NMJ formation.\",\n      \"evidence\": \"Genotype–phenotype correlation across RAPSN alleles of graded severity\",\n      \"pmids\": [\"18179903\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct biochemical reconstitution of each allele's residual activity\", \"Mechanism of fetal akinesia beyond NMJ failure not explored\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Reconstituting rapsyn LLPS in vitro demonstrated that multivalent TPR-domain interactions drive phase separation to form condensates that recruit AChRs and associated proteins, providing the biophysical mechanism underlying postsynaptic clustering.\",\n      \"evidence\": \"In vitro LLPS reconstitution, live-cell imaging, co-condensation assays, mutagenesis, and mouse genetic model with CMS-associated LLPS-deficient mutation\",\n      \"pmids\": [\"34033754\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How MuSK signaling biochemically modulates rapsyn LLPS (e.g., phosphorylation sites, kinetics) is not fully defined\", \"Whether LLPS is the sole clustering mechanism or acts in concert with classical scaffolding interactions remains open\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural basis of the rapsyn–AChR interface, the precise post-translational modifications by which MuSK signaling potentiates rapsyn LLPS, and whether rapsyn condensates undergo regulated disassembly during synaptic remodeling remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No high-resolution structure of a rapsyn–AChR complex\", \"Post-translational regulation of LLPS not mapped\", \"Role of rapsyn in activity-dependent synaptic plasticity at the NMJ unexplored\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 2, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 2, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CHRNA1\", \"MuSK\"],\n    \"other_free_text\": []\n  }\n}\n```"}