{"gene":"SMPX","run_date":"2026-06-10T07:46:36","timeline":{"discoveries":[{"year":2001,"finding":"SMPX (Smpx) mRNA is upregulated in skeletal muscle in response to 7 days of passive stretch in vivo, and the encoded protein contains a nuclear localization signal and two overlapping casein kinase II phosphorylation sites, suggesting a role in mechanosensing.","method":"In vivo stretch model of tibialis anterior muscle followed by mRNA differential display and sequence analysis of the novel cDNA","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean in vivo model with defined stretch stimulus and mRNA readout, single lab, single method","pmids":["11401441"],"is_preprint":false},{"year":1999,"finding":"SMPX encodes an 88-amino-acid protein (86 aa in rodents) expressed preferentially and abundantly in heart and skeletal muscle; the gene maps to Xp22.1 and consists of five exons spanning 52.1 kb.","method":"cDNA cloning, library screening, database alignment, complete genomic sequencing, and tissue expression analysis by reciprocal probing","journal":"Human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genomic structure and tissue expression established by multiple orthogonal methods (library screening, sequencing, expression profiling), single lab","pmids":["10598820"],"is_preprint":false},{"year":2005,"finding":"Chisel/Smpx localizes to focal adhesion complexes and co-immunoprecipitates with vinculin from C2C12 myoblasts and native muscle; it also co-localizes with paxillin, integrin β1, and Rac1 at peripheral membranes. Overexpression induces cell spreading and lamellipodia formation via a Rac1-dependent mechanism, increases Rac1 activity, and promotes vinculin–p34 (Arp2/3 subunit) association. Spreading is also dependent on p38 kinase activity downstream of Rac1.","method":"MYC-epitope-tagged overexpression in C2C12 cells, co-immunoprecipitation, immunofluorescence co-localization, Rac1 activity assay (pull-down), dominant-negative Rac1 rescue, p38 inhibitor treatment","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP from both cell line and native tissue, multiple orthogonal methods (Co-IP, co-localization, Rac1 activity assay, pharmacological inhibition), single lab","pmids":["15893749"],"is_preprint":false},{"year":2014,"finding":"In adult rat skeletal muscle fibers, SMPX-EGFP fusion protein localizes to repetitive double stripes flanking the Z-disc and is excluded from all nuclei, consistent with a sarcomeric/costameric mechanosensor role and inconsistent with a transcription factor role. Overexpression of ectopic SMPX in adult mouse skeletal muscle produces no significant changes in fiber type distribution or cross-sectional area.","method":"In vivo confocal imaging of SMPX-EGFP in adult rat muscle fibers; in vivo overexpression in adult mice with fiber-type and cross-sectional area measurements","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct in vivo localization with functional overexpression experiment, single lab, two orthogonal readouts","pmids":["24936977"],"is_preprint":false},{"year":2016,"finding":"The nuclear receptor NOR-1 directly transcriptionally activates SMPX through a non-consensus NBRE site in the human SMPX promoter. NOR-1 silencing in human skeletal muscle myoblasts (HSMM) prevents SMPX expression; NOR-1 overexpression in vascular smooth muscle cells upregulates SMPX. SMPX upregulation itself is dispensable for HSMM differentiation into myotubes, but NOR-1 silencing prevents both SMPX expression and myotube differentiation.","method":"NOR-1 overexpression in VSMC, NOR-1 siRNA knockdown in HSMM, transcriptional reporter assays, DNA-protein binding assays (EMSA/ChIP), RT-qPCR","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — DNA-protein binding assay plus loss-of-function and gain-of-function experiments, single lab, two orthogonal methods","pmids":["27181368"],"is_preprint":false},{"year":2021,"finding":"Missense mutations in SMPX increase the aggregation propensity of the SMPX protein; overexpressed mutant SMPX localizes to stress granules and slows their clearance in cell culture. Patient muscle biopsies show sarcoplasmic inclusions with amyloid-like characteristics and rimmed vacuoles.","method":"In silico aggregation prediction, cell culture overexpression of wild-type and mutant SMPX with stress granule marker co-localization, electron microscopy and histopathology of patient biopsies","journal":"Acta neuropathologica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell culture functional studies combined with patient biopsy histopathology and EM, single lab, multiple orthogonal methods","pmids":["33974137"],"is_preprint":false},{"year":2021,"finding":"In an Smpx knockout mouse model (CRISPR-Cas9), male null mice show progressive hearing loss starting at high frequencies from the 3rd month; hair cell stereocilia degenerate progressively from the shortest row, followed by cellular edema and cell death. Female knockouts show milder, later-onset loss. Fluorescently tagged Smpx expressed in living hair cells localizes to stereocilia, and noise exposure experiments indicate Smpx participates in maintaining hair cell bundle integrity.","method":"CRISPR-Cas9 Smpx knockout mouse; ABR testing; cochlear morphological analysis by confocal and electron microscopy; live-cell fluorescent Smpx expression in hair cells; noise exposure paradigm","journal":"Frontiers in cell and developmental biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean KO with defined auditory phenotype, direct localization in live hair cells, morphological time-course, multiple orthogonal methods in single study","pmids":["34722533"],"is_preprint":false},{"year":2021,"finding":"In Smpx-deficient zebrafish, inner ear hair cells show a marked decrease in kinocilia number and structural alterations of stereocilia and kinocilia, and mechanotransduction by hair cells is impaired. Additionally, muscle fiber organization and function are defective.","method":"Morpholino knockdown and CRISPR/Cas9 knockout in zebrafish; whole-mount immunofluorescence; mechanotransduction assays (FM1-43 dye uptake); muscle fiber morphology analysis","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two independent loss-of-function approaches (morpholino + CRISPR) with mechanotransduction functional readout, single lab","pmids":["34204426"],"is_preprint":false},{"year":2024,"finding":"Smpx is expressed in neuromast hair cells of the zebrafish posterior lateral line and localizes to the cytoplasm and primary cilium of these cells. Loss of Smpx (morpholino knockdown or CRISPR F0 knockout) results in fewer properly differentiated neuromasts, a smaller lateral line primordium, structurally and numerically altered kinocilia, and significantly reduced mechanotransduction activity in neuromast hair cells.","method":"Whole-mount in situ hybridization, immunofluorescence subcellular localization; morpholino knockdown; CRISPR/Cas9 F0 knockout; mechanotransduction assay (FM1-43 uptake)","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two independent loss-of-function strategies with functional mechanotransduction readout and subcellular localization, single lab","pmids":["38570547"],"is_preprint":false},{"year":2025,"finding":"MUSTN1 physically interacts with SMPX (identified as an SMPX-interacting protein), stabilizes the SMPX protein, and promotes myogenic differentiation through SMPX; SMPX's promotion of myogenic differentiation depends on MUSTN1. MUSTN1 knockout mice show reduced muscle mass, fiber cross-sectional area, exercise endurance, and delayed muscle regeneration.","method":"Co-immunoprecipitation (protein interaction), MUSTN1 knockout mice (phenotypic analysis), knockdown and overexpression in myoblasts, protein stability assays","journal":"Cell proliferation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP identifying interaction, KO mouse with defined phenotype, epistasis between MUSTN1 and SMPX in differentiation, single lab","pmids":["39828423"],"is_preprint":false},{"year":2011,"finding":"Loss-of-function nonsense mutations in SMPX (detected in two independent families) cause X-linked progressive hearing loss; heterologous overexpression studies of truncated SMPX proteins were compatible with a loss-of-function mechanism. SMPX protein is present in hair cells and supporting cells of the murine cochlea.","method":"Genome-wide linkage analysis, targeted DNA capture with high-throughput sequencing, Sanger validation, co-segregation analysis, heterologous overexpression of mutant proteins, mouse cochlea immunolocalization","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two independent families with co-segregating mutations, functional overexpression studies, direct cochlear localization; replicated in two papers (21549336, 21549342)","pmids":["21549336","21549342"],"is_preprint":false},{"year":2017,"finding":"A donor splice-site variant (c.132+1G>A) in SMPX leads to four aberrant RNA transcripts via alternative splicing including non-canonical splice sites, generating a frameshift and premature termination codon; the resulting mRNA is degraded by nonsense-mediated mRNA decay (NMD), establishing NMD as a loss-of-function mechanism for this allele.","method":"RT-PCR and Sanger sequencing of aberrant transcripts from patient-derived material; qPCR for mRNA quantification to confirm NMD","journal":"Molecular genetics & genomic medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct RNA analysis from patient material with two orthogonal methods (RT-PCR and qRT-PCR), single lab","pmids":["31478598"],"is_preprint":false}],"current_model":"SMPX encodes a small (~9 kDa), 88-amino-acid cytoskeleton-associated protein expressed predominantly in striated muscle and inner ear hair cells that functions as a mechanosensitive scaffold: it localizes to the costamere/Z-disc region and focal adhesion complexes (excluding nuclei) in muscle, and to stereocilia/kinocilia in cochlear and lateral-line hair cells; through association with vinculin and activation of the Rac1–p38 signaling axis it modulates actin dynamics and cell spreading; it is transcriptionally regulated by the nuclear receptor NOR-1; its interaction partner MUSTN1 stabilizes SMPX protein and channels its pro-myogenic activity; loss-of-function mutations cause progressive X-linked hearing loss by impairing stereocilia integrity and mechanotransduction in cochlear hair cells, while missense mutations increase protein aggregation propensity, sequester SMPX into stress granules, and cause a distinct X-linked distal myopathy."},"narrative":{"mechanistic_narrative":"SMPX encodes a small (~88-amino-acid) cytoskeleton-associated protein expressed preferentially in striated muscle and inner ear hair cells that functions as a mechanosensitive scaffold [PMID:10598820, PMID:11401441]. In muscle it localizes to focal adhesion complexes and to repetitive double stripes flanking the Z-disc while being excluded from nuclei, consistent with a sarcomeric/costameric mechanosensor rather than a transcription factor [PMID:15893749, PMID:24936977]. SMPX co-immunoprecipitates with vinculin and co-localizes with paxillin, integrin β1, and Rac1; its overexpression activates Rac1, promotes vinculin–Arp2/3 association, and drives cell spreading and lamellipodia formation through a Rac1–p38 signaling axis, linking it directly to actin dynamics [PMID:15893749]. Its expression is directly transcriptionally activated by the nuclear receptor NOR-1 [PMID:27181368], and its interaction partner MUSTN1 binds and stabilizes the SMPX protein, with SMPX's pro-myogenic activity depending on MUSTN1 [PMID:39828423]. In hair cells SMPX localizes to stereocilia, kinocilia, and the primary cilium, where it maintains hair bundle integrity and mechanotransduction [PMID:34722533, PMID:34204426, PMID:38570547]. Loss-of-function nonsense and splice-site mutations cause X-linked progressive hearing loss through stereocilia degeneration and impaired mechanotransduction [PMID:21549336, PMID:21549342, PMID:34722533], while missense mutations increase aggregation propensity, sequester SMPX into stress granules, and cause a distinct X-linked distal myopathy with amyloid-like sarcoplasmic inclusions [PMID:33974137].","teleology":[{"year":1999,"claim":"Establishing that SMPX is a small protein expressed abundantly in striated muscle defined the tissue context in which all later mechanistic work would be interpreted.","evidence":"cDNA cloning, genomic sequencing, and tissue expression profiling mapping the gene to Xp22.1","pmids":["10598820"],"confidence":"Medium","gaps":["No functional or subcellular role assigned","Hair-cell expression not yet recognized"]},{"year":2001,"claim":"The question of whether SMPX responds to mechanical input was addressed by showing its mRNA is induced by passive muscle stretch, framing it as a candidate mechanosensing gene.","evidence":"In vivo stretch model of tibialis anterior with mRNA differential display and sequence analysis","pmids":["11401441"],"confidence":"Medium","gaps":["Transcriptional induction does not establish protein-level mechanosensory function","Predicted NLS and CKII sites not functionally validated"]},{"year":2005,"claim":"The molecular mechanism was advanced from correlation to a defined signaling axis by placing SMPX at focal adhesions in physical association with vinculin and driving cytoskeletal remodeling via Rac1 and p38.","evidence":"Tagged overexpression in C2C12 cells with reciprocal Co-IP, co-localization, Rac1 pull-down activity assay, dominant-negative and pharmacological rescue","pmids":["15893749"],"confidence":"High","gaps":["Direct vs indirect vinculin binding not resolved","Effects shown by overexpression rather than endogenous loss-of-function","How mechanical stimulus couples to Rac1 activation unknown"]},{"year":2011,"claim":"The disease relevance of SMPX was established by linking nonsense loss-of-function mutations to X-linked progressive hearing loss and localizing the protein in cochlear hair and supporting cells.","evidence":"Linkage analysis, targeted sequencing, co-segregation in two families, heterologous mutant overexpression, and mouse cochlear immunolocalization","pmids":["21549336","21549342"],"confidence":"Medium","gaps":["Cellular mechanism of hearing loss not defined","No animal model of deafness yet"]},{"year":2014,"claim":"The localization question was resolved in adult muscle fibers, showing SMPX sits at the Z-disc/costamere and is excluded from nuclei, ruling out a transcription-factor role despite its predicted NLS.","evidence":"In vivo confocal imaging of SMPX-EGFP and in vivo overexpression with fiber-type and cross-sectional area measurements in adult muscle","pmids":["24936977"],"confidence":"Medium","gaps":["Ectopic overexpression produced no measurable phenotype, leaving the in vivo muscle function unresolved","Predicted NLS function unexplained"]},{"year":2016,"claim":"The upstream regulation of SMPX was defined by identifying the nuclear receptor NOR-1 as a direct transcriptional activator binding a non-consensus NBRE in its promoter.","evidence":"NOR-1 gain- and loss-of-function in VSMC and myoblasts, reporter assays, DNA-protein binding assays, and RT-qPCR","pmids":["27181368"],"confidence":"Medium","gaps":["SMPX itself dispensable for myotube differentiation, so its functional output downstream of NOR-1 remains unclear","Other transcriptional regulators not surveyed"]},{"year":2021,"claim":"Two distinct disease mechanisms were established: a knockout deafness model defining progressive stereocilia degeneration, and missense mutations defining a toxic aggregation myopathy.","evidence":"CRISPR Smpx knockout mouse with ABR, cochlear EM and live hair-cell localization; plus aggregation prediction, stress-granule co-localization, and patient biopsy histopathology/EM","pmids":["34722533","33974137"],"confidence":"High","gaps":["Molecular function of SMPX within stereocilia not defined","How missense aggregation links to muscle pathology mechanistically unresolved","Relationship between deafness loss-of-function and myopathy gain-of-toxicity within one protein not reconciled"]},{"year":2021,"claim":"The hair-cell role was extended cross-species by showing zebrafish Smpx loss impairs kinocilia/stereocilia structure and mechanotransduction, generalizing the mechanosensory function.","evidence":"Morpholino and CRISPR knockout in zebrafish with immunofluorescence and FM1-43 mechanotransduction assays plus muscle morphology","pmids":["34204426"],"confidence":"Medium","gaps":["Molecular interaction partners in hair cells not identified","Mechanism linking SMPX to ciliary maintenance unknown"]},{"year":2024,"claim":"The lateral-line study refined the mechanosensory role, localizing Smpx to the cytoplasm and primary cilium of neuromast hair cells and showing loss impairs hair-cell differentiation and mechanotransduction.","evidence":"In situ hybridization, immunofluorescence localization, morpholino and CRISPR F0 knockout, and FM1-43 mechanotransduction assay in zebrafish neuromasts","pmids":["38570547"],"confidence":"Medium","gaps":["Ciliary protein partners unidentified","Direct molecular activity at the cilium not established"]},{"year":2025,"claim":"A protein partner controlling SMPX stability was identified, with MUSTN1 binding and stabilizing SMPX and SMPX's pro-myogenic activity depending on MUSTN1.","evidence":"Reciprocal Co-IP, MUSTN1 knockout mouse phenotyping, and myoblast knockdown/overexpression with protein stability assays","pmids":["39828423"],"confidence":"Medium","gaps":["Structural basis of the MUSTN1–SMPX interaction unknown","How stabilization relates to the costameric/mechanosensor role not connected"]},{"year":null,"claim":"The core biochemical activity of SMPX and how a single small scaffold protein produces both loss-of-function deafness and gain-of-toxicity myopathy remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No defined enzymatic or direct molecular activity for SMPX","Mechanism coupling mechanical stimulus to Rac1/p38 signaling not reconstituted","No structural model of SMPX or its complexes"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[2]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[6,7,8]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[2,3]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[7,8]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-9709957","term_label":"Sensory Perception","supporting_discovery_ids":[6,7,8]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[4,9]}],"complexes":["focal adhesion complex","costamere/Z-disc"],"partners":["VCL","MUSTN1","RAC1","PXN","ITGB1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UHP9","full_name":"Small muscular protein","aliases":["Stretch-responsive skeletal muscle protein"],"length_aa":88,"mass_kda":9.6,"function":"Plays a role in the regulatory network through which muscle cells coordinate their structural and functional states during growth, adaptation, and repair","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q9UHP9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SMPX","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SMPX","total_profiled":1310},"omim":[{"mim_id":"301075","title":"MYOPATHY, DISTAL, 7, ADULT-ONSET, X-LINKED; MPD7","url":"https://www.omim.org/entry/301075"},{"mim_id":"300580","title":"MYOPATHY, CONGENITAL, WITH FIBER-TYPE DISPROPORTION, X-LINKED; CFTDX","url":"https://www.omim.org/entry/300580"},{"mim_id":"300226","title":"SMALL MUSCLE PROTEIN, X-LINKED; SMPX","url":"https://www.omim.org/entry/300226"},{"mim_id":"300066","title":"DEAFNESS, X-LINKED 4; DFNX4","url":"https://www.omim.org/entry/300066"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"heart muscle","ntpm":709.4},{"tissue":"skeletal muscle","ntpm":1108.9},{"tissue":"tongue","ntpm":1025.1}],"url":"https://www.proteinatlas.org/search/SMPX"},"hgnc":{"alias_symbol":["DFNX4","Chisel","Csl"],"prev_symbol":["DFN6"]},"alphafold":{"accession":"Q9UHP9","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UHP9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UHP9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UHP9-F1-predicted_aligned_error_v6.png","plddt_mean":67.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SMPX","jax_strain_url":"https://www.jax.org/strain/search?query=SMPX"},"sequence":{"accession":"Q9UHP9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UHP9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UHP9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UHP9"}},"corpus_meta":[{"pmid":"30770291","id":"PMC_30770291","title":"Stereotactic ablative radiotherapy versus standard radiotherapy in stage 1 non-small-cell lung cancer (TROG 09.02 CHISEL): a phase 3, open-label, randomised controlled trial.","date":"2019","source":"The Lancet. 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physics","url":"https://pubmed.ncbi.nlm.nih.gov/37087060","citation_count":5,"is_preprint":false},{"pmid":"33708524","id":"PMC_33708524","title":"A novel missense mutation in SMPX causes a rare form of X-linked postlingual sensorineural hearing loss in a Chinese family.","date":"2021","source":"Translational pediatrics","url":"https://pubmed.ncbi.nlm.nih.gov/33708524","citation_count":4,"is_preprint":false},{"pmid":"33927266","id":"PMC_33927266","title":"System for quantitative evaluation of DAB&H-stained breast cancer biopsy digital images (CHISEL).","date":"2021","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/33927266","citation_count":4,"is_preprint":false},{"pmid":"38570547","id":"PMC_38570547","title":"Differentiation and functioning of the lateral line organ in zebrafish require Smpx activity.","date":"2024","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/38570547","citation_count":1,"is_preprint":false},{"pmid":"34052664","id":"PMC_34052664","title":"Derivation of iPSC line UMi029-A bearing a hearing-loss associated variant in the SMPX gene.","date":"2021","source":"Stem cell research","url":"https://pubmed.ncbi.nlm.nih.gov/34052664","citation_count":1,"is_preprint":false},{"pmid":"39382099","id":"PMC_39382099","title":"A cost-effectiveness analysis of stereotactic ablative radiotherapy versus conventionally fractionated radiotherapy in the management of stage 1 non-small-cell lung cancer: Results from the TROG 09.02 CHISEL study.","date":"2024","source":"Journal of medical imaging and radiation oncology","url":"https://pubmed.ncbi.nlm.nih.gov/39382099","citation_count":1,"is_preprint":false},{"pmid":"40947416","id":"PMC_40947416","title":"[The pleiotropic role of X-linked SMPX gene mutations: Exploration of mechanism from deafness to myopathy].","date":"2025","source":"Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/40947416","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":18077,"output_tokens":3315,"usd":0.051978,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10903,"output_tokens":3562,"usd":0.071782,"stage2_stop_reason":"end_turn"},"total_usd":0.12376,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"SMPX (Smpx) mRNA is upregulated in skeletal muscle in response to 7 days of passive stretch in vivo, and the encoded protein contains a nuclear localization signal and two overlapping casein kinase II phosphorylation sites, suggesting a role in mechanosensing.\",\n      \"method\": \"In vivo stretch model of tibialis anterior muscle followed by mRNA differential display and sequence analysis of the novel cDNA\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean in vivo model with defined stretch stimulus and mRNA readout, single lab, single method\",\n      \"pmids\": [\"11401441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"SMPX encodes an 88-amino-acid protein (86 aa in rodents) expressed preferentially and abundantly in heart and skeletal muscle; the gene maps to Xp22.1 and consists of five exons spanning 52.1 kb.\",\n      \"method\": \"cDNA cloning, library screening, database alignment, complete genomic sequencing, and tissue expression analysis by reciprocal probing\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genomic structure and tissue expression established by multiple orthogonal methods (library screening, sequencing, expression profiling), single lab\",\n      \"pmids\": [\"10598820\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Chisel/Smpx localizes to focal adhesion complexes and co-immunoprecipitates with vinculin from C2C12 myoblasts and native muscle; it also co-localizes with paxillin, integrin β1, and Rac1 at peripheral membranes. Overexpression induces cell spreading and lamellipodia formation via a Rac1-dependent mechanism, increases Rac1 activity, and promotes vinculin–p34 (Arp2/3 subunit) association. Spreading is also dependent on p38 kinase activity downstream of Rac1.\",\n      \"method\": \"MYC-epitope-tagged overexpression in C2C12 cells, co-immunoprecipitation, immunofluorescence co-localization, Rac1 activity assay (pull-down), dominant-negative Rac1 rescue, p38 inhibitor treatment\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP from both cell line and native tissue, multiple orthogonal methods (Co-IP, co-localization, Rac1 activity assay, pharmacological inhibition), single lab\",\n      \"pmids\": [\"15893749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In adult rat skeletal muscle fibers, SMPX-EGFP fusion protein localizes to repetitive double stripes flanking the Z-disc and is excluded from all nuclei, consistent with a sarcomeric/costameric mechanosensor role and inconsistent with a transcription factor role. Overexpression of ectopic SMPX in adult mouse skeletal muscle produces no significant changes in fiber type distribution or cross-sectional area.\",\n      \"method\": \"In vivo confocal imaging of SMPX-EGFP in adult rat muscle fibers; in vivo overexpression in adult mice with fiber-type and cross-sectional area measurements\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct in vivo localization with functional overexpression experiment, single lab, two orthogonal readouts\",\n      \"pmids\": [\"24936977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The nuclear receptor NOR-1 directly transcriptionally activates SMPX through a non-consensus NBRE site in the human SMPX promoter. NOR-1 silencing in human skeletal muscle myoblasts (HSMM) prevents SMPX expression; NOR-1 overexpression in vascular smooth muscle cells upregulates SMPX. SMPX upregulation itself is dispensable for HSMM differentiation into myotubes, but NOR-1 silencing prevents both SMPX expression and myotube differentiation.\",\n      \"method\": \"NOR-1 overexpression in VSMC, NOR-1 siRNA knockdown in HSMM, transcriptional reporter assays, DNA-protein binding assays (EMSA/ChIP), RT-qPCR\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — DNA-protein binding assay plus loss-of-function and gain-of-function experiments, single lab, two orthogonal methods\",\n      \"pmids\": [\"27181368\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Missense mutations in SMPX increase the aggregation propensity of the SMPX protein; overexpressed mutant SMPX localizes to stress granules and slows their clearance in cell culture. Patient muscle biopsies show sarcoplasmic inclusions with amyloid-like characteristics and rimmed vacuoles.\",\n      \"method\": \"In silico aggregation prediction, cell culture overexpression of wild-type and mutant SMPX with stress granule marker co-localization, electron microscopy and histopathology of patient biopsies\",\n      \"journal\": \"Acta neuropathologica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell culture functional studies combined with patient biopsy histopathology and EM, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"33974137\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In an Smpx knockout mouse model (CRISPR-Cas9), male null mice show progressive hearing loss starting at high frequencies from the 3rd month; hair cell stereocilia degenerate progressively from the shortest row, followed by cellular edema and cell death. Female knockouts show milder, later-onset loss. Fluorescently tagged Smpx expressed in living hair cells localizes to stereocilia, and noise exposure experiments indicate Smpx participates in maintaining hair cell bundle integrity.\",\n      \"method\": \"CRISPR-Cas9 Smpx knockout mouse; ABR testing; cochlear morphological analysis by confocal and electron microscopy; live-cell fluorescent Smpx expression in hair cells; noise exposure paradigm\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined auditory phenotype, direct localization in live hair cells, morphological time-course, multiple orthogonal methods in single study\",\n      \"pmids\": [\"34722533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In Smpx-deficient zebrafish, inner ear hair cells show a marked decrease in kinocilia number and structural alterations of stereocilia and kinocilia, and mechanotransduction by hair cells is impaired. Additionally, muscle fiber organization and function are defective.\",\n      \"method\": \"Morpholino knockdown and CRISPR/Cas9 knockout in zebrafish; whole-mount immunofluorescence; mechanotransduction assays (FM1-43 dye uptake); muscle fiber morphology analysis\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two independent loss-of-function approaches (morpholino + CRISPR) with mechanotransduction functional readout, single lab\",\n      \"pmids\": [\"34204426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Smpx is expressed in neuromast hair cells of the zebrafish posterior lateral line and localizes to the cytoplasm and primary cilium of these cells. Loss of Smpx (morpholino knockdown or CRISPR F0 knockout) results in fewer properly differentiated neuromasts, a smaller lateral line primordium, structurally and numerically altered kinocilia, and significantly reduced mechanotransduction activity in neuromast hair cells.\",\n      \"method\": \"Whole-mount in situ hybridization, immunofluorescence subcellular localization; morpholino knockdown; CRISPR/Cas9 F0 knockout; mechanotransduction assay (FM1-43 uptake)\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two independent loss-of-function strategies with functional mechanotransduction readout and subcellular localization, single lab\",\n      \"pmids\": [\"38570547\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MUSTN1 physically interacts with SMPX (identified as an SMPX-interacting protein), stabilizes the SMPX protein, and promotes myogenic differentiation through SMPX; SMPX's promotion of myogenic differentiation depends on MUSTN1. MUSTN1 knockout mice show reduced muscle mass, fiber cross-sectional area, exercise endurance, and delayed muscle regeneration.\",\n      \"method\": \"Co-immunoprecipitation (protein interaction), MUSTN1 knockout mice (phenotypic analysis), knockdown and overexpression in myoblasts, protein stability assays\",\n      \"journal\": \"Cell proliferation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP identifying interaction, KO mouse with defined phenotype, epistasis between MUSTN1 and SMPX in differentiation, single lab\",\n      \"pmids\": [\"39828423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Loss-of-function nonsense mutations in SMPX (detected in two independent families) cause X-linked progressive hearing loss; heterologous overexpression studies of truncated SMPX proteins were compatible with a loss-of-function mechanism. SMPX protein is present in hair cells and supporting cells of the murine cochlea.\",\n      \"method\": \"Genome-wide linkage analysis, targeted DNA capture with high-throughput sequencing, Sanger validation, co-segregation analysis, heterologous overexpression of mutant proteins, mouse cochlea immunolocalization\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two independent families with co-segregating mutations, functional overexpression studies, direct cochlear localization; replicated in two papers (21549336, 21549342)\",\n      \"pmids\": [\"21549336\", \"21549342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A donor splice-site variant (c.132+1G>A) in SMPX leads to four aberrant RNA transcripts via alternative splicing including non-canonical splice sites, generating a frameshift and premature termination codon; the resulting mRNA is degraded by nonsense-mediated mRNA decay (NMD), establishing NMD as a loss-of-function mechanism for this allele.\",\n      \"method\": \"RT-PCR and Sanger sequencing of aberrant transcripts from patient-derived material; qPCR for mRNA quantification to confirm NMD\",\n      \"journal\": \"Molecular genetics & genomic medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct RNA analysis from patient material with two orthogonal methods (RT-PCR and qRT-PCR), single lab\",\n      \"pmids\": [\"31478598\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SMPX encodes a small (~9 kDa), 88-amino-acid cytoskeleton-associated protein expressed predominantly in striated muscle and inner ear hair cells that functions as a mechanosensitive scaffold: it localizes to the costamere/Z-disc region and focal adhesion complexes (excluding nuclei) in muscle, and to stereocilia/kinocilia in cochlear and lateral-line hair cells; through association with vinculin and activation of the Rac1–p38 signaling axis it modulates actin dynamics and cell spreading; it is transcriptionally regulated by the nuclear receptor NOR-1; its interaction partner MUSTN1 stabilizes SMPX protein and channels its pro-myogenic activity; loss-of-function mutations cause progressive X-linked hearing loss by impairing stereocilia integrity and mechanotransduction in cochlear hair cells, while missense mutations increase protein aggregation propensity, sequester SMPX into stress granules, and cause a distinct X-linked distal myopathy.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SMPX encodes a small (~88-amino-acid) cytoskeleton-associated protein expressed preferentially in striated muscle and inner ear hair cells that functions as a mechanosensitive scaffold [#1, #0]. In muscle it localizes to focal adhesion complexes and to repetitive double stripes flanking the Z-disc while being excluded from nuclei, consistent with a sarcomeric/costameric mechanosensor rather than a transcription factor [#2, #3]. SMPX co-immunoprecipitates with vinculin and co-localizes with paxillin, integrin \\u03b21, and Rac1; its overexpression activates Rac1, promotes vinculin\\u2013Arp2/3 association, and drives cell spreading and lamellipodia formation through a Rac1\\u2013p38 signaling axis, linking it directly to actin dynamics [#2]. Its expression is directly transcriptionally activated by the nuclear receptor NOR-1 [#4], and its interaction partner MUSTN1 binds and stabilizes the SMPX protein, with SMPX's pro-myogenic activity depending on MUSTN1 [#9]. In hair cells SMPX localizes to stereocilia, kinocilia, and the primary cilium, where it maintains hair bundle integrity and mechanotransduction [#6, #7, #8]. Loss-of-function nonsense and splice-site mutations cause X-linked progressive hearing loss through stereocilia degeneration and impaired mechanotransduction [#10, #6], while missense mutations increase aggregation propensity, sequester SMPX into stress granules, and cause a distinct X-linked distal myopathy with amyloid-like sarcoplasmic inclusions [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Establishing that SMPX is a small protein expressed abundantly in striated muscle defined the tissue context in which all later mechanistic work would be interpreted.\",\n      \"evidence\": \"cDNA cloning, genomic sequencing, and tissue expression profiling mapping the gene to Xp22.1\",\n      \"pmids\": [\"10598820\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional or subcellular role assigned\", \"Hair-cell expression not yet recognized\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"The question of whether SMPX responds to mechanical input was addressed by showing its mRNA is induced by passive muscle stretch, framing it as a candidate mechanosensing gene.\",\n      \"evidence\": \"In vivo stretch model of tibialis anterior with mRNA differential display and sequence analysis\",\n      \"pmids\": [\"11401441\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcriptional induction does not establish protein-level mechanosensory function\", \"Predicted NLS and CKII sites not functionally validated\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"The molecular mechanism was advanced from correlation to a defined signaling axis by placing SMPX at focal adhesions in physical association with vinculin and driving cytoskeletal remodeling via Rac1 and p38.\",\n      \"evidence\": \"Tagged overexpression in C2C12 cells with reciprocal Co-IP, co-localization, Rac1 pull-down activity assay, dominant-negative and pharmacological rescue\",\n      \"pmids\": [\"15893749\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs indirect vinculin binding not resolved\", \"Effects shown by overexpression rather than endogenous loss-of-function\", \"How mechanical stimulus couples to Rac1 activation unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"The disease relevance of SMPX was established by linking nonsense loss-of-function mutations to X-linked progressive hearing loss and localizing the protein in cochlear hair and supporting cells.\",\n      \"evidence\": \"Linkage analysis, targeted sequencing, co-segregation in two families, heterologous mutant overexpression, and mouse cochlear immunolocalization\",\n      \"pmids\": [\"21549336\", \"21549342\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cellular mechanism of hearing loss not defined\", \"No animal model of deafness yet\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"The localization question was resolved in adult muscle fibers, showing SMPX sits at the Z-disc/costamere and is excluded from nuclei, ruling out a transcription-factor role despite its predicted NLS.\",\n      \"evidence\": \"In vivo confocal imaging of SMPX-EGFP and in vivo overexpression with fiber-type and cross-sectional area measurements in adult muscle\",\n      \"pmids\": [\"24936977\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ectopic overexpression produced no measurable phenotype, leaving the in vivo muscle function unresolved\", \"Predicted NLS function unexplained\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"The upstream regulation of SMPX was defined by identifying the nuclear receptor NOR-1 as a direct transcriptional activator binding a non-consensus NBRE in its promoter.\",\n      \"evidence\": \"NOR-1 gain- and loss-of-function in VSMC and myoblasts, reporter assays, DNA-protein binding assays, and RT-qPCR\",\n      \"pmids\": [\"27181368\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SMPX itself dispensable for myotube differentiation, so its functional output downstream of NOR-1 remains unclear\", \"Other transcriptional regulators not surveyed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Two distinct disease mechanisms were established: a knockout deafness model defining progressive stereocilia degeneration, and missense mutations defining a toxic aggregation myopathy.\",\n      \"evidence\": \"CRISPR Smpx knockout mouse with ABR, cochlear EM and live hair-cell localization; plus aggregation prediction, stress-granule co-localization, and patient biopsy histopathology/EM\",\n      \"pmids\": [\"34722533\", \"33974137\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular function of SMPX within stereocilia not defined\", \"How missense aggregation links to muscle pathology mechanistically unresolved\", \"Relationship between deafness loss-of-function and myopathy gain-of-toxicity within one protein not reconciled\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The hair-cell role was extended cross-species by showing zebrafish Smpx loss impairs kinocilia/stereocilia structure and mechanotransduction, generalizing the mechanosensory function.\",\n      \"evidence\": \"Morpholino and CRISPR knockout in zebrafish with immunofluorescence and FM1-43 mechanotransduction assays plus muscle morphology\",\n      \"pmids\": [\"34204426\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular interaction partners in hair cells not identified\", \"Mechanism linking SMPX to ciliary maintenance unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The lateral-line study refined the mechanosensory role, localizing Smpx to the cytoplasm and primary cilium of neuromast hair cells and showing loss impairs hair-cell differentiation and mechanotransduction.\",\n      \"evidence\": \"In situ hybridization, immunofluorescence localization, morpholino and CRISPR F0 knockout, and FM1-43 mechanotransduction assay in zebrafish neuromasts\",\n      \"pmids\": [\"38570547\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ciliary protein partners unidentified\", \"Direct molecular activity at the cilium not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A protein partner controlling SMPX stability was identified, with MUSTN1 binding and stabilizing SMPX and SMPX's pro-myogenic activity depending on MUSTN1.\",\n      \"evidence\": \"Reciprocal Co-IP, MUSTN1 knockout mouse phenotyping, and myoblast knockdown/overexpression with protein stability assays\",\n      \"pmids\": [\"39828423\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of the MUSTN1\\u2013SMPX interaction unknown\", \"How stabilization relates to the costameric/mechanosensor role not connected\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The core biochemical activity of SMPX and how a single small scaffold protein produces both loss-of-function deafness and gain-of-toxicity myopathy remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No defined enzymatic or direct molecular activity for SMPX\", \"Mechanism coupling mechanical stimulus to Rac1/p38 signaling not reconstituted\", \"No structural model of SMPX or its complexes\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [6, 7, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [7, 8]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9709957\", \"supporting_discovery_ids\": [6, 7, 8]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [4, 9]}\n    ],\n    \"complexes\": [\"focal adhesion complex\", \"costamere/Z-disc\"],\n    \"partners\": [\"VCL\", \"MUSTN1\", \"RAC1\", \"PXN\", \"ITGB1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}