{"gene":"MYORG","run_date":"2026-04-29T11:37:56","timeline":{"discoveries":[{"year":2018,"finding":"MYORG mRNA is expressed specifically in S100β-positive astrocytes in mouse brain, and knockout of Myorg in mice induces brain calcification by 9 months of age, establishing loss-of-function as the disease mechanism.","method":"In situ hybridization/immunostaining for cell-type specificity; Myorg knockout mouse model with CT/histological phenotype readout","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 — clean KO mouse with defined calcification phenotype, cell-type localization confirmed; foundational paper with 141 citations","pmids":["29910000"],"is_preprint":false},{"year":2009,"finding":"NET37 (MYORG) is a nuclear envelope/ER transmembrane protein whose glycosidase homology domain faces the lumen of the nuclear envelope/ER; mutation of a conserved catalytic residue abolishes its ability to support myoblast differentiation, indicating enzymatic activity is required for function.","method":"Protease protection mapping for domain topology; RNAi knockdown of NET37 in C2C12 myoblasts; rescue with catalytically-dead mutant; co-immunoprecipitation with pro-IGF-II","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods (topology mapping, mutagenesis, RNAi rescue, co-IP) in single rigorous study","pmids":["19706595"],"is_preprint":false},{"year":2009,"finding":"NET37 (MYORG) co-immunoprecipitates with pro-IGF-II and is required for IGF-II secretion and downstream Akt activation during myoblast differentiation, placing it in the IGF-II maturation pathway in the secretory pathway.","method":"Co-immunoprecipitation; IGF-II secretion assay; Akt phosphorylation immunoblot after RNAi knockdown","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP plus functional pathway readouts (Akt activation, IGF-II secretion) in same study","pmids":["19706595"],"is_preprint":false},{"year":2022,"finding":"MYORG is an α-galactosidase (not an α-glucosidase as previously assumed), residing in the lumen of the endoplasmic reticulum; high-resolution crystal structures in complex with substrate and inhibitor reveal the active site and show that disease-associated mutations map to positions that would disrupt catalytic activity.","method":"In vitro enzymatic activity assay with defined substrates; X-ray crystallography of MYORG–substrate and MYORG–inhibitor complexes; active-site mutagenesis informed by structure; thermal stabilization by pharmacological chaperone","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with substrate/inhibitor, in vitro catalytic assay, mutagenesis, and chaperone stabilization all in one study","pmids":["36129849"],"is_preprint":false},{"year":2022,"finding":"Morpholino-mediated knockdown of myorg in zebrafish causes multiple brain calcifications detectable by calcein staining at 2–4 days post-fertilization, and this phenotype is rescued by myorg cDNA replenishment, confirming a conserved loss-of-function mechanism.","method":"Morpholino antisense oligonucleotide knockdown at splice and translation sites; calcein staining of zebrafish brain; mRNA rescue experiment","journal":"Molecular brain","confidence":"High","confidence_rationale":"Tier 2 — knockdown with defined phenotype rescued by cDNA, replicated across two morpholino designs","pmids":["35870928"],"is_preprint":false},{"year":2024,"finding":"Autopsy of a MYORG-PFBC patient revealed calcifications predominantly in capillaries and arterioles, shortened astrocyte foot processes in calcified regions, and decreased AQP4 immunoreactivity, implicating astrocyte dysfunction and blood-brain barrier disruption as downstream pathophysiological consequences of MYORG loss.","method":"Neuropathological autopsy with immunohistochemistry (AQP4), morphological analysis of astrocyte foot processes, neuroimaging (perfusion)","journal":"Acta neuropathologica communications","confidence":"Medium","confidence_rationale":"Tier 2/3 — direct pathological examination linking MYORG loss to astrocyte morphology and AQP4 changes, but single autopsy case","pmids":["39180105"],"is_preprint":false},{"year":2018,"finding":"Phylogenetic profiling of MYORG protein sequence across hundreds of species reveals co-evolution with calcium channels and proteins regulating anion transmembrane transport, and with PDCD6IP (a PDGFR-β interactor), suggesting MYORG functions in ion homeostasis and a PDGFR-β-related pathway.","method":"Phylogenetic profiling / co-evolution analysis across species; bioinformatic pathway enrichment","journal":"Annals of clinical and translational neurology","confidence":"Low","confidence_rationale":"Tier 4 — computational co-evolution analysis only, no direct experimental validation","pmids":["30656188"],"is_preprint":false}],"current_model":"MYORG (NET37) is an ER-luminal α-galactosidase of the GH31 family whose catalytic activity—defined by crystal structures with substrate/inhibitor and in vitro assays—is essential for function; it is expressed predominantly in astrocytes, where loss-of-function (by knockout or disease mutations that disrupt the active site) causes brain calcification in mice and zebrafish, likely through disruption of astrocyte BBB integrity, and in non-neural contexts it participates in pro-IGF-II maturation to support Akt-dependent myoblast differentiation."},"narrative":{"teleology":[{"year":2009,"claim":"Establishing that MYORG (NET37) is an ER/nuclear-envelope transmembrane protein with a lumen-facing glycosidase domain whose catalytic activity is required for myoblast differentiation, answering the basic question of where the protein acts and whether its enzymatic fold is functionally relevant.","evidence":"Protease protection topology mapping, RNAi knockdown in C2C12 myoblasts with catalytic-dead mutant rescue, co-immunoprecipitation with pro-IGF-II, IGF-II secretion and Akt phosphorylation assays","pmids":["19706595"],"confidence":"High","gaps":["The endogenous substrate cleaved by MYORG in the ER lumen was not identified","Whether the IGF-II interaction is direct or bridged by other ER-resident factors was not resolved","Relevance of the myoblast differentiation function to in vivo physiology was unknown"]},{"year":2018,"claim":"Demonstrating that MYORG is astrocyte-specific in brain and that its loss of function causes brain calcification in mice, establishing MYORG as a causal gene for primary familial brain calcification and shifting attention from muscle to the nervous system.","evidence":"In situ hybridization and immunostaining for cell-type localization; Myorg knockout mouse model with CT and histological analysis of brain calcification","pmids":["29910000"],"confidence":"High","gaps":["The molecular mechanism linking ER-luminal glycosidase activity to calcium deposition was not determined","Whether calcification arises cell-autonomously in astrocytes or involves non-cell-autonomous effects on neighboring cells was unclear","The endogenous glycoprotein substrates in astrocytes were not identified"]},{"year":2022,"claim":"Defining MYORG's precise enzymatic specificity as an α-galactosidase (not α-glucosidase) and solving its crystal structure with substrate and inhibitor, which revealed that disease mutations cluster at the active site and that pharmacological chaperones can stabilize pathogenic variants.","evidence":"In vitro enzymatic assays with defined substrates; X-ray crystallography of MYORG–substrate and MYORG–inhibitor complexes; active-site mutagenesis; thermal shift assay with pharmacological chaperone","pmids":["36129849"],"confidence":"High","gaps":["The physiological galactose-containing glycan substrate in the ER remains unidentified","Whether chaperone-mediated stabilization of mutant MYORG restores function in vivo has not been tested","The structural basis for MYORG's interaction with pro-IGF-II or other client proteins is unknown"]},{"year":2022,"claim":"Confirming evolutionary conservation of the MYORG loss-of-function brain calcification phenotype by showing that morpholino knockdown in zebrafish produces brain calcification that is rescued by cDNA replenishment.","evidence":"Morpholino knockdown at splice and translation sites in zebrafish; calcein staining; mRNA rescue","pmids":["35870928"],"confidence":"High","gaps":["The specific cell types affected in zebrafish brain were not determined","Whether zebrafish calcification involves the same astrocyte-BBB mechanism as in mammals is unknown"]},{"year":2024,"claim":"Linking MYORG loss to astrocyte foot process retraction and reduced AQP4 at brain capillaries in a human autopsy, implicating blood–brain barrier disruption as a downstream pathophysiological consequence.","evidence":"Neuropathological autopsy with AQP4 immunohistochemistry and astrocyte morphometry in a MYORG-PFBC patient","pmids":["39180105"],"confidence":"Medium","gaps":["Based on a single autopsy case; independent replication in additional patients or animal models is needed","Whether AQP4 loss and foot process changes are a direct consequence of MYORG enzymatic deficiency or secondary to calcification is unresolved","The molecular link between ER-luminal α-galactosidase activity and astrocyte endfeet maintenance is unknown"]},{"year":null,"claim":"The identity of MYORG's physiological glycan or glycoprotein substrate in astrocytes remains the central open question; without it, the causal chain from α-galactosidase deficiency to calcium deposition cannot be mechanistically completed.","evidence":"","pmids":[],"confidence":"High","gaps":["No endogenous ER-luminal substrate has been identified in astrocytes","The cell-autonomous versus non-cell-autonomous basis of calcification has not been resolved","Whether MYORG's role in IGF-II processing is relevant to astrocyte function or brain calcification is untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[1,3]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[1,3]},{"term_id":"GO:0005635","term_label":"nuclear envelope","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[1,3]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2]}],"complexes":[],"partners":["IGF2"],"other_free_text":[]},"mechanistic_narrative":"MYORG (NET37) is an ER-luminal α-galactosidase of the GH31 glycoside hydrolase family that functions in astrocyte biology and brain calcium homeostasis, with loss-of-function causing primary familial brain calcification. Crystal structures with substrate and inhibitor define its active site, and disease-associated mutations map to catalytically critical residues; mutation of the conserved catalytic residue abolishes enzymatic function and the protein's ability to support cellular differentiation [PMID:36129849, PMID:19706595]. MYORG is expressed specifically in S100β-positive astrocytes, and knockout in mice or knockdown in zebrafish produces brain calcification, with human autopsy revealing shortened astrocyte foot processes and reduced AQP4 at the blood–brain barrier as downstream consequences of MYORG loss [PMID:29910000, PMID:35870928, PMID:39180105]. In myoblasts, MYORG associates with pro-IGF-II and is required for IGF-II secretion and Akt activation during differentiation, indicating a role in glycoprotein processing within the ER lumen [PMID:19706595]."},"prefetch_data":{"uniprot":{"accession":"Q6NSJ0","full_name":"Alpha-galactosidase MYORG","aliases":["Myogenesis regulating glycosidase","Nuclear envelope transmembrane protein 37"],"length_aa":714,"mass_kda":81.1,"function":"Alpha-galactosidase with unusual specificity for the Gal-alpha1,4-Glc structure, whose in vivo substrate is still unknown (PubMed:36129849). Promotes myogenesis by activating AKT signaling through the maturation and secretion of IGF2 (By similarity)","subcellular_location":"Nucleus membrane; Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/Q6NSJ0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MYORG","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":77,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MYORG","total_profiled":1310},"omim":[{"mim_id":"620786","title":"BASAL GANGLIA CALCIFICATION, IDIOPATHIC, 9, AUTOSOMAL RECESSIVE; IBGC9","url":"https://www.omim.org/entry/620786"},{"mim_id":"618317","title":"BASAL GANGLIA CALCIFICATION, IDIOPATHIC, 7, AUTOSOMAL RECESSIVE; IBGC7","url":"https://www.omim.org/entry/618317"},{"mim_id":"618255","title":"MYOGENESIS-REGULATING GLYCOSIDASE; MYORG","url":"https://www.omim.org/entry/618255"},{"mim_id":"614246","title":"N-ALPHA-ACETYLTRANSFERASE 60, NatF CATALYTIC SUBUNIT; NAA60","url":"https://www.omim.org/entry/614246"},{"mim_id":"213600","title":"BASAL GANGLIA CALCIFICATION, IDIOPATHIC, 1; IBGC1","url":"https://www.omim.org/entry/213600"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Mitochondria","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"skeletal muscle","ntpm":78.8}],"url":"https://www.proteinatlas.org/search/MYORG"},"hgnc":{"alias_symbol":["NET37"],"prev_symbol":["KIAA1161"]},"alphafold":{"accession":"Q6NSJ0","domains":[{"cath_id":"2.60.40","chopping":"91-301","consensus_level":"high","plddt":93.1862,"start":91,"end":301}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6NSJ0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6NSJ0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6NSJ0-F1-predicted_aligned_error_v6.png","plddt_mean":90.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MYORG","jax_strain_url":"https://www.jax.org/strain/search?query=MYORG"},"sequence":{"accession":"Q6NSJ0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6NSJ0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6NSJ0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6NSJ0"}},"corpus_meta":[{"pmid":"29910000","id":"PMC_29910000","title":"Biallelic Mutations in MYORG Cause Autosomal Recessive Primary Familial Brain Calcification.","date":"2018","source":"Neuron","url":"https://pubmed.ncbi.nlm.nih.gov/29910000","citation_count":141,"is_preprint":false},{"pmid":"31009047","id":"PMC_31009047","title":"Biallelic MYORG mutation carriers exhibit primary brain calcification with a distinct phenotype.","date":"2019","source":"Brain : a journal of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/31009047","citation_count":62,"is_preprint":false},{"pmid":"19706595","id":"PMC_19706595","title":"NET37, a nuclear envelope transmembrane protein with glycosidase homology, is involved in myoblast differentiation.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19706595","citation_count":37,"is_preprint":false},{"pmid":"30656188","id":"PMC_30656188","title":"MYORG is associated with recessive primary familial brain calcification.","date":"2018","source":"Annals of clinical and translational neurology","url":"https://pubmed.ncbi.nlm.nih.gov/30656188","citation_count":29,"is_preprint":false},{"pmid":"30589467","id":"PMC_30589467","title":"Evaluation of MYORG mutations as a novel cause of primary familial brain calcification.","date":"2018","source":"Movement disorders : official journal of the Movement Disorder Society","url":"https://pubmed.ncbi.nlm.nih.gov/30589467","citation_count":27,"is_preprint":false},{"pmid":"32211515","id":"PMC_32211515","title":"MYORG-related disease is associated with central pontine calcifications and atypical parkinsonism.","date":"2020","source":"Neurology. Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/32211515","citation_count":21,"is_preprint":false},{"pmid":"36129849","id":"PMC_36129849","title":"The primary familial brain calcification-associated protein MYORG is an α-galactosidase with restricted substrate specificity.","date":"2022","source":"PLoS biology","url":"https://pubmed.ncbi.nlm.nih.gov/36129849","citation_count":19,"is_preprint":false},{"pmid":"31440850","id":"PMC_31440850","title":"MYORG Mutations: a Major Cause of Recessive Primary Familial Brain Calcification.","date":"2019","source":"Current neurology and neuroscience reports","url":"https://pubmed.ncbi.nlm.nih.gov/31440850","citation_count":18,"is_preprint":false},{"pmid":"30895394","id":"PMC_30895394","title":"Primary familial brain calcification caused by a novel homozygous MYORG mutation in a consanguineous Italian family.","date":"2019","source":"Neurogenetics","url":"https://pubmed.ncbi.nlm.nih.gov/30895394","citation_count":17,"is_preprint":false},{"pmid":"31951047","id":"PMC_31951047","title":"MYORG Mutation Heterozygosity Is Associated With Brain Calcification.","date":"2020","source":"Movement disorders : official journal of the Movement Disorder Society","url":"https://pubmed.ncbi.nlm.nih.gov/31951047","citation_count":16,"is_preprint":false},{"pmid":"32873236","id":"PMC_32873236","title":"Brain hypoperfusion and nigrostriatal dopaminergic dysfunction in primary familial brain calcification caused by novel MYORG variants: case report.","date":"2020","source":"BMC neurology","url":"https://pubmed.ncbi.nlm.nih.gov/32873236","citation_count":10,"is_preprint":false},{"pmid":"32451491","id":"PMC_32451491","title":"The first Japanese case of primary familial brain calcification caused by an MYORG variant.","date":"2020","source":"Journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/32451491","citation_count":9,"is_preprint":false},{"pmid":"35870928","id":"PMC_35870928","title":"Knockdown of myorg leads to brain calcification in zebrafish.","date":"2022","source":"Molecular brain","url":"https://pubmed.ncbi.nlm.nih.gov/35870928","citation_count":8,"is_preprint":false},{"pmid":"33958240","id":"PMC_33958240","title":"First pediatric case with primary familial brain calcification due to a novel variant on the MYORG gene and review of the literature.","date":"2021","source":"Brain & development","url":"https://pubmed.ncbi.nlm.nih.gov/33958240","citation_count":8,"is_preprint":false},{"pmid":"39180105","id":"PMC_39180105","title":"Genetic and pathophysiological insights from autopsied patient with primary familial brain calcification: novel MYORG variants and astrocytic implications.","date":"2024","source":"Acta neuropathologica communications","url":"https://pubmed.ncbi.nlm.nih.gov/39180105","citation_count":7,"is_preprint":false},{"pmid":"34745211","id":"PMC_34745211","title":"Mutation Analysis of MYORG in a Chinese Cohort With Primary Familial Brain Calcification.","date":"2021","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/34745211","citation_count":7,"is_preprint":false},{"pmid":"35530931","id":"PMC_35530931","title":"Primary familial brain calcification in a patient with a novel compound heterozygous mutation in MYORG presenting with an acute ischemic stroke: a case report.","date":"2022","source":"Annals of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35530931","citation_count":7,"is_preprint":false},{"pmid":"33372568","id":"PMC_33372568","title":"A novel mutation in MYORG leads to primary familial brain calcification and cerebral infarction.","date":"2021","source":"The International journal of neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/33372568","citation_count":6,"is_preprint":false},{"pmid":"34540974","id":"PMC_34540974","title":"Idiopathic basal ganglia calcification associated with new MYORG mutation site: A case report.","date":"2021","source":"World journal of clinical cases","url":"https://pubmed.ncbi.nlm.nih.gov/34540974","citation_count":6,"is_preprint":false},{"pmid":"36252969","id":"PMC_36252969","title":"Ischemic stroke in a patient with Fahr's disease carrying biallelic mutations in the MYORG gene.","date":"2022","source":"Neurosciences (Riyadh, Saudi Arabia)","url":"https://pubmed.ncbi.nlm.nih.gov/36252969","citation_count":4,"is_preprint":false},{"pmid":"36816548","id":"PMC_36816548","title":"A novel MYORG mutation causes primary familial brain calcification with migraine: Case report and literature review.","date":"2023","source":"Frontiers in neurology","url":"https://pubmed.ncbi.nlm.nih.gov/36816548","citation_count":3,"is_preprint":false},{"pmid":"36690225","id":"PMC_36690225","title":"Report of a young patient with brain calcifications with a novel homozygous MYORG variant.","date":"2023","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/36690225","citation_count":3,"is_preprint":false},{"pmid":"40373456","id":"PMC_40373456","title":"A patient with a MYORG variant in primary brain calcification has rapid clinical course and increased calcification volume on an image analyzer.","date":"2025","source":"Clinical neurology and neurosurgery","url":"https://pubmed.ncbi.nlm.nih.gov/40373456","citation_count":2,"is_preprint":false},{"pmid":"35266134","id":"PMC_35266134","title":"Primary Familial Brain Calcification Caused by a Novel Compound Heterozygous Mutation in the MYORG Gene.","date":"2022","source":"Acta neurologica Taiwanica","url":"https://pubmed.ncbi.nlm.nih.gov/35266134","citation_count":2,"is_preprint":false},{"pmid":"37680026","id":"PMC_37680026","title":"Fahr's disease linked to a novel mutation in MYORG variants manifesting as paroxysmal limb stiffness and dysarthria: Case report and literature review.","date":"2023","source":"Molecular genetics & genomic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/37680026","citation_count":2,"is_preprint":false},{"pmid":"40120050","id":"PMC_40120050","title":"Mutation spectrum and clinical features of MYORG in Iranian patients with Primary Familial Brain Calcification (PFBC).","date":"2025","source":"Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology","url":"https://pubmed.ncbi.nlm.nih.gov/40120050","citation_count":0,"is_preprint":false},{"pmid":"40555662","id":"PMC_40555662","title":"[A case report of a family with Primary familial brain calcification caused by a novel MYORG gene variants].","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/40555662","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13777,"output_tokens":1674,"usd":0.033221},"stage2":{"model":"claude-opus-4-6","input_tokens":4967,"output_tokens":1818,"usd":0.105427},"total_usd":0.138648,"stage1_batch_id":"msgbatch_012ZiiCUBfB62NCTQd4Bk1Jo","stage2_batch_id":"msgbatch_01Xuqq9aUHoQouS7UVGZNStZ","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2018,\n      \"finding\": \"MYORG mRNA is expressed specifically in S100β-positive astrocytes in mouse brain, and knockout of Myorg in mice induces brain calcification by 9 months of age, establishing loss-of-function as the disease mechanism.\",\n      \"method\": \"In situ hybridization/immunostaining for cell-type specificity; Myorg knockout mouse model with CT/histological phenotype readout\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO mouse with defined calcification phenotype, cell-type localization confirmed; foundational paper with 141 citations\",\n      \"pmids\": [\"29910000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"NET37 (MYORG) is a nuclear envelope/ER transmembrane protein whose glycosidase homology domain faces the lumen of the nuclear envelope/ER; mutation of a conserved catalytic residue abolishes its ability to support myoblast differentiation, indicating enzymatic activity is required for function.\",\n      \"method\": \"Protease protection mapping for domain topology; RNAi knockdown of NET37 in C2C12 myoblasts; rescue with catalytically-dead mutant; co-immunoprecipitation with pro-IGF-II\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods (topology mapping, mutagenesis, RNAi rescue, co-IP) in single rigorous study\",\n      \"pmids\": [\"19706595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"NET37 (MYORG) co-immunoprecipitates with pro-IGF-II and is required for IGF-II secretion and downstream Akt activation during myoblast differentiation, placing it in the IGF-II maturation pathway in the secretory pathway.\",\n      \"method\": \"Co-immunoprecipitation; IGF-II secretion assay; Akt phosphorylation immunoblot after RNAi knockdown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP plus functional pathway readouts (Akt activation, IGF-II secretion) in same study\",\n      \"pmids\": [\"19706595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MYORG is an α-galactosidase (not an α-glucosidase as previously assumed), residing in the lumen of the endoplasmic reticulum; high-resolution crystal structures in complex with substrate and inhibitor reveal the active site and show that disease-associated mutations map to positions that would disrupt catalytic activity.\",\n      \"method\": \"In vitro enzymatic activity assay with defined substrates; X-ray crystallography of MYORG–substrate and MYORG–inhibitor complexes; active-site mutagenesis informed by structure; thermal stabilization by pharmacological chaperone\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with substrate/inhibitor, in vitro catalytic assay, mutagenesis, and chaperone stabilization all in one study\",\n      \"pmids\": [\"36129849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Morpholino-mediated knockdown of myorg in zebrafish causes multiple brain calcifications detectable by calcein staining at 2–4 days post-fertilization, and this phenotype is rescued by myorg cDNA replenishment, confirming a conserved loss-of-function mechanism.\",\n      \"method\": \"Morpholino antisense oligonucleotide knockdown at splice and translation sites; calcein staining of zebrafish brain; mRNA rescue experiment\",\n      \"journal\": \"Molecular brain\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — knockdown with defined phenotype rescued by cDNA, replicated across two morpholino designs\",\n      \"pmids\": [\"35870928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Autopsy of a MYORG-PFBC patient revealed calcifications predominantly in capillaries and arterioles, shortened astrocyte foot processes in calcified regions, and decreased AQP4 immunoreactivity, implicating astrocyte dysfunction and blood-brain barrier disruption as downstream pathophysiological consequences of MYORG loss.\",\n      \"method\": \"Neuropathological autopsy with immunohistochemistry (AQP4), morphological analysis of astrocyte foot processes, neuroimaging (perfusion)\",\n      \"journal\": \"Acta neuropathologica communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — direct pathological examination linking MYORG loss to astrocyte morphology and AQP4 changes, but single autopsy case\",\n      \"pmids\": [\"39180105\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Phylogenetic profiling of MYORG protein sequence across hundreds of species reveals co-evolution with calcium channels and proteins regulating anion transmembrane transport, and with PDCD6IP (a PDGFR-β interactor), suggesting MYORG functions in ion homeostasis and a PDGFR-β-related pathway.\",\n      \"method\": \"Phylogenetic profiling / co-evolution analysis across species; bioinformatic pathway enrichment\",\n      \"journal\": \"Annals of clinical and translational neurology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 — computational co-evolution analysis only, no direct experimental validation\",\n      \"pmids\": [\"30656188\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MYORG (NET37) is an ER-luminal α-galactosidase of the GH31 family whose catalytic activity—defined by crystal structures with substrate/inhibitor and in vitro assays—is essential for function; it is expressed predominantly in astrocytes, where loss-of-function (by knockout or disease mutations that disrupt the active site) causes brain calcification in mice and zebrafish, likely through disruption of astrocyte BBB integrity, and in non-neural contexts it participates in pro-IGF-II maturation to support Akt-dependent myoblast differentiation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"MYORG (NET37) is an ER-luminal α-galactosidase of the GH31 glycoside hydrolase family that functions in astrocyte biology and brain calcium homeostasis, with loss-of-function causing primary familial brain calcification. Crystal structures with substrate and inhibitor define its active site, and disease-associated mutations map to catalytically critical residues; mutation of the conserved catalytic residue abolishes enzymatic function and the protein's ability to support cellular differentiation [PMID:36129849, PMID:19706595]. MYORG is expressed specifically in S100β-positive astrocytes, and knockout in mice or knockdown in zebrafish produces brain calcification, with human autopsy revealing shortened astrocyte foot processes and reduced AQP4 at the blood–brain barrier as downstream consequences of MYORG loss [PMID:29910000, PMID:35870928, PMID:39180105]. In myoblasts, MYORG associates with pro-IGF-II and is required for IGF-II secretion and Akt activation during differentiation, indicating a role in glycoprotein processing within the ER lumen [PMID:19706595].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Establishing that MYORG (NET37) is an ER/nuclear-envelope transmembrane protein with a lumen-facing glycosidase domain whose catalytic activity is required for myoblast differentiation, answering the basic question of where the protein acts and whether its enzymatic fold is functionally relevant.\",\n      \"evidence\": \"Protease protection topology mapping, RNAi knockdown in C2C12 myoblasts with catalytic-dead mutant rescue, co-immunoprecipitation with pro-IGF-II, IGF-II secretion and Akt phosphorylation assays\",\n      \"pmids\": [\"19706595\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The endogenous substrate cleaved by MYORG in the ER lumen was not identified\",\n        \"Whether the IGF-II interaction is direct or bridged by other ER-resident factors was not resolved\",\n        \"Relevance of the myoblast differentiation function to in vivo physiology was unknown\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrating that MYORG is astrocyte-specific in brain and that its loss of function causes brain calcification in mice, establishing MYORG as a causal gene for primary familial brain calcification and shifting attention from muscle to the nervous system.\",\n      \"evidence\": \"In situ hybridization and immunostaining for cell-type localization; Myorg knockout mouse model with CT and histological analysis of brain calcification\",\n      \"pmids\": [\"29910000\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The molecular mechanism linking ER-luminal glycosidase activity to calcium deposition was not determined\",\n        \"Whether calcification arises cell-autonomously in astrocytes or involves non-cell-autonomous effects on neighboring cells was unclear\",\n        \"The endogenous glycoprotein substrates in astrocytes were not identified\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defining MYORG's precise enzymatic specificity as an α-galactosidase (not α-glucosidase) and solving its crystal structure with substrate and inhibitor, which revealed that disease mutations cluster at the active site and that pharmacological chaperones can stabilize pathogenic variants.\",\n      \"evidence\": \"In vitro enzymatic assays with defined substrates; X-ray crystallography of MYORG–substrate and MYORG–inhibitor complexes; active-site mutagenesis; thermal shift assay with pharmacological chaperone\",\n      \"pmids\": [\"36129849\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The physiological galactose-containing glycan substrate in the ER remains unidentified\",\n        \"Whether chaperone-mediated stabilization of mutant MYORG restores function in vivo has not been tested\",\n        \"The structural basis for MYORG's interaction with pro-IGF-II or other client proteins is unknown\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Confirming evolutionary conservation of the MYORG loss-of-function brain calcification phenotype by showing that morpholino knockdown in zebrafish produces brain calcification that is rescued by cDNA replenishment.\",\n      \"evidence\": \"Morpholino knockdown at splice and translation sites in zebrafish; calcein staining; mRNA rescue\",\n      \"pmids\": [\"35870928\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The specific cell types affected in zebrafish brain were not determined\",\n        \"Whether zebrafish calcification involves the same astrocyte-BBB mechanism as in mammals is unknown\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linking MYORG loss to astrocyte foot process retraction and reduced AQP4 at brain capillaries in a human autopsy, implicating blood–brain barrier disruption as a downstream pathophysiological consequence.\",\n      \"evidence\": \"Neuropathological autopsy with AQP4 immunohistochemistry and astrocyte morphometry in a MYORG-PFBC patient\",\n      \"pmids\": [\"39180105\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Based on a single autopsy case; independent replication in additional patients or animal models is needed\",\n        \"Whether AQP4 loss and foot process changes are a direct consequence of MYORG enzymatic deficiency or secondary to calcification is unresolved\",\n        \"The molecular link between ER-luminal α-galactosidase activity and astrocyte endfeet maintenance is unknown\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The identity of MYORG's physiological glycan or glycoprotein substrate in astrocytes remains the central open question; without it, the causal chain from α-galactosidase deficiency to calcium deposition cannot be mechanistically completed.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No endogenous ER-luminal substrate has been identified in astrocytes\",\n        \"The cell-autonomous versus non-cell-autonomous basis of calcification has not been resolved\",\n        \"Whether MYORG's role in IGF-II processing is relevant to astrocyte function or brain calcification is untested\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [1, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0005635\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"IGF2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}