Pathway-Based Molecular Subtyping Reveals a GABA-Collapsed Autism Subgroup and Cross-Disease Convergence with Schizophrenia in Human Cerebral Cortex

Pathway-Based Molecular Subtyping Reveals a GABA-Collapsed Autism Subgroup and Cross-Disease Convergence with Schizophrenia in Human Cerebral Cortex | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Pathway-Based Molecular Subtyping Reveals a GABA-Collapsed Autism Subgroup and Cross-Disease Convergence with Schizophrenia in Human Cerebral Cortex Rohit Chauhan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8913089/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Autism spectrum disorder (ASD) and schizophrenia are genetically heterogeneous, with hundreds of risk genes contributing to single clinical diagnoses. This heterogeneity has frustrated molecular subtyping efforts and contributed to inconsistent drug trial outcomes. We present the Pathway Subtyping Framework, an open-source pipeline that shifts analysis from individual genes to biological pathways, and validate it on 444 postmortem brain transcriptome samples across three independent datasets spanning two diseases. In the discovery cohort (GSE28521, Voineagu et al., 2011; frontal cortex, n=32), pathway-based GMM clustering revealed a GABA-Collapsed subtype containing 56% of ASD samples and zero controls (Cohen's d = 3.21 for GABA signaling, p < 0.001). Independent validation on GSE64018 (Gupta et al., 2014; RNA-seq, temporal cortex, n=24) replicated the finding: a disease-enriched subtype (83% ASD) with the same top 3 disrupted pathways and overlapping driver genes (NRXN1, CNTNAP2, PTBP1), despite a different brain region and sequencing platform. Cross-disease analysis on GSE80655 (Ramaker et al., 2017; 141 schizophrenia + control samples, 3 brain regions) identified three molecular subtypes — Dopamine-Hyperactive (28%), Neurodevelopment-Activated (65%), and Synaptic-Collapsed (7%) — and was the first real-data analysis to pass all 3 validation gates (bootstrap ARI = 0.923). ASD and schizophrenia pathway sets produced 87% identical subtypes on the same data (ARI = 0.870). Multi-diagnosis pooled clustering of all 281 samples (schizophrenia, bipolar, depression, controls) showed that molecular subtypes are independent of clinical diagnosis (chi-squared p = 0.72) but strongly associated with brain region (p = 3 x 10^-16). These findings suggest that molecular subtypes cut across traditional diagnostic boundaries, with direct implications for precision drug selection in psychiatric clinical trials. The framework (v0.3.0, 968 tests) is available on PyPI ('pip install pathway-subtyping') Cellular & Molecular Neuroscience Molecular Genetics Computational Biology autism spectrum disorder schizophrenia molecular subtyping GABA signaling dopamine signaling pathway analysis precision medicine unsupervised clustering transcriptomics cross-disease open-source Full Text Additional Declarations The authors declare no competing interests. Supplementary Files supplementarymaterials.zip Supplementary Materials: Pathway-Based Molecular Subtyping Reveals a GABA-Collapsed Autism Subgroup and Cross-Disease Convergence with Schizophrenia in Human Cerebral Cortex Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8913089","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":593599676,"identity":"4764a766-8370-4c67-9071-1f46df3b9695","order_by":0,"name":"Rohit Chauhan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYFACHoYDQDIBxGBgqACJMDYQ1nIAruWMAUTLAQJaGOBaGNsMIIL4tJi39x48/IHBLo9/Ru7Bz4Xz/siZix1u/gwUkdPF4T6ZM+cSgA5LLpa4kZcsPXObgbHl7MQ2CaCIsRkOqyQkcgyAWg4kNtzIMZDm3WaQuOF2YhsDSGQbIS3zb+QY/+adA9bS/IEoLRtu5JhJ8zaAtTRI4NXCc8bgwBmD5MSNZ96YWfMcM4b4BSiC2y/sPcYfKirsEucdzzG+zVMjJ2cunf4YJCKHSwsEGGCwDbArJKx9FIyCUTAKRgEIAADsLmNXSiSEhgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0003-9895-4629","institution":"Topmist LLC","correspondingAuthor":true,"prefix":"","firstName":"Rohit","middleName":"","lastName":"Chauhan","suffix":""}],"badges":[],"createdAt":"2026-02-19 01:30:04","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-8913089/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8913089/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104779128,"identity":"088d0d8b-b086-4564-a376-ed1af5fa85e4","added_by":"auto","created_at":"2026-03-17 07:35:25","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3467159,"visible":true,"origin":"","legend":"","description":"","filename":"biorxivpathwaysubtypingframework2026.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8913089/v1_covered_b6fa6b7d-62ec-4b56-8473-d5e15bcceb92.pdf"},{"id":103284622,"identity":"7fd7f9e2-884c-4818-bd1f-4a2eb6e83f2d","added_by":"auto","created_at":"2026-02-24 04:19:04","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":163423,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Materials: Pathway-Based Molecular Subtyping Reveals a GABA-Collapsed Autism Subgroup and Cross-Disease Convergence with Schizophrenia in Human Cerebral Cortex\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"supplementarymaterials.zip","url":"https://assets-eu.researchsquare.com/files/rs-8913089/v1/c743d64b3b376652ee6e5ae7.zip"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003ePathway-Based Molecular Subtyping Reveals a GABA-Collapsed Autism Subgroup and Cross-Disease Convergence with Schizophrenia in Human Cerebral Cortex\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Topmist LLC","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"autism spectrum disorder, schizophrenia, molecular subtyping, GABA signaling, dopamine signaling, pathway analysis, precision medicine, unsupervised clustering, transcriptomics, cross-disease, open-source","lastPublishedDoi":"10.21203/rs.3.rs-8913089/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8913089/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAutism spectrum disorder (ASD) and schizophrenia are genetically heterogeneous, with hundreds of risk genes contributing to single clinical diagnoses. This heterogeneity has frustrated molecular subtyping efforts and contributed to inconsistent drug trial outcomes. We present the Pathway Subtyping Framework, an open-source pipeline that shifts analysis from individual genes to biological pathways, and validate it on 444 postmortem brain transcriptome samples across three independent datasets spanning two diseases.\u003c/p\u003e\n\u003cp\u003eIn the discovery cohort (GSE28521, Voineagu et al., 2011; frontal cortex, n=32), pathway-based GMM clustering revealed a GABA-Collapsed subtype containing 56% of ASD samples and zero controls (Cohen's d = 3.21 for GABA signaling, p \u0026lt; 0.001). Independent validation on GSE64018 (Gupta et al., 2014; RNA-seq, temporal cortex, n=24) replicated the finding: a disease-enriched subtype (83% ASD) with the same top 3 disrupted pathways and overlapping driver genes (NRXN1, CNTNAP2, PTBP1), despite a different brain region and sequencing platform.\u003c/p\u003e\n\u003cp\u003eCross-disease analysis on GSE80655 (Ramaker et al., 2017; 141 schizophrenia + control samples, 3 brain regions) identified three molecular subtypes — Dopamine-Hyperactive (28%), Neurodevelopment-Activated (65%), and Synaptic-Collapsed (7%) — and was the first real-data analysis to pass all 3 validation gates (bootstrap ARI = 0.923). ASD and schizophrenia pathway sets produced 87% identical subtypes on the same data (ARI = 0.870). Multi-diagnosis pooled clustering of all 281 samples (schizophrenia, bipolar, depression, controls) showed that molecular subtypes are independent of clinical diagnosis (chi-squared p = 0.72) but strongly associated with brain region (p = 3 x 10^-16).\u003c/p\u003e\n\u003cp\u003eThese findings suggest that molecular subtypes cut across traditional diagnostic boundaries, with direct implications for precision drug selection in psychiatric clinical trials. 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