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This study aimed to investigate potential relationship between genetically predicted cerebrospinal fluid (CSF), metabolite levels and ASD liability, and to prioritize regulatory genes, key pathways, and candidate environmental toxicants. Methods: Using two ASD GWAS datasets (exploration data: 18,381 ASD cases\/27,969 controls; validation data: 18,235 ASD cases\/36,741 controls), we applied multi-omics approaches to prioritize ASD-associated CSF metabolites, regulatory SNPs, and genes. Enrichment analysis and protein\u2013protein interaction (PPI) network analysis were performed on these metabolite-related genes to explore the potential mechanisms linking CSF metabolic disturbances to ASD. Finally, candidate environmental neurotoxicants were screened through protein-chemical interaction analysis, with binding relationships assessed via molecular docking prediction. Results: Two-sample Mendelian randomization (MR) analysis prioritized adenine and proline as candidate CSF metabolites with potential risk associations with ASD. Summary-data-based MR (SMR) prioritized 39 brain-specific quantitative trait loci (QTL) involving 35 candidate regulatory genes, including dual-metabolite modulator GRM8. Functional enrichment analyses suggested potential associations with mitochondrial dysfunction, Hippo signaling pathway, and microtubule dynamics impairment, with protein\u2013protein interaction networks highlighting KATNA1\/KATNAL2 as hubs. Protein-chemical interaction screening nominated 14 candidate environmental toxicants, including established chemicals (acetaminophen, valproic acid, estradiol) and novel candidates (SB-431542, K 7174, benzo[a]pyrene), with docking affinity assessed computationally. Conclusions: Our study provides suggestive evidence that elevated adenine and proline may be potential risk factors for ASD and suggests possible involvement of the mitochondrial\u2013Hippo\u2013microtubule pathway. We also propose benzo[a]pyrene as a candidate environmental toxicant that may perturb CSF metabolism. However, given the limited statistical significance, these findings require further validation.<\/jats:p>","DOI":"10.3390\/genes17080874","type":"journal-article","created":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T16:11:56Z","timestamp":1785168716000},"page":"874","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Multi-Omic Analysis of Cerebrospinal Fluid Metabolites in Autism Spectrum Disorder: Biomarker Identification, Metabolic Genetics Insights, and Network Toxicology"],"prefix":"10.3390","volume":"17","author":[{"given":"Dan","family":"Zhao","sequence":"first","affiliation":[{"name":"Department of Medical Genetics and Developmental Biology, School of Basic Medical Science, Capital Medical University, Beijing 100069, China"},{"name":"Laboratory for Clinical Medicine, Capital Medical University, Beijing 100069, China"},{"name":"Beijing Key Laboratory of Cell and Gene Therapy in Otology, Beijing 100069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-8272-6230","authenticated-orcid":false,"given":"Junzhi","family":"Guo","sequence":"additional","affiliation":[{"name":"Department of Medical Genetics and Developmental Biology, School of Basic Medical Science, Capital Medical University, Beijing 100069, China"},{"name":"Laboratory for Clinical Medicine, Capital Medical University, Beijing 100069, China"},{"name":"Beijing Key Laboratory of Cell and Gene Therapy in Otology, Beijing 100069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ying","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Medical Genetics and Developmental Biology, School of Basic Medical Science, Capital Medical University, Beijing 100069, China"},{"name":"Laboratory for Clinical Medicine, Capital Medical University, Beijing 100069, China"},{"name":"Beijing Key Laboratory of Cell and Gene Therapy in Otology, Beijing 100069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuanfeng","family":"Lan","sequence":"additional","affiliation":[{"name":"Department of Medical Genetics and Developmental Biology, School of Basic Medical Science, Capital Medical University, Beijing 100069, China"},{"name":"Laboratory for Clinical Medicine, Capital Medical University, Beijing 100069, China"},{"name":"Beijing Key Laboratory of Cell and Gene Therapy in Otology, Beijing 100069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tian","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Medical Genetics and Developmental Biology, School of Basic Medical Science, Capital Medical University, Beijing 100069, China"},{"name":"Laboratory for Clinical Medicine, Capital Medical University, Beijing 100069, China"},{"name":"Beijing Key Laboratory of Cell and Gene Therapy in Otology, Beijing 100069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-5142-9790","authenticated-orcid":false,"given":"Yiliang","family":"Xu","sequence":"additional","affiliation":[{"name":"Department of Medical Genetics and Developmental Biology, School of Basic Medical Science, Capital Medical University, Beijing 100069, China"},{"name":"Laboratory for Clinical Medicine, Capital Medical University, Beijing 100069, China"},{"name":"Beijing Key Laboratory of Cell and Gene Therapy in Otology, Beijing 100069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qizhou","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of Medical Genetics and Developmental Biology, School of Basic Medical Science, Capital Medical University, Beijing 100069, China"},{"name":"Laboratory for Clinical Medicine, Capital Medical University, Beijing 100069, China"},{"name":"Department of Neurocritical Care, Beijing Anzhen Hospital, Capital 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