Identification of potential key targets and mechanisms underlying cleft palate induced by tobacco smoke exposure through multi-omics integrated Mendelian randomization analysis

Tobacco smoke (TS), a major indoor air pollutant, poses a significant health threat, notably increasing the incidence of congenital malformations, particularly cleft palate (CP). Although the mechanisms underlying TS-induced CP remain incompletely understood, this study provides novel insights by integrating multi-omics with Mendelian Randomization (MR) analyses. By utilizing two-sample MR and sensitivity analyses, we identified protective metabolites associated with CP, including glycerate, glycochenodeoxycholate, and isobutyrylcarnitine. Metabolomics analysis of human embryonic palatal mesenchymal (HEPM) cells exposed to cigarette smoke extract (CSE) revealed that downregulated 3-Phospho-D-glycerate (3-PG) and its involvement in glycine, serine, and threonine metabolism, as well as the pentose phosphate pathway, highlighting their importance in TS-induced CP. Further proteomics analysis unveiled functional abnormalities at the protein level affecting 3-PG metabolism, such as lysosomal function, extracellular matrix-receptor interaction, and carbohydrate metabolism. Gene Set Variation Analysis of transcriptome sequencing data identified significant downregulation of platelet-derived growth factor binding and glycosaminoglycan (GAG) degradation, which are closely related to the protein functions involved in 3-PG metabolism. Integrative transcriptome-MR analysis pinpointed CP-associated genes downregulated by CSE and positively correlated with GAG degradation, including TMTC1, CYP1B1, and IL32, with summary-data-based MR analysis highlighting TMTC1 as a key gene in TS-induced CP. Notably, we validated the correlation between 3-PG and TMTC1 and confirmed the downregulation of TMTC1 by CSE through expression correlation analysis and quantitative polymerase chain reaction experiments. In conclusion, this groundbreaking multi-omics study, combined with MR analysis, not only elucidates biomarkers and key dysfunction related to TS-induced CP but also provides new perspectives and theoretical support for future research in this field.