Integrative analysis of bulk and single-nucleus transcriptomes suggests proteostasis- and metabolism-related alterations in the right ventricular outflow tract of non-syndromic Tetralogy of Fallot.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease, among which non-syndromic TOF (nsTOF) represents the most common subtype; however, the molecular mechanisms underlying right ventricular outflow tract (RVOT) remodeling in nsTOF remain incompletely understood. Bulk transcriptomic and single-nucleus RNA sequencing (snRNA-seq) datasets derived from fetal and infant RVOT tissues were integrated for analysis. Differential expression, miRNA-mRNA regulatory network construction, protein-protein interaction analysis, functional enrichment, and pseudotime trajectory analyses were performed to identify candidate hub genes and dynamic transcriptional alterations associated with nsTOF. A total of 842 differentially expressed mRNAs and 66 differentially expressed miRNAs were identified. Integration analyses yielded a regulatory network containing 54 DE-miRNAs and 538 DE-mRNAs. Fourteen hub genes, including PSMD14, NDUFA5, RPS27L, MRPS16, FOS, and SNRNP70, were identified through consensus topological filtering. Functional analyses suggested potential involvement of pathways related to protein homeostasis, ribosome-associated quality control, RNA splicing, mitochondrial metabolism, and stress-response signaling. snRNA-seq analysis demonstrated cell type-specific expression patterns of hub genes. Pseudotime analysis further suggested stage-dependent transcriptional alterations during RVOT remodeling. Integrated multi-omics analysis identified 14 candidate hub genes potentially involved in RVOT remodeling in nsTOF. These findings suggest that dysregulation of protein homeostasis, RNA-processing pathways, and mitochondrial metabolic processes may contribute to nsTOF pathology. Further experimental validation is required to confirm these observations.