Comprehensive methylome-wide association study with methylation-based WGCNA profiling and functional enrichment analyses in drug-induced liver injury

Background Idiosyncratic drug-induced liver injury (DILI) is a rare, unpredictable and host-specific immune-mediated adverse reaction to drugs, herbal and dietary supplements. DILI is a complex disease whose presentation is influenced by multiple host and environmental factors. The latter modulate gene expression through epigenetic regulatory mechanisms, such as DNA methylation. This methylome-wide association study aims to characterise DILI differential methylation profile and to determine the disease mechanisms affected by DNA methylation dysregulation. Methods Whole blood DNA samples from 32 DILI patients and 33 healthy controls included in the Spanish DILI Registry were analysed using the Infinium MethylationEPIC 850k BeadChip microarray to screen differentially methylated CpG positions. After methylation data quality control, differentially methylated CpG sites or positions (DMPs) and regions (DMRs) analyses were conducted. Additionally, a DNA methylation-based weighted gene correlation network analysis (WGCNA) was performed with CpG sites nearby the gene transcription start site (TSS) to characterise clusters of functionally related genes based on their DNA methylation profiles. Functional and tissue enrichment analyses were performed to identify the processes and the target tissues affected by differential methylation in DILI. Results Global hypomethylation was observed in DILI. Hypomethylated DMR1 in ENSG00000298543 was the most significant (HMFDR = 3.43\(\times{10}^{-5}\)). ENSG00000298543 encodes a long non-coding RNA antisense to CD58 and IGSF3, indicating DMR1 potential role in enhancing the immune response. Two gene modules with significant differential methylation were identified. ME1 was the most significant and the main methylation pattern in DILI (FDR = 1.29 \(\times{10}^{-10}\)). Meanwhile, ME0 (FDR = 7.13\(\times{10}^{-5}\)) was a male-specific DILI module. Hypermethylated MIR342 in ME1 suggested possible shared mechanisms with metabolic dysfunction-associated steatotic liver disease (MASLD). Functional enrichment analyses have shown the relevance of the sensory receptor signalling pathways and the immune response activation. Conclusions Altered DNA methylation was observed across samples, indicating genomic instability as a disease determinant in DILI. Additionally, DNA methylation profiling near the TSS region revealed DNA methylation participation in the regulation of the sensory signalling pathways and in the immune system overactivation, and evidenced the interaction between DNA methylation and non-coding RNAs. Moreover, this study demonstrated possible shared epigenetic features with other liver diseases, such as MASLD.