Contribution of m6A subtype classification on heterogeneity of sepsis

Sepsis is a highly heterogeneous syndrome with diverse immune status and varied bioprocesses among individuals. The heterogeneity of sepsis could be associated with N6-methyladenosine (m6A) RNA methylation, due to m6A as a common and reversible posttranscriptional RNA modification involved in the regulation of whole bioprocesses. Therefore, we aim to identify m6A induced molecular subtypes of sepsis and furthermore explore the probable mechanism.

Gene expression datasets with 479 consecutive patients admitted for sepsis to the intensive care unit (ICU) in the Amsterdam Academic Medical Center were included in present study at first. Secondly, twelve m6A methylation regulatory genes were determined via systematic review in published researches. Furthermore, we utilized unsupervised clustering (consensus k means clustering) to identify m6A induced molecular subtypes in sepsis based on m6A prognostic molecular, and assess the association of these subtypes with clinical traits and survival outcomes. Moreover, the probable mechanism and regulatory relationship of m6A in sepsis was also explored through Gene Set Enrichment Analysis (GSEA), Weighted gene co-expression network analysis (WGCNA), Gene Ontology (GO) analysis and Co-expression analysis.

Three m6A subtypes with different outcome were identified in sepsis cohort through unsupervised clustering on m6A prognostic molecular, designated Cluster 1/2/3 (log-rank P=0.004). The best outcome was found for patients classified as having cluster 3, and at 28 days, 21 of 144 people with cluster 3 had died [hazard ratio (HR) vs. all other clusters 5.42 (95% CI: 0.359-0.819); P=0.011], compared with 57 of 224 people with cluster 1 (HR 0.579, 95% CI: 0.364-0.920; P=0.037), and 36 of 112 people with cluster 2 (HR 0.477, 95% CI: 0.272-0.833; P=0.003). For exploration of the relationship between m6A subtypes and immunity, the GSEA found that patients in cluster 1 suffered from hyper-activated immunocompetent status; patients in cluster 2 indicated immunosuppressive status; and patients in cluster 3 showed the moderate immune activity (P<0.05). Co-expression analysis furthermore identified 82 immune molecules and 40 autophagy-related molecules could be regulated by prognostic m6A RNA methylation regulators (P<0.05) and correlation coefficient >0.6. In addition, WGCNA and GO analysis indicated that autophagy was significantly and widely activated in patients with cluster 3 (P<0.05).

According to the heterogeneity in m6A methylation regulatory genes, three distinct subtypes in sepsis were identified with different RNA epigenetics, immune status, biological processes and outcomes, which initially uncovered that heterogeneity of sepsis may be largely caused by m6A RNA methylation.