Aberrant DNA methylation within circadian entrainment and clock genes two years after pediatric critical illness is associated with impaired physical and neurocognitive development.

BACKGROUND: Pediatric critical illness can disrupt circadian rhythms, potentially leading to long-term deficits in growth, neurocognition, and behavior. Disturbances in circadian rhythms have been associated with altered gene expression and DNA methylation. We investigated long-term DNA methylation alterations in circadian entrainment and clock genes in children previously admitted to the pediatric intensive care unit (PICU), the influence of parenteral nutrition (PN) timing hereon and their associations with long-term health outcomes. METHODS: This study is a secondary analysis of the PEPaNIC randomized controlled trial (RCT) and its two-year follow-up. The PEPaNIC-RCT randomized critically ill children to early initiation of supplemental PN within the first 24 h or its omission in the first week of PICU admission. DNA methylation of 127 circadian entrainment and clock genes was studied in buccal mucosa DNA of former PICU patients (n = 818) and matched healthy children (n = 392) at the two-year follow-up using Infinium® HumanMethylation EPIC BeadChips. Multivariable linear models were used to identify differential methylation between former patients and controls and their association with randomization group and outcomes at two-year follow-up. RESULTS: Former PICU patients showed 61 differentially methylated CpG sites (DMPs) within 34 of the genes, with 60 (98.4%) showing hypomethylation as compared with healthy children. Omitting early PN during PICU stay did not affect CpG site methylation. 702 associations (34.9%) were observed between abnormal DNA methylation and adverse long-term health and developmental outcomes, most notably visual-motor integration (47 DMPs, 77%), height (46 DMPs, 75%) and total IQ (46 DMPs, 75%). CONCLUSION: Former PICU patients exhibited altered DNA methylation in circadian entrainment and clock genes compared to healthy children, and this was associated with impaired growth, neurocognition and behavioral problems at two-year follow-up. Disruptions in circadian entrainment and clock gene methylation may contribute to the adverse long-term health consequences that children experience after critical illness.