Assessment of aberrant DNA methylation two years after paediatric critical illness: a pre-planned secondary analysis of the international PEPaNIC trial

Critically ill children requiring intensive care suffer from impaired physical/neurocognitive development 2 y later, partially preventable by omitting early use of parenteral nutrition (early-PN) in the paediatric intensive-care-unit (PICU). Altered methylation of DNA from peripheral blood during PICU-stay provided a molecular basis hereof. Whether DNA-methylation of former PICU patients, assessed 2 y after critical illness, is different from that of healthy children remained unknown. In a pre-planned secondary analysis of the PEPaNIC-RCT (clinicaltrials.gov-NCT01536275) 2-year follow-up, we assessed buccal-mucosal DNA-methylation (Infinium-HumanMethylation-EPIC-BeadChip) of former PICU-patients (N = 406 early-PN; N = 414 late-PN) and matched healthy children (N = 392). CpG-sites differentially methylated between groups were identified with multivariable linear regression and differentially methylated DNA-regions via clustering of differentially methylated CpG-sites using kernel-estimates. Analyses were adjusted for technical variation and baseline risk factors, and corrected for multiple testing (false-discovery-rate <0.05). Differentially methylated genes were functionally annotated (KEGG-pathway database), and allocated to three classes depending on involvement in physical/neurocognitive development, critical illness and intensive medical care, or pre-PICU-admission disorders. As compared with matched healthy children, former PICU-patients showed significantly different DNA-methylation at 4047 CpG-sites (2186 genes) and 494 DNA-regions (468 genes), with most CpG-sites being hypomethylated (90.3%) and with an average absolute 2% effect-size, irrespective of timing of PN initiation. Of the differentially methylated KEGG-pathways, 41.2% were related to physical/neurocognitive development, 32.8% to critical illness and intensive medical care and 26.0% to pre-PICU-admission disorders. Two years after critical illness in children, buccal-mucosal DNA showed abnormal methylation of CpG-sites and DNA-regions located in pathways known to be important for physical/neurocognitive development.

需要重症监护的危重儿童在患病2年后会出现躯体/神经认知发育受损，而在儿科重症监护病房（pediatric intensive-care-unit, PICU）中避免早期启用肠外营养（early-PN）可部分预防该不良结局。患儿在PICU住院期间外周血DNA甲基化（DNA-methylation）改变为此提供了分子机制基础。目前尚不清楚，在危重疾病发生2年后对既往PICU患者进行评估时，其DNA甲基化水平是否与健康儿童存在差异。

本研究针对PEPaNIC随机对照试验（RCT，clinicaltrials.gov-NCT01536275）的2年随访数据开展预先计划的二次分析，对既往PICU患者（早期肠外营养组n=406，晚期肠外营养组n=414）及匹配的健康儿童（n=392）的颊黏膜DNA甲基化（Infinium-HumanMethylation-EPIC-BeadChip）水平进行了检测。采用多变量线性回归（multivariable linear regression）识别组间差异甲基化CpG位点（CpG-sites），并通过核估计（kernel-estimates）对差异甲基化CpG位点进行聚类，以鉴定差异甲基化DNA区域。分析校正了技术变异与基线危险因素，并针对多重检验进行了校正，错误发现率（false-discovery-rate）<0.05。对差异甲基化基因采用KEGG通路数据库（KEGG-pathway database）进行功能注释，并根据其参与躯体/神经认知发育、危重疾病与重症医疗，或PICU入院前疾病的情况分为三类。

与匹配的健康儿童相比，既往PICU患者在4047个CpG位点（涉及2186个基因）及494个DNA区域（涉及468个基因）上的DNA甲基化水平存在显著差异。其中大多数CpG位点呈低甲基化状态（90.3%），平均绝对效应量为2%，且与肠外营养启动时机无关。在差异甲基化的KEGG通路中，41.2%与躯体/神经认知发育相关，32.8%与危重疾病及重症医疗相关，26.0%与PICU入院前疾病相关。

儿童罹患危重疾病2年后，其颊黏膜DNA在已知与躯体/神经认知发育密切相关的通路上的CpG位点及DNA区域出现异常甲基化。