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

Abstract

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.

Abnormal DNA methylation within genes of the steroidogenesis pathway two years after paediatric critical illness and association with stunted growth in height further in time

“…As previously described [ 29 ], DNA was extracted from all available buccal mucosa swabs from patients and healthy children (DDK DNA isolation kit, Isohelix). DNA concentrations were quantified with the Qubit® 3.0 fluorometer (Thermo Fisher Scientific, Waltham, MA).…”

“…This package contains R functions based on the Minfi pipeline [ 32 – 34 ] to exclude low-quality samples (not showing the typical bi-peak curve of the methylation value distribution in the low- and high-end range on the sample histograms) and low-quality probes (probes that did not exceed the background signal and probes spanning known single nucleotide polymorphisms), and to normalise the methylation data, adjust for batch effect and find differentially methylated positions and regions, as described below. Non-biological or technical variation due to experimental conditions was corrected for by including the first 30 principal components (PCs) of the control probes located on the Infinium® HumanMethylation EPIC BeadChip, excluding the negative control probes, as covariates in all multivariable linear regression models [ 29 , 35 ].…”

“…DMRs in former PICU patients as compared with the healthy children were identified with the DMRcate package, as previously described [ 29 , 38 ]. This procedure involved calculation of a t-statistic with multivariable regression modelling comparing patients with the healthy children (adjusted for the above-mentioned baseline risk factors and for batch effect [ 35 ]), calculation of a weighted average for every CpG site (kernel estimate), comparison of the kernel estimates against a null comparison to assess statistical significance, and final determination of a differentially methylated region by grouping all significantly different kernel estimates not further than 1000 base pairs separated from one another.…”

The epigenetic legacy of ICU feeding and its consequences

“…Further corroboration of plausible involvement of an altered DNA-methylome in the developmental impairments of former PICU-patients was provided by a recent study on buccal-mucosa DNA collected at the PEPaNIC 2-year follow-up. Former patients, as compared with healthy children, showed abnormal methylation of DNA-positions and DNA-regions among which many located within genes of pathways known to be important for physical and neurocognitive development [91 ▪ ]. A few other studies also focused on samples obtained long after the critical illness phase.…”

Nutrition and autophagy deficiency in critical illness

“…The long-term impact of nutritional management in ICU on autophagy in relation to the long-term adverse legacy after critical illness [104–110,131 ▪ ,132] remains unclear. Epigenetic abnormalities induced by critical illness or its nutritional management provide a plausible molecular basis for their adverse effects on long-term outcomes [133–136,137 ▪ ]. Autophagy is prone to epigenetic regulation, and abnormal DNA methylation of genes involved in autophagy was observed in the muscles of critically ill patients versus matched controls [137 ▪ ].…”