1 Clinical and Experimental Nutrition, Department of Nutrition, Exercise and Sports, Faculty of Science, Københavns Universitet2 Section for Metabolic Genetics, Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, Københavns Universitet3 Genome Research and Molecular Bio Medicine, Department of Biology, Faculty of Science, Københavns Universitet4 Department of Science & Technology, BGI-Shenzhen, Shenzhen5 Comparative Paediatrics and Nutrition, Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, Københavns Universitet6 Clinical an Experimental Nutrition, Department of Human Nutrition, Faculty of Life Sciences, Københavns Universitet7 Comparative Paediatrics and Nutrition, Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, Københavns Universitet8 Clinical an Experimental Nutrition, Department of Human Nutrition, Faculty of Life Sciences, Københavns Universitet9 Genome Research and Molecular Bio Medicine, Department of Biology, Faculty of Science, Københavns Universitet
BACKGROUND: The serious feeding- and microbiota-associated intestinal disease, necrotizing enterocolitis (NEC), occurs mainly in infants born prematurely (5-10% of all newborns) and most frequently after formula-feeding. We hypothesized that changes in gene methylation is involved in the prenatal maturation of the intestine and its response to the first days of formula feeding, potentially leading to NEC in preterm pigs used as models for preterm infants. RESULTS: Reduced Representation Bisulfite Sequencing (RRBS) was used to assess if changes in intestinal DNA methylation are associated with formula-induced NEC outbreak and advancing age from 10 days before birth to 4 days after birth. Selected key genes with differentially methylated gene regions (DMRs) between groups were further validated by HiSeq-based bisulfite sequencing PCR and RT-qPCR to assess methylation and expression levels. Consistent with the maturation of many intestinal functions in the perinatal period, methylation level of most genes decreased with advancing pre- and postnatal age. The highest number of DMRs was identified between the newborn and 4 d-old preterm pigs. There were few intestinal DMR differences between unaffected pigs and pigs with initial evidence of NEC. In the 4 d-old formula-fed preterm pigs, four genes associated with intestinal metabolism (CYP2W1, GPR146, TOP1MT, CEND1) showed significant hyper-methylation in their promoter CGIs, and thus, down-regulated transcription. Methylation-driven down-regulation of such genes may predispose the immature intestine to later metabolic dysfunctions and severe NEC lesions. CONCLUSIONS: Pre- and postnatal changes in intestinal DNA methylation may contribute to high NEC sensitivity in preterm neonates. Optimizing gene methylation changes via environmental stimuli (e.g. diet, nutrition, gut microbiota), may help to make immature newborn infants more resistant to gut dysfunctions, both short and long term.