Abstract
BackgroundThe 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.ResultsReduced 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.ConclusionsPre- 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-716) contains supplementary material, which is available to authorized users.
Highlights
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
Representation Bisulfite Sequencing (RRBS) data generation and characteristics of porcine intestinal methylome To characterize the intestinal methylome of preterm piglets associated with the adaptation around birth and development of NEC, we collected empty, 2 cm full-wall sections from the mid intestine (50% intestinal length from the stomach) from four groups of newborn piglets, all delivered by caesarean section
The other two groups consisted of preterm piglets fed with infant formula for four days following the rearing and feeding protocol specified previously [16], but showing severe symptoms of NEC in regions of the intestine other than the mid intestine that was selected for the analyses (4d-preterm-NEC) or being healthy (4d-preterm, each n = 3; Additional file 1: Table S1 and S2)
Summary
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. Epigenetics represent stable and heritable changes in gene expression without changing the DNA sequence, providing a mechanism whereby environmental factors can affect functions of specific cells, tissues and organs [1]. Preterm delivery at 88–95% gestation is associated with organ immaturities and clinical complications similar to those in infants born at 70–90% gestation [5]. Preterm infants and pigs lack the final maturation of the GIT and suffer from a series of intestinal defects, leading to increased risk of intestinal complications, including necrotizing enterocolitis (NEC). Studies of the intestinal proteome suggest that the intestinal maladaptation in preterm neonates can be explained by a feeding-induced decrease in intestinal metabolism and stress response [8,10], but the molecular mechanisms remain unknown
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