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Updated: Nov 12, 2025
A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis
Published on: April 10, 2019
DNA Methylation of TLR4, VEGFA, and DEFA5 Is Associated With Necrotizing Enterocolitis in Preterm Infants
Daphne H Klerk1, Torsten Plösch2, Rikst Nynke Verkaik-Schakel2
1Division of Neonatology, Beatrix Children's Hospital, University Medical Center Groningen, University of Groningen, Groningen, Netherlands.
Insights
Epigenetic changes in DNA methylation of TLR4, VEGFA, and DEFA5 genes are linked to necrotizing enterocolitis (NEC) risk in preterm infants. Stool DNA analysis offers a novel, non-invasive method for NEC research.
Area of Science:
- Epigenetics
- Molecular Biology
- Neonatal Medicine
Background:
- Necrotizing enterocolitis (NEC) is a severe gastrointestinal condition in preterm infants.
- Epigenetic modifications, specifically DNA methylation, are implicated in NEC pathogenesis.
- Investigating methylation patterns in NEC-associated genes may reveal susceptibility factors.
Purpose of the Study:
- To assess DNA methylation differences in five NEC-associated genes (EPO, VEGFA, ENOS, DEFA5, TLR4) between preterm infants with NEC and controls.
- To examine temporal methylation changes in NEC-associated genes in relation to NEC onset.
- To evaluate the utility of stool DNA analysis for non-invasive assessment of the infant gut.
Main Methods:
- Observational cohort study comparing 24 preterm infants with NEC (≥Bell Stage IIA) and 45 matched controls.
- DNA isolation from stool samples followed by pyrosequencing to measure DNA methylation.
- Analysis of methylation patterns prior to NEC, and in NEC infants, long before, shortly before, and after NEC onset.
Main Results:
- Increased TLR4 CpG 2 methylation was observed in NEC infants prior to disease onset compared to controls (p=0.025).
- In NEC infants, VEGFA CpG 3 and DEFA5 CpG 1 methylation levels increased significantly over time, preceding and following NEC onset (p<0.05).
- No significant methylation changes were found for EPO and ENOS genes.
Conclusions:
- Epigenetic alterations in TLR4, VEGFA, and DEFA5 are associated with NEC in preterm infants.
- Temporal changes in DNA methylation of these genes may influence gene expression and contribute to NEC development.
- Human DNA extraction from stool samples provides a novel, non-invasive approach for studying NEC and the preterm infant gut.
Abstract:
Background: Epigenetic changes, such as DNA methylation, may contribute to an increased susceptibility for developing necrotizing enterocolitis (NEC) in preterm infants. We assessed DNA methylation in five NEC-associated genes, selected from literature: EPO, VEGFA, ENOS, DEFA5, and TLR4 in infants with NEC and controls. Methods: Observational cohort study including 24 preterm infants who developed NEC (≥Bell Stage IIA) and 45 matched controls. DNA was isolated from stool samples and methylation measured using pyrosequencing. We investigated differences in methylation prior to NEC compared with controls. Next, in NEC infants, we investigated methylation patterns long before, a short time before NEC onset, and after NEC. Results: Prior to NEC, only TLR4 CpG 2 methylation was increased in NEC infants (median = 75.4%, IQR = 71.3-83.8%) versus controls (median = 69.0%, IQR = 64.5-77.4%, p = 0.025). In NEC infants, VEGFA CpG 3 methylation was 0.8% long before NEC, increasing to 1.8% a short time before NEC and 2.0% after NEC (p = 0.011; p = 0.021, respectively). A similar pattern was found in DEFA5 CpG 1, which increased from 75.4 to 81.4% and remained 85.3% (p = 0.027; p = 0.019, respectively). These changes were not present for EPO, ENOS, and TLR4. Conclusion: Epigenetic changes of TLR4, VEGFA, and DEFA5 are present in NEC infants and can differ in relation to the time of NEC onset. Differences in DNA methylation of TLR4, VEGFA, and DEFA5 may influence gene expression and increase the risk for developing NEC. This study also demonstrates the use of human DNA extraction from stool samples as a novel non-invasive method for exploring the bowel of preterm infants and which can also be used for necrotizing enterocolitis patients.
