Low birth weight, DNA methylation patterns in cord blood, and inflammation at birth

Low birth weight (LBW) is a significant global health issue associated with an increased risk of long-term chronic diseases, yet its underlying molecular mechanisms remain unclear. DNA methylation serves as a crucial epigenetic mechanism for investigating these associations. Our study employed a unique model of birth weight-discordant monozygotic (MZ) twin neonates and a multi-level validation strategy to elucidate the mechanisms by which LBW affects immune function through DNA methylation at birth.

We performed genome-wide DNA methylation profiling using methylated DNA immunoprecipitation sequencing (MeDIP-seq) on cord blood mononuclear cells (CBMCs) from monozygotic (MZ) twin pairs discordant for birth weight. After identifying the most significantly differentially methylated genes (DMGs), gene set linkage analysis (GSLA) was conducted to reveal key functional networks. Key DMGs were validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to quantify transcript levels in CBMCs obtained from an independent neonatal cohort. Furthermore, we quantified the production of eight cytokines through in vitro immune stimulation assays in an additional independent neonatal cohort. We isolated lymphocytes and monocytes from cord blood and stimulated lymphocytes with phytohemagglutinin (PHA) and monocytes with lipopolysaccharide (LPS), respectively.

We identified top 50 DMGs significantly associated with LBW, among which 11 DMGs formed functional networks enriched in ten biological processes, with the interferon (IFN)-γ mediated immune response emerging as the predominant theme. We validated the messenger RNA (mRNA) levels of 11 DMGs in CMBCs and found significant changes in eight of these DMGs in LBW neonates. Regarding lymphocyte function in neonatal cord blood, baseline levels of IFN-γ mRNA and protein, as well as mRNA levels of IP-10, interleukin (IL)-4 and IL-10, were comparable between the LBW and NBW groups. After PHA stimulation, lymphocytes from LBW neonates produced significantly higher levels of IFN-γ and IP-10, but lower levels of IL-4 and IL-10, compared to those from NBW neonates (P<0.01). Additionally, monocytes from cord blood of LBW neonates consistently exhibited higher mRNA levels of IL-1β (P<0.001) and tumor necrosis factor (TNF)-α (P<0.05) than NBW neonates, both before and after LPS stimulation.

A distinct genomic DNA methylation pattern in LBW neonates may underlie hyperresponsive IFN-γ signaling and a persistent pro-inflammatory immune phenotype. These findings indicate that epigenetic mechanisms that affect the immune response in individuals born with LBW may contribute to an increased long-term susceptibility to chronic inflammatory diseases. Future work is needed to establish the causal links among the observed methylation pattern associated with LBW, IFN‑γ hyperresponsiveness, and long‑term disease risk.