DNA methylation patterns at birth predict health outcomes in young adults born very low birthweight

**Background:** Individuals born very low birthweight (VLBW, <1500 g) are at increased risk for cardiovascular and respiratory disorders in adulthood. The mechanisms are poorly understood, but epigenetic processes such as DNA methylation may link early-life exposures to later disease. This study aimed to identify methylation markers at birth that predict adult health outcomes in VLBW individuals.

**Methods:** The study analyzed the New Zealand 1986 VLBW cohort (all infants born <1500 g in NZ in 1986). Genomic DNA was extracted from archived newborn heel-prick blood spots (109 VLBW, 51 controls) and from peripheral blood at ~28 years (215 VLBW, 96 controls). Samples were analyzed on Illumina Infinium MethylationEPIC 850K arrays. After quality assurance and normalization, methylation levels were compared between VLBW cases and controls at both ages by linear regression, with genome-wide significance set to p<0.05 adjusted for false discovery rate (FDR, Benjamini-Hochberg). Differentially methylated regions (DMRs) were identified using DMRcate. Pathway analysis was performed using Ingenuity Pathway Analysis. Associations between neonatal methylation and adult cardiovascular and respiratory outcomes were tested using general linear models adjusted for case-control status, sex, and ethnicity.

**Key Results:** In neonates, over 16,400 CpG sites differed between VLBW cases and controls at FDR-adjusted significance. The canonical pathway most enriched was Cardiac Hypertrophy Signaling (p=3.44E−11). The top 20 CpGs featured clusters in ARID3A, SPATA33, and PLCH1. Fifteen of the top 20 CpGs in neonates showed associations between methylation at birth and adult cardiovascular traits, particularly LnRHI (reactive hyperemic index, a measure of endothelial function). In 28-year-old adults, 12 CpGs differed between VLBW cases and controls at FDR-adjusted significance, including hypermethylation in EBF4 (4 CpGs), CFI and UNC119B, and hypomethylation in HIF3A (3 CpGs) and KCNQ1. DNAm GrimAge scores at 28 years were significantly greater in VLBW cases vs. controls (38.12±4.83 vs. 36.38±4.87, p=0.003). Four CpGs overlapped between neonates and adults, with three reversing direction with age (two CpGs in EBF4 and one in SNAI1 were hypomethylated in neonates, hypermethylated in adults). CpG cg16426670 in EBF4 at birth showed associations with several cardiovascular traits in adults.

**Clinical Implications:** This is the first study to demonstrate that altered methylation at birth in VLBW infants is associated with health outcomes in adulthood, particularly cardiovascular health. The findings suggest that neonatal methylation patterns, especially in genes like EBF4, may serve as biomarkers to identify VLBW individuals at highest risk for future cardiovascular and respiratory disease. Such markers could guide early preventative interventions, including optimized nutrition, blood pressure monitoring, and lifestyle modifications. The dynamic methylation of EBF4 from hypomethylation at birth to hypermethylation in adulthood may reflect compensatory cardiovascular development and could be a target for future research. Limitations include the use of archived blood spots (30-year storage) vs. fresh adult blood, and limited data on perinatal confounders in controls.