Epigenome-wide association study of bronchopulmonary dysplasia in preterm infants: results from the discovery-BPD program

Abstract

BackgroundBronchopulmonary dysplasia (BPD) is a lung disease in premature infants caused by therapeutic oxygen supplemental and characterized by impaired pulmonary development which persists into later life. While advances in neonatal care have improved survival rates of premature infants, cases of BPD have been increasing with limited therapeutic options for prevention and treatment. This study was designed to explore the relationship between gestational age (GA), birth weight, and estimated blood cell-type composition in premature infants and to elucidate early epigenetic biomarkers associated with BPD.MethodsCord blood DNA from preterm neonates that went on to develop BPD (n = 14) or not (non-BPD,n = 93) was applied to Illumina 450 K methylation arrays. Blood cell-type compositions were estimated using DNA methylation profiles. Multivariable robust regression analysis elucidated CpGs associated with BPD risk. cDNA microarray analysis of cord blood RNA identified differentially expressed genes in neonates who later developed BPD.ResultsThe development of BPD and the need for oxygen supplementation were strongly associated with GA (BPD,p < 1.0E−04; O2supplementation,p < 1.0E−09) and birth weight (BPD,p < 1.0E−02; O2supplementation,p < 1.0E−07). The estimated nucleated red blood cell (NRBC) percent was negatively associated with birth weight and GA, positively associated with hypomethylation of the tobacco smoke exposure biomarker cg05575921, and high-NRBC blood samples displayed a hypomethylation profile. Epigenome-wide association study (EWAS) identified 38 (Bonferroni) and 275 (false discovery rate 1%) differentially methylated CpGs associated with BPD. BPD-associated CpGs in cord blood were enriched for lung maturation and hematopoiesis pathways. Stochastic epigenetic mutation burden at birth was significantly elevated among those who developed BPD (adjustedp = 0.02). Transcriptome changes in cord blood cells reflected cell cycle, development, and pulmonary disorder events in BPD.ConclusionsWhile results must be interpreted with caution because of the small size of this study, NRBC content strongly impacted DNA methylation profiles in preterm cord blood and EWAS analysis revealed potential insights into biological pathways involved in BPD pathogenesis.Graphical Abstract

Prospective epigenome and transcriptome analyses of cord and peripheral blood from preterm infants at risk of bronchopulmonary dysplasia

“…In addition to the immunological biomarkers, we also observed development-and alveolarization-related epigenetic and transcriptomic markers in cord blood of infants who developed BPD 30,31,40 . Concurrently with BPD epigenome data 22 , we identified SPOCK2 and AGER among the BPD-DEGs in cord blood. SPOCK2, a BPD www.nature.com/scientificreports/ susceptibility gene identified from GWAS 7,30,31 , was reported to be deleterious in BPD development as its overexpression in the lung altered neonatal mouse lung alveolarization and exacerbated hyperoxia-caused alveolar simplification while anti-SPOCK2 antibody treatment in mice improved it 7,30 .…”

“…DNA methylation microarray analysis. Aliquots (100-500 ng) of genomic DNA from whole peripheral blood were bisulfite-converted using EZ-96 DNA Methylation MagPrep kits (Zymo Research, Irvine, CA) following the manufacturer's instructions as described previously 22 . Bisulfite-converted DNAs were applied to Human MethylationEPIC BeadChip (Illumina, San Diego, CA), which covers over 850,000 CpG sites in the human genome, at the NIEHS Molecular Genomics Core Laboratory.…”

“…*Genes significant at false discovery rate 10%. † Genes overlapped with those annotated to epigenome-wide association study previously published 22 . www.nature.com/scientificreports/ BPD-CpG annotated genes observed on day 28 (Fig.…”

DNA methylation in necrotizing enterocolitis

“…In addition, an epigenome-wide association study found significant differences in the DNA methylation patterns in cord blood of preterm neonates who subsequently developed BPD compared with those who did not develop lung disease (Ref. 11).…”

“…In this review, we will discuss DNA methylation, which has been linked to early development and neonatal disease (Refs 8, 9, 10, 11, 12, 13, 14, 15, 16). We will specifically focus on the intestinal disease of prematurity, necrotizing enterocolitis (NEC).…”