Analysis of two birth tissues provides new insights into the epigenetic landscape of neonates born preterm

Yonghui Wu, Xinyi Lin, Ives Yubin Lim, Li Chen, Ai Ling Teh, Julia L MacIsaac, Kok Hian Tan, Michael S Kobor, Yap Seng Chong, Peter D Gluckman, Neerja Karnani, Yonghui Wu, Xinyi Lin, Ives Yubin Lim, Li Chen, Ai Ling Teh, Julia L MacIsaac, Kok Hian Tan, Michael S Kobor, Yap Seng Chong, Peter D Gluckman, Neerja Karnani

Abstract

Background: Preterm birth (PTB), defined as child birth before completion of 37 weeks of gestation, is a major challenge in perinatal health care and can bear long-term medical and financial burden. Over a million children die each year due to PTB complications, and those who survive can face developmental delays. Unfortunately, our understanding of the molecular pathways associated with PTB remains limited. There is a growing body of evidence suggesting the role of DNA methylation (DNAm) in mediating the effects of PTB on future health outcomes. Thus, epigenome-wide association studies (EWAS), where DNAm sites are examined for associations with PTB, can help shed light on the biological mechanisms linking the two.

Results: In an Asian cohort of 1019 infants (68 preterm, 951 full term), we examined and compared the associations between PTB and genome-wide DNAm profiles using both cord tissue (n = 1019) and cord blood (n = 332) samples on Infinium HumanMethylation450 arrays. PTB was significantly associated (P < 5.8e-7) with DNAm at 296 CpGs (209 genes) in the cord blood. Over 95% of these CpGs were replicated in other PTB/gestational age EWAS conducted in (cord) blood. This replication was apparent even across populations of different ethnic origin (Asians, Caucasians, and African Americans). More than a third of these 296 CpGs were replicated in at least 4 independent studies, thereby identifying a robust set of PTB-linked epigenetic signatures in cord blood. Interrogation of cord tissue in addition to cord blood provided novel insights into the epigenetic status of the neonates born preterm. Overall, 994 CpGs (608 genes, P < 3.7e-7) associated with PTB in cord tissue, of which only 10 of these CpGs were identified in the analysis using cord blood. Genes from cord tissue showed enrichment of molecular pathways related to fetal growth and development, while those from cord blood showed enrichment of immune response pathways. A substantial number of PTB-associated CpGs from both the birth tissues were also associated with gestational age.

Conclusions: Our findings provide insights into the epigenetic landscape of neonates born preterm, and that its status is captured more comprehensively by interrogation of more than one neonatal tissue in tandem. Both these neonatal tissues are clinically relevant in their unique ways and require careful consideration in identification of biomarkers related to PTB and gestational age.

Trial registration: This birth cohort is a prospective observational study designed to study the developmental origins of health and disease, and was retrospectively registered on 1 July 2010 under the identifier NCT01174875 .

Keywords: DNA methylation; Epigenome wide association study; Gestational age; Neonate; Preterm birth; Tissue specificity.

Conflict of interest statement

Ethics approval and consent to participate

Written informed consent was obtained from all women who participated in the study. Approval for the study was granted by the ethics boards of both KK Women’s and Children’s Hospital (KKH) and National University Hospital (NUH), which are the Centralised Institute Review Board (CIRB) and the Domain Specific Review Board (DSRB) respectively.

Consent for publication

Not applicable.

Competing interests

YSC, PDG, and NK have received reimbursement for speaking at conferences sponsored by companies selling nutritional products. They are part of an academic consortium that has received research funding from Abbott Nutrition, Nestec and Danone. The other authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Preterm births (PTB) were associated with global alterations in infants’ cord tissue DNA methylation. a Manhattan plot and b volcano plot illustrating the relationship of the 134,676 infant cord tissue CpGs analyzed with respect to PTB. The top 10 CpGs with the smallest P values are indicated on both plots and labeled with the gene it is associated with or CpG identifier if the CpG lies within an intergenic region. Points on each plot represent individual CpGs which in a have genomic locations on the horizontal axis with alternating colors representing different chromosomes and in b have the change in DNA methylation Z-score on the horizontal axis. The red horizontal line in a represents the Bonferroni threshold (P < 3.7 × 10−7). Nine hundred ninety-four infant cord tissue CpGs were found to significantly associate with PTB and are indicated as red points in b. In both plots, the vertical axis represents the negative log10 P values with respect to PTB, adjusted for infant sex, ethnicity, cell-type proportions, bisulfite conversion batch, and DNA extraction batch
Fig. 2
Fig. 2
Preterm births (PTB) were associated with global alterations in infants’ cord blood DNA methylation. a Manhattan plot and b volcano plot illustrating the relationship of the 85,624 infant cord blood CpGs analyzed with respect to PTB. The top 10 CpGs with the smallest P values are indicated on both plots and labeled with the gene it is associated with or CpG identifier if the CpG lies within an intergenic region. Points on each plot represent individual CpGs which in a have genomic locations on the horizontal axis with alternating colors representing different chromosomes and in b have the change in DNA methylation Z-score on the horizontal axis. The red horizontal line in a represents the Bonferroni threshold (P < 5.8 × 10−7). Two hundred ninety-six infant cord blood CpGs were found to significantly associate with PTB and are indicated as red points in b. In both plots, the vertical axis represents the negative log10 P values with respect to preterm birth status, adjusted for infant sex, ethnicity, cell-type proportions, and bisulfite conversion batch
Fig. 3
Fig. 3
Cord blood CpGs previously reported in association with gestational age (GA) or preterm births (PTB). a The Venn diagram shows the relationship between cord blood CpGs previously reported to be significantly associated with gestational age or PTB in relation to PTB-associated CpGs in the current study. b The bar graph shows the reproducibility of the 296 PTB-associated cord blood CpGs in the current study. The vertical axis gives the number of PTB-associated cord blood CpGs in the current study, while the horizontal axis gives the number of earlier GA/PTB epigenome-wide association studies (EWAS) our PTB-associated cord blood CpGs are replicated in. Bar graph colors are representative of the number of earlier studies our PTB-associated CpGs replicated in black (0), green (1), purple (2), orange (3), blue (4), pink (5), and brown (6). c This UpSet plot further breaks down the replication of our PTB-associated CpGs in the earlier studies. Each column represents the number of CpGs, for each unique intersection of the current study (GUSTO) with other studies, as indicated by the gray dot and connecting line. Intersection sets with no CpGs are not shown on the plot
Fig. 4
Fig. 4
a, b REVIGO summarized Gene Ontology Clusters with respect to preterm birth (PTB)-associated CpGs in both cord tissue and cord blood. Gene ontology (GO) enrichment was performed on PTB-associated CpGs in both cord tissue and cord blood for each tissue separately using missMethyl. REVIGO was then used to reclassify the biological process-related enriched GO terms (parent GO term containing under 300 genes, semantic similarity measure between each GO term < 0.7). Cord tissue CpGs had 10 GO clusters from 41 unique GO terms, while cord blood CpGs had 10 GO clusters from 43 unique GO terms. GO clusters with 5 or more genes are represented by the bar graphs, with plots on the left and right corresponding to cord tissue and cord blood respectively. The vertical axis of the bar graphs represents the REVIGO cluster names, while the horizontal axis represents the number of genes in the REVIGO cluster containing at least one significantly associated CpG

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