title: Analysis of epigenetic changes in survivors of preterm birth reveals the effect of gestational age and evidence for a long term legacy.
abstract: BACKGROUND
  Preterm birth confers a high risk of adverse long term health outcomes for survivors, yet the underlying molecular mechanisms are unclear. We hypothesized that effects of preterm birth can be mediated through measurable epigenomic changes throughout development. We therefore used a longitudinal birth cohort to measure the epigenetic mark of DNA methylation at birth and 18 years comparing survivors of extremely preterm birth with infants born at term.
  
  
  METHODS
  Using 12 extreme preterm birth cases and 12 matched, term controls, we extracted DNA from archived neonatal blood spots and blood collected in a similar way at 18 years of age. DNA methylation was measured at 347,789 autosomal locations throughout the genome using Infinium HM450 arrays. Representative methylation differences were confirmed by Sequenom MassArray EpiTYPER.
  
  
  RESULTS
  At birth we found 1,555 sites with significant differences in methylation between term and preterm babies. At 18 years of age, these differences had largely resolved, suggesting that DNA methylation differences at birth are mainly driven by factors relating to gestational age, such as cell composition and/or maturity. Using matched longitudinal samples, we found evidence for an epigenetic legacy associated with preterm birth, identifying persistent methylation differences at ten genomic loci. Longitudinal comparisons of DNA methylation at birth and 18 years uncovered a significant overlap between sites that were differentially-methylated at birth and those that changed with age. However, we note that overlapping sites may either differ in the same (300/1,555) or opposite (431/1,555) direction during gestation and aging respectively.
  
  
  CONCLUSIONS
  We present evidence for widespread methylation differences between extreme preterm and term infants at birth that are largely resolved by 18 years of age. These results are consistent with methylation changes associated with blood cell development, cellular composition, immune induction and age at these time points. Finally, we identified ten probes significantly associated with preterm individuals and with greater than 5% methylation discordance at birth and 18 years that may reflect a long term epigenetic legacy of preterm birth.
authors.affiliation: Early Life Epigenetics Group, Murdoch Childrens Research Institute (MCRI), Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia ; Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia ; Present address: Telethon Institute for Child Health Research, University of Western Australia, 100 Roberts Road, Subiaco, WA 6008, Australia.
authors.forename: Mark N
authors.initials: MN
authors.lastname: Cruickshank
authors.affiliation: Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia ; Bioinformatics Group, MCRI, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia.
authors.forename: Alicia
authors.initials: A
authors.lastname: Oshlack
authors.affiliation: Early Life Epigenetics Group, Murdoch Childrens Research Institute (MCRI), Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia ; Neonatal Services, Royal Women's Hospital, Parkville, Victoria 3052, Australia ; Department of Obstetrics and Gynaecology, University of Melbourne, Royal Women's Hospital, Parkville, Victoria 3052, Australia.
authors.forename: Christiane
authors.initials: C
authors.lastname: Theda
authors.affiliation: Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia ; Neonatal Services, Royal Women's Hospital, Parkville, Victoria 3052, Australia ; Department of Obstetrics and Gynaecology, University of Melbourne, Royal Women's Hospital, Parkville, Victoria 3052, Australia.
authors.forename: Peter G
authors.initials: PG
authors.lastname: Davis
authors.affiliation: Cancer and Developmental Epigenetics Group, MCRI, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia.
authors.forename: David
authors.initials: D
authors.lastname: Martino
authors.affiliation: Department of Obstetrics and Gynaecology, University of Melbourne, Royal Women's Hospital, Parkville, Victoria 3052, Australia.
authors.forename: Penelope
authors.initials: P
authors.lastname: Sheehan
authors.affiliation: Early Life Epigenetics Group, Murdoch Childrens Research Institute (MCRI), Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia.
authors.forename: Yun
authors.initials: Y
authors.lastname: Dai
authors.affiliation: Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia ; Cancer and Developmental Epigenetics Group, MCRI, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia.
authors.forename: Richard
authors.initials: R
authors.lastname: Saffery
authors.affiliation: Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia ; Neonatal Services, Royal Women's Hospital, Parkville, Victoria 3052, Australia ; Department of Obstetrics and Gynaecology, University of Melbourne, Royal Women's Hospital, Parkville, Victoria 3052, Australia.
authors.forename: Lex W
authors.initials: LW
authors.lastname: Doyle
authors.affiliation: Early Life Epigenetics Group, Murdoch Childrens Research Institute (MCRI), Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia ; Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia.
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Craig