Intake of mother's milk by very-low-birth-weight infants and variation in DNA methylation of genes involved in neurodevelopment at 5.5 years of age

Am J Clin Nutr. 2022 Oct 6;116(4):1038-1048. doi: 10.1093/ajcn/nqac221.


Background: Mechanisms responsible for associations between intake of mother's milk in very-low-birth-weight (VLBW, <1500 g) infants and later neurodevelopment are poorly understood. It is proposed that early nutrition may affect neurodevelopmental pathways by altering gene expression through epigenetic modification. Variation in DNA methylation (DNAm) at cytosine-guanine dinucleotides (CpGs) is a commonly studied epigenetic modification.

Objectives: We aimed to assess whether early mother's milk intake by VLBW infants is associated with variations in DNAm at 5.5 y, and whether these variations correlate with neurodevelopmental phenotypes.

Methods: This cohort study was a 5.5-y follow-up (2016-2018) of VLBW infants born in Ontario, Canada who participated in the Donor Milk for Improved Neurodevelopmental Outcomes trial. We performed an epigenome-wide association study (EWAS) to test whether percentage mother's milk (not including supplemental donor milk) during hospitalization was associated with DNAm in buccal cells during early childhood (n = 143; mean ± SD age: 5.7 ± 0.2 y; birth weight: 1008 ± 517 g). DNAm was assessed with the Illumina Infinium MethylationEPIC array at 814,583 CpGs. In secondary analyses, we tested associations between top-ranked CpGs and measures of early childhood neurodevelopment, e.g., total surface area of the cerebral cortex (n = 41, MRI) and Full-Scale IQ (n = 133, Wechsler Preschool and Primary Scale of Intelligence-IV).

Results: EWAS analysis demonstrated percentage mother's milk intake by VLBW infants during hospitalization was associated with DNAm at 2 CpGs, cg03744440 [myosin XVB (MYO15B)] and cg00851389 [metallothionein 1A (MT1A)], at 5.5 y (P < 9E-08). Gene set enrichment analysis indicated that top-ranked CpGs (P < 0.001) were annotated to genes enriched in neurodevelopmental biological processes. Corroborating these findings, DNAm at several top identified CpGs from the EWAS was associated with cortical surface area and IQ at 5.5 y (P < 0.05).

Conclusions: In-hospital percentage mother's milk intake by VLBW infants was associated with variations in DNAm of neurodevelopmental genes at 5.5 y; some of these DNAm variations are associated with brain structure and IQ.This trial was registered at as ISRCTN35317141 and at as NCT02759809.

Keywords: DNA methylation; epigenome-wide association study; mother's milk; neurodevelopment; very-low-birth-weight infants.

Publication types

  • Clinical Trial
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Child, Preschool
  • Cohort Studies
  • Cytosine
  • DNA Methylation*
  • Female
  • Guanine
  • Humans
  • Infant, Newborn
  • Infant, Very Low Birth Weight
  • Metallothionein
  • Milk, Human
  • Mothers*
  • Mouth Mucosa
  • Myosins
  • Ontario


  • Guanine
  • Cytosine
  • Metallothionein
  • Myosins

Associated data

  • ISRCTN/ISRCTN35317141