Enhanced Preprints

The influence of prenatal alcohol exposure and maternal diet on offspring DNA methylation: a cross-species study

  1. Mitchell Bestry
  2. Alexander N. Larcombe
  3. Nina Kresoje
  4. Emily K Chivers
  5. Chloe Bakker
  6. James P Fitzpatrick
  7. Elizabeth J Elliott
  8. Jeffrey M Craig
  9. Evelyne Muggli
  10. Jane Halliday
  11. Delyse Hutchinson
  12. Sam Buckberry
  13. Ryan Lister
  14. Martyn Symons
  15. David Martino author has email address
  1. Telethon Kids Institute, The University of Western Australia, Perth, Australia
  2. Respiratory Environmental Health, Wal-yan Respiratory Research Centre, Telethon Kids Institute, Nedlands, Western Australia, Australia
  3. Occupation, Environment and Safety, School of Population Health, Curtin University, Perth, Western Australia
  4. Telethon Kids Institute, 15 Hospital Ave, Nedlands, Western Australia, Australia
  5. School of Psychological Sciences, University of Western Australia, Perth, Australia
  6. University of Sydney, Faculty of Medicine and Health, Specialty of Child and Adolescent Health, Sydney, NSW 2006, Australia
  7. Sydney Children’s Hospitals Network (Westmead) and Kids Research, Sydney, NSW 2145, Australia
  8. Deakin University, IMPACT – the Institute for Mental and Physical Health and Clinical Translation, School of Medicine, Geelong, Australia
  9. Murdoch Children’s Research Institute, Royal Children’s Hospital, Flemington Road, Parkville, Victoria 3052, Australia
  10. Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Flemington Road, Parkville, Victoria 3052, Australia
  11. Public Health Genetics, Murdoch Children’s Research Institute, Parkville, VIC, Australia
  12. Deakin University, School of Psychology, Faculty of Health, Geelong, Australia
  13. Murdoch Children’s Research Institute, Centre for Adolescent Health, Royal Children’s Hospital, Melbourne, Australia
  14. University New South Wales, National Drug and Alcohol Research Centre, Sydney, Australia
  15. Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, WA 6009, Australia
  16. ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, WA 6009, Australia
  17. National Drug Research Institute, enAble Institute, Curtin University.
  18. Wal-yan Respiratory Research Centre, Telethon Kids Institute, Nedlands, Western Australia, Australia

Peer review process

Not revised: This Reviewed Preprint includes the authors’ original preprint (without revision), an eLife assessment, and public reviews.

Read more about eLife’s peer review process.

Editors

  • Reviewing Editor
    Weiwei Dang
    Baylor College of Medicine, Houston, United States of America
  • Senior Editor
    Carlos Isales
    Augusta University, Augusta, United States of America

Reviewer #1 (Public Review):

Summary:
This manuscript examined the impact of prenatal alcohol exposure on genome-wide DNA methylation in the brain and liver, comparing ethanol-exposed mice to unexposed controls. They also investigated whether a high-methyl diet (HMD) could prevent the DNA methylation alterations caused by alcohol. Using bisulfite sequencing (n=4 per group), they identified 78 alcohol-associated differentially methylated regions (DMRs) in the brain and 759 DMRs in the liver, of which 85% and 84% were mitigated by the HMD group, respectively. The authors further validated 7 DMRs in humans using previously published data from a Canadian cohort of children with FASD.

Overall, the findings from this study provide new insight into the impact of prenatal alcohol exposure, while also showing evidence for methyl-rich diets as an intervention to prevent the effects of alcohol on the epigenome. However, several methodological concerns limit the robustness of these results and should be addressed to further strengthen the conclusions of this study and its applicability to broader settings.

Strengths:
- The use of whole genome bisulfite sequencing allowed for the interrogation of the entire DNA methylome and DMR analysis, rather than a subset of CpGs.
- The combination of data from animal models and humans allowed the authors to make stronger inferences regarding their findings.
- The authors investigated a potential mechanism (high methyl diet) to buffer against the effects of prenatal alcohol exposure, which increases the relevance and applicability of this research.

Weaknesses:
- Mistakes and discontinuities in the reporting of results and methods made the manuscript difficult to follow. There was also some overuse of causal language and overinterpretation of differences.
- The authors provide insufficient details to replicate their analyses, particularly for data quality control steps and statistical analyses.
- The sample size was very small for the epigenetic analyses, which limits the robustness of the findings. This limitation is further emphasized by the cutoffs used to identify DMRs, which did not include multiple test corrections and used a delta cutoff that was not supported by the sequencing depth.
- The authors do not account for potential confounders in their analyses, including birthweight, alcohol levels, and sex. This is a particular problem for the high-methyl diet analyses, in which the alcohol-exposed mice seemed to consume less alcohol than their non-diet counterparts.

Reviewer #2 (Public Review):

Summary:
Bestry et al. investigated the effects of prenatal alcohol exposure (PAE) and high methyl donor diet (HMD) on offspring DNA methylation and behavioral outcomes using a mouse model that mimics common patterns of alcohol consumption in pregnancy in humans. The researchers employed whole-genome bisulfite sequencing (WGBS) for unbiased assessment of the epigenome in the newborn brain and liver, two organs affected by ethanol, to explore tissue-specific effects and to determine any "tissue-agnostic" effects that may have arisen prior to the germ-layer commitment during early gastrulation. The authors found that PAE induces measurable changes in offspring DNA methylation. DNA methylation changes induced by PAE coincide with non-coding regions, including enhancers and promoters, with the potential to regulate gene expression. Though the majority of the alcohol-sensitive differentially methylated regions (DMRs) were not conserved in humans, the ones that were conserved were associated with clinically relevant traits such as facial morphology, educational attainment, intelligence, autism, and schizophrenia. Finally, the study provides evidence that maternal dietary support with methyl donors alleviates the effects of PAE on DNA methylation, suggesting a potential prenatal care option.

Strengths:
The strengths of the study include the use of a mouse model where confounding factors such as genetic background and diet can be well controlled. The study performed whole-genome bisulfite sequencing which allows a comprehensive analysis of the effects of PAE on DNA methylation. However, some weaknesses and limitations of the study are detected.

Weaknesses:
1. The low generalizability between mouse and human data alerts the validity of the mouse model designed in the study. On the same note, the authors failed to detect any significant effect on PAE-induced behavioral outcomes. I recognize that it is difficult to model all possible conditions of PAE in mice because the amount, frequency, and duration of alcohol consumption in humans vary significantly. Therefore, if the authors only focus on moderate PAE, it should be emphasized in the title and throughout the paper to avoid misinterpretation. In addition, is it possible to stratify the human data based on the level of PAE and compare it to the mouse data?
2. A major finding of the study is that PAE affects non-coding genomic regions in mice including enhancers and promoters. To improve the significance of the study, the authors need to back up this finding with transcriptome analysis and determine if these DMRs indeed affect gene expression.
3. The low generalizability between mouse and human data suggests that the regions affected by PAE may be species-specific. It is critical to analyze if PAE-induced DMRs in humans are also enriched in non-coding genomic regions. Considering the huge difference between mouse and human development, particularly in the brain, it is not surprising that different genomic loci are affected, but the affected loci may share similar features.
4. The specific brain regions and the lobes of the liver where the samples were taken should be clearly indicated.
5. I don't fully agree with the authors' interpretation that the two shared genomic regions affected in the brain and the liver "must have arisen before the germ layers separated". To claim so, the authors need to exclude the possibility that the two regions are just a coincidence due to the stochastic effect of PAE on DNA methylation.

  1. Howard Hughes Medical Institute
  2. Wellcome Trust
  3. Max-Planck-Gesellschaft
  4. Knut and Alice Wallenberg Foundation