Differential DNA methylation analysis optimally requires purified cell populations


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Volume 106, Issue 3, Page 551


Michael K. Skinner, Ph.D.


Reflections on "Assisted reproductive technologies alter DNA methylation profiles in bloodspots of newborn infants" by Estill et al.

Read the full text here.

Fertility and Sterility

Editorial Office, American Society for Reproductive Medicine

Fertility and Sterility® is an international journal for obstetricians, gynecologists, reproductive endocrinologists, urologists, basic scientists and others who treat and investigate problems of infertility and human reproductive disorders. The journal publishes juried original scientific articles in clinical and laboratory research relevant to reproductive endocrinology, urology, andrology, physiology, immunology, genetics, contraception, and menopause. Fertility and Sterility® encourages and supports meaningful basic and clinical research, and facilitates and promotes excellence in professional education, in the field of reproductive medicine.


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Molly Estill about 4 years ago

Abstract: The present study identified genomic loci influenced by ART highlighting the potential contribution of metastable epialleles and other alleles as they respond to cryopreservation. The precise determination of the state of DNA methylation in any tissue is dependent on the composition of the population cells as highlighted in blood. This is a particular challenge when using newborn bloodspot resources but provides a window to examining a host of mechanisms.

Main text:
The editorial by Dr. Skinner (1), addresses the impact of blood cell populations on epigenetics, particularly methylation, as one of the factors responsible for directing the cellular phenotype. Archived newborn bloodspots (Guthrie cards) are a unique resource for use in epidemiological studies, allowing retrospective analysis of DNA, RNA, and pre-natal chemical exposures. They are particularly relevant in context of understanding the influence of fetal experience on adult health.

In the present study (2), blood cell proportions were inferred using the methods employed by Jaffe and Irizarry (3). This method, which is currently implemented in the minfi R package, was designed using sorted blood cell populations of adults. This is the only method available to infer blood cell composition from methylation microarrays (e.g. Infinium HumanMethylation450 Beadchip) of newborn samples in the absence of a verified methylome of the different blood cell types present in infants.

One must consider that discordance between the inferred populations and observed populations in very young children has been observed (4). Despite this challenge, the 450k methylation profiles of the samples used in this study were not significantly influenced by the inferred blood cell composition (2). Notably, the metastable epialleles (MEs) described are established in the early embryo, prior to tissue differentiation and their methylation is known to be consistent across diverse tissues ranging from blood to hair follicles (5, 6). Hence, any uncertainties in leukocyte composition do not appear to underlie the group differences in ME methylation observed.

On the practical use of archived bloodspots, it is important to note that the efficiency of DNA extraction from bloodspots, particularly those archived for many years, can be poor. Therefore, it may be untenable to experimentally measure the blood cell proportions measured using a gold standard technique, such as direct cell count (7). Concentrated efforts characterizing the methylome of newborn whole blood are required to develop bioinformatics methods that best infer blood cell populations from methylation data of newborn whole blood. This will serve as a prelude to clinical trials that will address the influence of various aspects of ART and infertility on the epigenome of children impacting fetal, childhood and adult health.

1. Skinner MK. Differential DNA Methylation Analysis Optimally Requires Purified Cell Populations. Fertility and Sterility 2016;In Press.
2. Estill M, Bolnick J, Waterland R, Bolnick A, Diamond M, Krawetz S. Assisted Reproductive Technologies Alter DNA Methylation Profiles in Bloodspots of Newborn Infants. Fertility and Sterility 2016;In press.
3. Jaffe AE, Irizarry RA. Accounting for cellular heterogeneity is critical in epigenome-wide association studies. Genome Biology 2014;15:1-9.
4. Yousefi P, Huen K, Quach H, Motwani G, Hubbard A, Eskenazi B et al. Estimation of blood cellular heterogeneity in newborns and children for epigenome-wide association studies. Environmental and Molecular Mutagenesis 2015;56:751-8.
5. Dominguez-Salas P, Moore SE, Baker MS, Bergen AW, Cox SE, Dyer RA et al. Maternal nutrition at conception modulates DNA methylation of human metastable epialleles. Nature Communications 2014;5:3746.
6. Silver M, Hennig B, Dominguez-Salas P, Waterland R. Periconceptional environment affects epigenetic metastability at VTRNA2-1, a non-coding RNA with tumor suppressor activity. Genome Biology 2015;In Press.
7. Zaccaria A, Celso B, Raspadori D, Motta M, Testoni N, Rizzi S. Comparative evaluation of differential leukocyte counts by Coulter VCS cytometer and direct microscopic observation. Haematologica 1990;75:412-9.