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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Genet. | doi: 10.3389/fgene.2019.00816

In epigenomic studies including cell type adjustments in regression models can introduce multicollinearity, resulting in apparent reversal of direction of association

  • 1MRC Lifecourse Epidemiology Unit (MRC), United Kingdom
  • 2Centre for Genetic Origins of Health and Disease, University of Western Australia, Australia
  • 3School of Pharmacy and Biomedical Sciences, Faculty of Health Sciences, Curtin University, Australia
  • 4Human Development & Health, Faculty of Medicine, University of Southampton, United Kingdom
  • 5Telethon Kids Institute, Australia
  • 6Faculty of Natural and Environmental Sciences, University of Southampton, United Kingdom
  • 7NIHR Southampton Biomedical Research Centre, University Hospital Southampton NHS Foundation Trust, United Kingdom

Background
Association studies of epigenome-wide DNA methylation and disease can inform biological mechanisms. DNA methylation is often measured in peripheral blood, with heterogeneous cell types with different methylation profiles. Influences such as adiposity-associated inflammation can change cell type proportions, altering measured blood methylation levels. To determine whether associations between loci-specific methylation and outcomes result from cellular heterogeneity many studies adjust for estimated blood cell proportions, but high correlations between methylation and cell type proportions could violate the statistical assumption of no multicollinearity. We examined these assumptions in a population-based study.
Methods
CDKN2A promoter CpG methylation was measured in peripheral blood from 812 adolescents aged 17-years (Western Australian RAINE mother-offspring cohort). Loge adolescent BMI was used as the outcome in a regression analysis with DNA methylation as predictor, adjusting for age/sex. Further regression analyses additionally adjusted for estimated cell type proportions using the reference-based Houseman method, and simulations modelled the effects of varying levels of correlation between cell proportions and methylation. Correlations between estimated cell proportions and CpG methylation from Illumina 450K were measured.
Results
Lower DNA methylation was associated with higher BMI when cell type adjustment was not included; for CpG4 β=-0.004 logeBMI/%methylation (95%CI -0.0065, -0.001; p=0.003). The direction of association reversed when adjustment for 6 cell types was made; for CpG4 β=0.004 logeBMI/%methylation (-0.0002, 0.0089; p=0.06). Correlations between CpG methylation and cell type proportions were high, and Variance Inflation Factors (VIFs) were extremely high (25 to 113.7). Granulocyte count was correlated with BMI, and removing granulocytes from the regression model reduced all VIFs to <3.1, with persistence of a positive association between methylation and BMI (CpG4 β=0.004 logeBMI/%methylation (-0.0002, 0.0088; p=0.06)). Simulations supported major effects of multicollinearity on regression results.
Conclusions
Where cell types are highly correlated with other covariates in regression models the statistical assumption of no multicollinearity may be violated. This can result in reversal of direction of association, particularly when examining associations with phenotypes related to inflammation, as CpG methylation may associate with changes in cell type proportions. Removing predictors with high correlations from regression models may remove the multicollinearity. However this might hinder biological interpretability.

Keywords: Epigenomics, Houseman cell-type adjustments , statistical assumptions, multicollinearity, reversal of direction of association, Illumina 450K - Illumina Infinium HumanMethylation450 array

Received: 05 Mar 2019; Accepted: 07 Aug 2019.

Copyright: © 2019 Barton, Melton, Titcombe, Murray, Rauschert, Lillycrop, Huang, Holbrook and Godfrey. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Dr. Sheila J. Barton, MRC Lifecourse Epidemiology Unit (MRC), Southampton, United Kingdom, S.J.Barton@soton.ac.uk