Increased Correlation Between Methylation Sites In Epigenome-Wide Replication Studies: Impact on Analysis And Results

Abstract

Aim: To show that an increased correlation between CpGs after selection through an epigenome-wide association studies (EWAS) might translate into biased replication results. Methods: Pairwise correlation coefficients between CpGs selected in two published EWAS, the top hits replication, Bonferroni p-values, Benjamini–Hochberg (BH) false discovery rate (FDR) and directional FDR r-values were calculated in the NINFEA cohort data. Exposures’ random permutations were performed to show the empirical p-value distributions. Results: The average pairwise correlation coefficients between CpGs were enhanced after selection for the replication (e.g., from 0.12 at genome-wide level to 0.26 among the selected CpGs), affecting the empirical p-value distributions and the usual multiple testing control. Conclusion: Bonferroni and Benjamini–Hochberg FDR are inappropriate for the EWAS replication phase, and methods that account for the underlying correlation need to be used.

Keywords::

• bias
• correlation
• discovery study
• epigenetics
• EWAS
• replication study

Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at: https://www.tandfonline.com/doi/full/10.2217/epi-2017-0073

Acknowledgements

The authors are grateful to all the participants of the NINFEA birth cohort. The authors thank S Polidoro for her contributions to the epigenome-wide association study nested in the NINFEA cohort, and G Fiorito for his comments on an earlier version of the manuscript.

Financial & competing interest disclosure

The NINFEA study was partially funded by the Compagnia San Paolo Foundation. L Richiardi received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 733206, LIFE-CYCLE project. M Popovic was supported by the Erasmus Mundus for Western Balkans (ERAWEB II) program (reference number: E2.D2.14.270) and the present work is a part of her PhD thesis. A Biggeri was partially supported by MIUR ex 60% funds. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Disclaimer

This publication reflects only the author’s views and the funders are not liable for any use that may be made of the information contained therein.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Additional information

Funding

The NINFEA study was partially funded by the Compagnia San Paolo Foundation. L Richiardi received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 733206, LIFE-CYCLE project. M Popovic was supported by the Erasmus Mundus for Western Balkans (ERAWEB II) program (reference number: E2.D2.14.270) and the present work is a part of her PhD thesis. A Biggeri was partially supported by MIUR ex 60% funds. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.