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. 2025 Apr 8;194(4):1012-1022.
doi: 10.1093/aje/kwae254.

Epigenome-wide differential methylation and differential variability as predictors of high-grade cervical intraepithelial neoplasia (CIN2+)

Alexandra Bukowski  1 , Cathrine Hoyo  2 , Misa Graff  1 , Nadja A Vielot  3 , Michael R Kosorok  4 , Wendy R Brewster  1   5 , Rachel L Maguire  2   6 , Susan K Murphy  6 , Belinda Nedjai  7 , Efthymios Ladoukakis  7 , Kari E North  1 , Jennifer S Smith  1   8
Affiliations

Epigenome-wide differential methylation and differential variability as predictors of high-grade cervical intraepithelial neoplasia (CIN2+)

Alexandra Bukowski et al. Am J Epidemiol. .

Abstract

CpG site methylation patterns have potential to improve differentiation of high-grade screening-detected cervical abnormalities. We assessed CpG differential methylation (DM) and differential variability (DV) in high-grade (CIN2+) vs low-grade (≤ CIN1) lesions. In ≤ CIN1 (n = 117) and CIN2+ (n = 31) samples, cervical sample DNA underwent testing with Illumina HumanMethylation arrays. We assessed DM and DV of CpG methylation M-values among 9 cervical cancer-associated genes. We fit CpG-specific linear models and estimated empirical Bayes standard errors and false discovery rates (FDRs). An exploratory epigenome-wide association study (EWAS) aimed to detect novel DM and DV CpGs (FDR < 0.05) and Gene Ontology (GO) term enrichment. Compared to ≤ CIN1, CIN2+ exhibited greater methylation at CCNA1 cluster 1 (M-value difference 0.24; 95% CI, 0.04-0.43) and RARB cluster 2 (0.16; 95% CI, 0.05-0.28), and lower methylation at CDH1 cluster 1 (-0.15; 95% CI, -0.26 to -0.04). CIN2+ exhibited lower variability at CDH1 cluster 2 (variation difference -0.24; 95% CI, -0.41 to -0.05) and FHIT cluster 1 (-0.30; 95% CI, -0.50 to -0.09). EWAS detected 3534 DM and 270 DV CpGs. Forty-four GO terms were enriched with DM CpGs related to transcriptional, structural, developmental, and neuronal processes. Methylation patterns may help triage screening-detected cervical abnormalities and inform US screening algorithms. This article is part of a Special Collection on Gynecological Cancer.

Keywords: biomarkers; cervical cancer; cervical intraepithelial neoplasia; epigenome-wide association study; methylation.

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Conflict of interest statement

J.S.S. has received research grants and consultancies from Hologic and Becton Dickinson for the past 5 years. The remaining authors (A.B., C.H., N.V., M.G., M.R.K., W.R.B., R.L.M., S.K.M., B.N., E.L., and K.E.N.) have no conflicts to disclose.

Figures

Figure 1
Figure 1
Flowchart for 148 CINCS participants included in analytic sample. Eligibility and inclusion criteria for a secondary data analysis of the Cervical Intraepithelial Neoplasia Cohort Study (CINCS), based in Durham, North Carolina, USA. Samples with greater amounts of stored sample were prioritized for methylation testing.
Figure 2
Figure 2
Epigenome-wide association study (EWAS) analysis: Manhattan plot of EWAS results for differentially methylated CpG sites between CIN2+ vs ≤ CIN1 samples. Unadjusted P values for association between continuous methylation levels at epigenome-wide CpGs and CIN2+ vs ≤ CIN1. Dashed horizontal line indicates the Bonferroni epigenome-wide significance level.
Figure 3
Figure 3
Epigenome-wide association study (EWAS) and Gene Ontology (GO) term enrichment: CpG sites and GO terms detected in differential methylation (DM) and differential variability (DV) EWAS analyses with false discovery rate < 0.05.
See this image and copyright information in PMC

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