156 Chapter 7 and vaccinated LSIL/HSIL cases. For the viral DNA methylation sites, only a few individuals had methylation of HPV33 L2 gene detected among vaccinated HSIL and control groups, 18.06 (SD ±11.37) and 14.15 (SD ±23.73), respectively. For the remaining HPV types (16, 18 and 31), no viral methylation was recorded. Figure 7.1 illustrates the differences in methylation levels of host-cell genes FAM19A4 and miR124-2 (Figure 7.1A and 7.1B); and EPB41L3 host-cell gene and the S5-classifier panel (Figure 7.1C and 7.1D). In HPV-vaccinated controls and LSIL/HSIL cases compared to the unvaccinated population, a dotted reference line was added to each figure to Methylation level (S5-classifier score) 0.0 0.5 1.0 5 10 15 Control LSIL HSIL 1.5 FAM19A4 (A) EPB41L3 (C) S5-classifier (D) miR124-2 (B) Methylation level (sqrt transformed ΔΔCq ratio) 0.0 0.5 1.0 5 10 15 Control LSIL HSIL 1.5 Methylation level (sqrt transformed ΔΔCq ratio) 0.0 0.5 1.0 5 10 15 Control LSIL HSIL 1.5 Methylation level (average) 0.0 0.5 1.0 5 10 15 Control LSIL HSIL 1.5 Figure 7.1 Methylation levels of FAM19A4, miR124-2, EPB41L3 and S5-classifier in cervical samples of HPV-vaccinated women. Methylation levels represented by boxplots of the square-root transformed ΔΔCq-ratios (y-axis) of A) FAM19A4 and B) miR124-2, and mean with standard deviation of C) the average EPB41L3 methylation and D) S5 methylation panel score (y-axis) in cervical samples stratified by study endpoint. The dotted lines in the figures illustrate the mean reference methylation level among unvaccinated women, the light pink dotted line for LSIL and dark pink dashed line for HSIL. Abbreviations: LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; sqrt, square-root; Cq, quantification cycle
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