Additional file 10 of Gender-affirming hormone therapy induces specific DNA methylation changes in blood
Creators: name: Shepherd, Rebecca
name: Bretherton, Ingrid
name: Pang, Ken
name: Mansell, Toby
name: Czajko, Anna
name: Kim, Bowon
name: Vlahos, Amanda
name: Zajac, Jeffrey D.
name: Saffery, Richard
name: Cheung, Ada
name: Novakovic, Boris
Publisher: name: figshare
Year: 2022
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 10: Fig. S1. Summary of covariates used in the EWAS analysis. A. PCA loadings in B. Heatmap highlighting the contribution of different variables to each principal component. Technical variation, such as location of sample in the EPIC array, was the biggest contributor to variation, followed by cell composition. The top number in each box within the heatmap is the correlation between principal component and data traits and underneath in brackets are the p values. Covariates with a high correlation and low p value are highlighted in red for positive correlations and blue for negative correlations. Fig. S2. A. Bar plot showing change in DNA methylation (Δβ) relative to baseline for top DMPs in the baseline vs 6 months or baseline vs 12 months comparison for transgender women. B. Bar plot showing change in DNA methylation (Δβ) or top DMPs in transgender men. C-F. Line plot showing DNA methylation changes per donor (different colour per donor) over time for selected DMPs. The mean DNA methylation level is shown as a bar plot. The X-axis refers to the GAHT group and time: TW = Transgender women, TM = Transgender men. Fig. S3. DNA methylation signature separates baseline from 6-month and 12-month GAHT samples. A. Scatter plot of change in DNA methylation relative to baseline for DMPs that are significant at both 6m and 12m following feminizing GAHT. There is a positive correlation between 6m-baseline Δβ (x axis) and 12m-baseline Δβ (y axis). B. Scatter plot of change in DNA methylation relative to baseline for DMPs that are significant at both 6m and 12m following masculinizing GAHT. There is a positive correlation between 6m-baseline Δβ (x axis) and 12m-baseline Δβ (y axis). C. Direction-of-change Venn diagram depicting overlap of 6m and 12m gain or loss of methylation DMPs in transgender women (TW). D. Direction-of-change Venn diagram depicting overlap of 6m and 12m gain or loss of methylation DMPs in transgender men (TM). In C. and D., we observe that overlapping DMPs show the same pattern of change (gain or loss). E. PCA Plot showing separation of samples based on DMPs in transgender women. A clear separation of 6m and 12m samples from baseline is observed on PC3. F. PCA Plot showing separation of samples based on DMPs in transgender men. A clear separation of 6m and 12m samples from baseline is observed on PC2. Fig. S4. DMP clusters identify temporal DNA methylation dynamics during GAHT. A. Boxplots showing DNA methylation level (z-score) for feminizing GAHT DMP clusters. B. Boxplots showing DNA methylation level (z-score) for masculinizing GAHT DMP clusters. K-means clustering identified 8 feminizing GAHT clusters and 6 masculinizing GAHT clusters. The dynamic nature of each cluster is labelled at the top of each boxplot and the number of DMPs in each cluster is displayed under the cluster description. The gene ontology terms associated with these clusters are shown in Table S3, while the DMPs that belong to each cluster are labelled in Table S1 and S2. TM = transgender men, TW = transgender women. Fig. S5. GAHT-associated DNA methylation change generally trends towards the profile of opposite assigned sex. A. PCA plot based on all DMPs identified in transgender women or transgender men comparisons. Individual donors are shown, as are the trajectory of group averages (open circles and triangles). In general, the two GAHT groups move in opposite directions on PC2, similarly to the pattern observed when only the 39 anti-correlating DMPs are used in Fig. S2. B. Scatter plot showing correlation between change in DNA methylation in transgender women 12m – baseline (x axis) and people assigned female at birth – people assigned male at birth (y axis). C. Scatter plot showing correlation between change in DNA methylation in transgender men at 12m – baseline (x axis) and people assigned male at birth – people assigned female at birth (y axis). Fig. S6. Overlap between GAHT DMPs and pregnancy and autoimmune disease-related DMPs. A. Overlap between GAHT DMPs identified in our study and a pregnancy cohort. Out of a total of 15 validated pregnancy-associated DMPs, 3 (20%) are significant in the feminizing GAHT group, while none are significant in the masculinizing GAHT group. This confirms that these DMPs are sensitive to oestrogen in both GAHT and third trimester pregnancy. B. Overlap between GAHT DMPs and rheumatoid arthritis-associated DMPs, only a 0.1% overlap was identified. Fig. S7. Summary of Differentially Methylated Regions (DMRs) associated with GAHT. A. Strategy for identifying DMRs using DMRcate. B. Summary of DMRs identified at 6 months and 12 months following GAHT in feminizing and masculinizing groups. C. Bar plots of 12-month DMRs in transgender men. Bar plots show change in DNA methylation (Δβ) after 6 months (light blue) and 12 months (dark blue) of masculinizing GAHT relative to baseline in transgender men. D. Bar plots of 6-month DMRs in transgender women. Bar plots show change in DNA methylation (Δβ) after 6 months (pink) and 12 months (red) of feminizing GAHT relative to baseline in transgender women. E. Bar plots of 12-month DMRs in transgender women. No DMRs were identified in the 6-month comparison in transgender men. Fig. S8. Effect of GAHT on sex-specific DNA methylation patterns on autosomes and X chromosome. Boxplots of DNA methylation (z-scored) of sex-specific autosomal DMPs (A), and X chromosome DMPs (baseline transgender men vs baseline transgender women Δβ &gt; 0.05 or &lt; -0.05 and p value &lt;0.01) (B and C). The first panels (left) are DMPs that are not influenced by GAHT, while centre and right panels are DMPs that are significant in transgender women and transgender men 12-month vs baseline comparisons, respectively (Δβ &gt; 0.02 or &lt; -0.02, and p value &lt; 0.05). Fig. S9. Effect of GAHT on DNA methylation levels at the XIST locus. Bar plot shows the mean level of DNA methylation in transgender women and transgender men at baseline, 6 months, and 12 months into GAHT. Both feminizing and masculinizing GAHT had no effect on XIST DNA methylation. Fig. S10. Direction of change at sex-specific DMPs that are affected by GAHT. Bar plots showing change in DNA methylation (Δβ) for (A) sex-specific DMPs that show gain of methylation in people assigned female at birth (red) and are significant in the transgender men (12-month masculinizing GAHT) comparison (blue) and (B) sex-specific DMPs that show gain of methylation in people assigned female at birth and are significant in the transgender women (12-month feminizing GAHT) comparison (pink). C. Change in DNA methylation (Δβ) for sex-specific DMPs that show loss of methylation in people assigned female at birth and are significant in the transgender men (12-month masculinizing GAHT) comparison and (D) sex-specific DMPs that show loss of methylation in people assigned female and are significant in the transgender women (12-month feminizing GAHT) comparison. Common probes are highlighted, with the corresponding gene name.