Milea Timbergen

101 and T41A samples. Most genes, with the exception of DUX4L6, present with DMRs in the gene body. The DMRs and their location were visualized by loading the MeD-seq data into the Integrative Genomics Viewer (IGV) (Supplemental Figure 4A-E). The IGV graphs present the methylation patterns in a quantitative way in each DTF sample that was analysed. It was observed that the methylation profiles within a CTNNB1 mutant class may differ considerable, with samples displaying almost a complete absence of methylation whereas other samples appear (partly) methylated. Figure 2 depicts the actual normalized read counts of the DMRs and associated genes detected between S45F and T41A DTF samples. A more stringent statistical analysis of the raw data revealed that only the DMR associated with CCDC6 remained statistically significant (p = 0.04). Table 2. Overview of DMRs with a fold change ≥ 1.5 between S45F and T41A DTF. Chromosome Fold change Position (start – end) Methylation status Over- lapping genes Position S45F T41A 19 2.68 55850028-55850071 + - NLRP4 postTSS1KB-TES 17_GL383563v3_alt 2.27 130411-131133 - + - 17 1.85 82585430-82587461 - + FOXK2 postTSS1KB-TES 16 1.79 85410202-85410284 + - - 1 1.77 977947-977974 - + PERM1 postTSS1KB-TES 21 42956670-42958541 + - - 10 1.70 59882022-59883084 - + CCDC6 postTSS1KB-TES 10 1.69 42089321-42090999 + - - 12 1.65 132149685-132150607 + - NOC4L postTSS1KB-TES 4 1.50 190075267-133745860 + - DUX4L6 TSS DMRs located on sex (X- and Y) chromosomes were excluded; TSS, Transcription Start Site; TES, Transcription End Site; postTSS1KB-TES, indicates the region starting at 1 Kb after the TSS till the TES thus corresponding to the gene body without promoter region. - hypomethylation, + hypermethylation Effects of DMRs on gene expression levels Next, the effect of the initially detected DMRs with a fold change ≥ 1.5 on the expression of the associated genes was examined. DMR associated genes NLRP4, FOXK2, PERM1, CCDC6, NOC4L and DUX4L6 were identified in a publicly available mRNA expression dataset of 34 S45F and 45 T41A DTF samples. It was noted that the same genes are detected by multiple probes on the Affymetrix platform used (Table 3). A Mann-Whitney U test indicated that no significant expression differences were observed between S45F and T41A mutant DTF for most genes with the exception of CCDC6 (p = 0.034, 1 out of 2 probes) and FOXK2 (p = 0.004, 1 out of 4 probes) (Table 3). It was noted that the expression of CCDC6 4