DNA methylation profiling in breast cancer discordant identical twins identifies DOK7 as novel epigenetic biomarker

Holger Heyn, F Javier Carmona, Antonio Gomez, Humberto J Ferreira, Jordana T Bell, Sergi Sayols, Kirsten Ward, Olafur A Stefansson, Sebastian Moran, Juan Sandoval, Jorunn E Eyfjord, Tim D Spector, Manel Esteller, Holger Heyn, F Javier Carmona, Antonio Gomez, Humberto J Ferreira, Jordana T Bell, Sergi Sayols, Kirsten Ward, Olafur A Stefansson, Sebastian Moran, Juan Sandoval, Jorunn E Eyfjord, Tim D Spector, Manel Esteller

Abstract

Using whole blood from 15 twin pairs discordant for breast cancer and high-resolution (450K) DNA methylation analysis, we identified 403 differentially methylated CpG sites including known and novel potential breast cancer genes. Confirming the results in an independent validation cohort of 21 twin pairs determined the docking protein DOK7 as a candidate for blood-based cancer diagnosis. DNA hypermethylation of the promoter region was also seen in primary breast cancer tissues and cancer cell lines. Hypermethylation of DOK7 occurs years before tumor diagnosis, suggesting a role as a powerful epigenetic blood-based biomarker as well as providing insights into breast cancer pathogenesis.

Figures

Fig. 1.
Fig. 1.
Differentially methylated CpG sites within MZ twin pairs discordant for breast cancer. (A) DNA methylation level of CpG sites identified by the Infinium 450K DNA methylation assay. Displayed are normalized beta-values of one representative example of discordant twins (990836 and 989697). (B) 403 differentially methylated CpG sites (bcDMP) within twins discordant for breast cancer identified by Wilcoxon signed rank test (P < 0.05) and represented as median beta-value differences (cancer-healthy twin). bcDMP were ranked by median beta-value difference. (C) Genomic distribution of bcDMPs regarding their respective location to genes and CpG context. (D) Delta methylation level (cancer-healthy twin) of a differentially methylated promoter CpG site of FGFR2 (cg12835048). (E) bcDMP varying epigenetically despite the identical genetic background of the twins identified by multivariate filter analysis. The red dot identifies a healthy sample within the cancer cluster. The bcDMP DNA methylation level is color coded (yellow: sample with lowest methylation level; red: sample with highest methylation level). (F) Hierarchical cluster of bcDMPs in six primary breast cancer pairs analyzed on the Infinium HumanMethylation450 BeadChip platform.
Fig. 2.
Fig. 2.
bcDMP associated genes are enriched in cancer associated pathways. Network analysis using GeneMANIA and ClueGO identified cancer-related KEGG pathways enriched in bcDMP associated genes.
Fig. 3.
Fig. 3.
DOK7 is hypermethylated in different breast cancer contexts. (A) Schematic overview of the DOK7 gene variants and associated features. Differentially methylated position (DMP; cg15652666) and associated region (DMR) are indicated. Asterisks are indicating transcription factor binding sites. (B) Intra-pair difference (cancer-healthy) of the DOK7 associated bcDMP in eight twins (identification set) postdiagnosis assessed by pyrosequencing. (C) Intra-pair difference (cancer-healthy) of the DOK7 associated bcDMP in 16 twins (validation set) postdiagnosis assessed by pyrosequencing. (D) Differences of DNA methylation (all 24 twin pairs postdiagnosis) of CpG site upstream of the bcDMP. Significant consistent differences comparing all twin pairs are indicated (*P < 0.05). The bcDMP highlighted (red box). (E) Unpaired analysis of twin samples comparing healthy and breast cancer blood samples. DNA methylation data were assessed by pyrosequencing, and significance between the groups is indicated (*P < 0.05). (F) Unpaired analysis of twin samples comparing CpG sites upstream of the bcDMP in healthy (white) and breast cancer (gray) blood samples. DNA methylation data were assessed by pyrosequencing, and significance between the groups is indicated (*P < 0.05). The bcDMP highlighted (red box) and outliers (black circles) were identified by the Tukey test. (G) Intra-pair difference (cancer-healthy) of the DOK7 associated bcDMP in all 11 twin pairs prediagnosis assessed by pyrosequencing. (H) Intra-pair difference (cancer-normal) of the DOK7 associated bcDMP in primary breast tumor samples and matched normal control tissue assessed by pyrosequencing. (I) Differences in DNA methylation of six breast cancer cell line displayed relative to the median level of six normal breast samples assessed by the Infinium HumanMethylation450 BeadChip platform.

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