Abstract P1-04-01: Epigenome-wide association study for breast cancer risk using whole genome and target captured bisulphite sequencing: A pooled case-control study nested in the breakthrough generations study
Authors: Flanagan JM , Curry E , Stirling L , Flower K , Orr N , Tomczyk K , Coulson P , Jones M , Ashworth A , Swerdlow A , Brown R , Garcia-Closas M 
Published: March 2017

Abstract
Background: The field of epigenetic epidemiology has rapidly advanced and recent work has discovered epigenetic markers of breast cancer risk in white blood cell (WBC) DNA. Using Epigenome-Wide Association Studies (EWAS) on the Illumina 450k methylation array, we and others have shown epigenome-wide hypomethylation (-0.2%, p&lt;2.2x10-16) in incident breast cancer cases compared with controls in several prospective cohorts. We have proposed a mechanism that involves cancer risk exposures, lifetime and environmental events, that alter the epigenome and stably modifies an individual's cancer risk. However, more work is needed to establish the clinical utility of this observation, the underlying causes of this variation and to determine whether the 1.7% of CpG sites targeted by the 450k array are representative of the remaining 98% of the epigenome that has not yet been interrogated. Our overall aim is to identify epigenetic traits within the epigenome that are associated with the risk of developing breast cancer.
Methods: We conducted an EWAS using whole genome bisulphite sequencing (WGBS) of WBC DNA from incident breast cancer cases (n=548) compared to matched controls (n=548) from a prospective cohort (Breakthrough Generations Study) using a DNA pooling approach. Eight DNA pools were prepared in sequencing libraries and sequenced on the Hiseq2500 at PE100bp reads, resulting in ~10-fold coverage per CpG, per library, across ~20 million mappable CpG sites. Each pooled sample was also analysed in triplicate on the Illumina 450k methylation array for validation. We used Agilent target capture bisulphite sequencing (TCBS) for technical validation in a subset of breast cancer cases (n=48) and matched controls (n=48), individually barcoded and sequenced on the MiSeq at PE150bp, aiming for &gt;1000 fold coverage of 425 kb targeted sequence.
Results: Interrogation of specific genomic regions showed that gene-body methylation averages tended to be hypomethylated in cases, while CpG island averages identified both hypo- and hypermethylation. We have validated the same direction of change in 40/51 CpG islands that were covered by the Illumina 450K methylation array and have developed a target capture panel for validation of 960 gene body regions and 224 CpG island regions that were identified as significantly different between cases and controls (average -11%, FDR&lt;5%). Analysis of TCBS data is ongoing.
Conclusions: Results indicate that epigenome-wide hypomethylation and methylation in specific sites, particularly gene bodies, measured in pre-diagnostic blood samples may be predictive of breast cancer risk, and may thus be useful as a risk biomarker.
Citation Format: Flanagan JM, Curry E, Stirling L, Flower K, Orr N, Tomczyk K, Coulson P, Jones M, Ashworth A, Swerdlow A, Brown R, Garcia-Closas M. Epigenome-wide association study for breast cancer risk using whole genome and target captured bisulphite sequencing: A pooled case-control study nested in the breakthrough generations study [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-04-01.