Abstract
BackgroundThe number of tumor suppressor genes for which germline mutations have been linked to cancer risk is steadily increasing. However, while recent reports have linked constitutional normal tissue promoter methylation of BRCA1 and MLH1 to ovarian and colon cancer risk, the role of epigenetic alterations as cancer risk factors remains largely unknown, presenting an important area for future research. Currently, we lack fast and sensitive methods for assessment of promoter methylation status across known tumor suppressor genes.ResultsIn this paper, we present a novel NGS-based approach assessing promoter methylation status across a large panel of defined tumor suppressor genes to base-pair resolution. The method omits the limitations related to commonly used array-approaches. Our panel includes 565 target regions covering the promoters of 283 defined tumor suppressors, selected by pre-specified criteria, and was applied for rapid targeted methylation-specific NGS. The feasibility of the method was assessed by analyzing normal tissue DNA (white blood cells, WBC) samples from 34 healthy postmenopausal women and by performing preliminary assessment of the methylation landscape of tumor suppressors in these individuals. The mean target coverage was 189.6x providing a sensitivity of 0.53%, sufficient for promoter methylation assessment of low-level methylated genes like BRCA1. Within this limited test-set, we detected 206 regions located in the promoters of 149 genes to be differentially methylated (hyper- or hypo-) at > 99% confidence level. Seven target regions in gene promoters (CIITA, RASSF1, CHN1, PDCD1LG2, GSTP1, XPA, and ZNF668) were found to be hyper-methylated in a minority of individuals, with a > 20 percent point difference in mean methylation across the region between individuals. In an exploratory hierarchical clustering analysis, we found that the individuals analyzed may be grouped into two main groups based on their WBC methylation profile across the 283 tumor suppressor gene promoters.ConclusionsMethylation-specific NGS of our tumor suppressor panel, with detailed assessment of differential methylation in healthy individuals, presents a feasible method for identification of novel epigenetic risk factors for cancer.
Highlights
The number of tumor suppressor genes for which germline mutations are linked to elevated cancer risk is steadily increasing [1,2,3]
Some of the best described “classical” high penetrance genes include BRCA1/2, for which germline mutations are associated with an elevated risk of ovarian and breast cancer [6], MLH1/MSH2 linked to colorectal cancer [7], CDKN2A and RB1, associated with melanoma and retinoblastoma, respectively [8,9,10], as well as TP53, associated with the Li-Fraumeni syndrome with an elevated risk for multiple cancer forms [11]
We show the feasibility of the method by depicting promoter methylation variation across the promoter panel in a set of white blood cell (WBC) DNA obtained from 34 healthy individuals
Summary
The number of tumor suppressor genes for which germline mutations are linked to elevated cancer risk is steadily increasing [1,2,3]. Some of the best described “classical” high penetrance genes include BRCA1/2, for which germline mutations are associated with an elevated risk of ovarian and breast cancer [6], MLH1/MSH2 linked to colorectal cancer [7], CDKN2A and RB1, associated with melanoma and retinoblastoma, respectively [8,9,10], as well as TP53, associated with the Li-Fraumeni syndrome with an elevated risk for multiple cancer forms [11]. The number of tumor suppressor genes for which germline mutations have been linked to cancer risk is steadily increasing. We lack fast and sensitive methods for assessment of promoter methylation status across known tumor suppressor genes
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
