Methylation profiling of 48 candidate genes in tumor and matched normal tissues from breast cancer patients

Abstract

Gene-specific methylation alterations in breast cancer have been suggested to occur early in tumorigenesis and have the potential to be used for early detection and prevention. The continuous increase in worldwide breast cancer incidences emphasizes the urgent need for identification of methylation biomarkers for early cancer detection and patient stratification. Using microfluidic PCR-based target enrichment and next-generation bisulfite sequencing technology, we analyzed methylation status of 48 candidate genes in paired tumor and normal tissues from 180 Chinese breast cancer patients. Analysis of the sequencing results showed 37 genes differentially methylated between tumor and matched normal tissues. Breast cancer samples with different clinicopathologic characteristics demonstrated distinct profiles of gene methylation. The methylation levels were significantly different between breast cancer subtypes, with basal-like and luminal B tumors having the lowest and the highest methylation levels, respectively. Six genes (ACADL, ADAMTSL1, CAV1, NPY, PTGS2, and RUNX3) showed significant differential methylation among the 4 breast cancer subtypes and also between the ER +/ER- tumors. Using unsupervised hierarchical clustering analysis, we identified a panel of 13 hypermethylated genes as candidate biomarkers that performed a high level of efficiency for cancer prediction. These 13 genes included CST6, DBC1, EGFR, GREM1, GSTP1, IGFBP3, PDGFRB, PPM1E, SFRP1, SFRP2, SOX17, TNFRSF10D, and WRN. Our results provide evidence that well-defined DNA methylation profiles enable breast cancer prediction and patient stratification. The novel gene panel might be a valuable biomarker for early detection of breast cancer.

Next-Generation Sequencing in Oncology: Genetic Diagnosis, Risk Prediction and Cancer Classification

“…DNA methylation has been assessed by ChIP-seq using chromatin methyl-binding-domain immunoprecipitation [304], or by applying NGS directly to bisulphite-treated DNA, as recently used to characterise the methylation profile in breast cancer patients and controls [305]. Additional epigenetic events like microRNA expression can be characterised by NGS [306] as also demonstrated by The Cancer Genome Atlas [225].…”

Integrated analysis of promoter methylation and expression of telomere related genes in breast cancer

“…In addition to the 29 genes we studied here, 6 more telomere related genes ( CTC1, MRE11, TRF2, UPF, TIN2 , and TNKS2 ) were beyond the online software's parameters for promoter methylation primer design and were excluded from the study. The other two crucial genes TERT and WRN have been studied in our previous methylation and expression analysis [40, 41]. To our knowledge, this is the first comprehensive study of methylation on a large number (29) of telomere related genes in a relative large number of samples (184 breast cancer patients).…”

“…DNA and total RNA was extracted from fresh frozen tissue samples as previously described [40, 41]. Sodium bisulfite conversion of 500 ng genomic DNA was carried out using the EZ DNA Methylation-Lightning TM Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's instruction.…”

“…These genes involved in telomere maintenance, as well as genes that are transiently associated with the telomere, and genes involved in DNA repair and helicase genes [56–60]. TERT and WRN have been studied in our previous methylation/expression studies [40, 41] and excluded from analysis here. The 5′ promoter sequence of candidate genes was obtained from UCSC genome browser (http://genome.ucsc.edu/cgi-bin/hgGateway).…”

“…As our previous studies, the 48.48 Access Array TM system (Fluidigm, South San Franciso, CA, USA) and Access Array Barcode Library was used for target enrichment and sequencing libraries construction [40, 41]. The product size distribution was examined by Agilent Bioanalyzer 2100.…”