DNA methylation profiling to assess pathogenicity of BRCA1 unclassified variants in breast cancer
ABSTRACT: The Illumina Infinium 450k Human DNA methylation Beadchip was used to obtain DNA methylation profiles across 485,577 CpGs . Samples were restored FFPE DNA extracted from breast tumours in 3 groups; BRCA1 germline mutated tumours (BRCA1), BRCA1 germline wildtype tumours from women from high risk families (BRCAx) and the designated test variant tumours (BRCA1UV). Bisulphite converted DNA was hybridised to the Illumina Infinium 450k Human Methylation Beadchip
Project description:DNA methylation is the best known epigenetic mark. Cancer and other pathologies show an altered DNA methylome. However, delivering complete DNA methylation maps is compromised by the price and labor-intensive interpretation of single nucleotide methods.Following the success of the HumanMethylation450 BeadChip (Infinium) methylation microarray (450K), we report the technical and biological validation of the newly developed MethylationEPIC BeadChip (Infinium) microarray that covers over 850,000 CpG methylation sites (850K). The 850K microarray contains >90% of the 450K sites, but adds 333,265 CpGs located in enhancer regions identified by the ENCODE and FANTOM5 projects.The 850K array demonstrates high reproducibility at the 450K CpG sites, is consistent among technical replicates, is reliable in the matched study of fresh frozen versus formalin-fixed paraffin-embeded samples and is also useful for 5-hydroxymethylcytosine. These results highlight the value of the MethylationEPIC BeadChip as a useful tool for the analysis of the DNA methylation profile of the human genome.
Project description:Breast cancers arising in women carrying a germline mutation in BRCA1 are typically high-grade, early-onset and have distinct morphological features (BRCA1-like). However, the majority of early-onset breast cancers of this morphological type are not associated with germline BRCA1 mutations or constitutional BRCA1 promoter methylation. We aimed to assess DNA methylation across the genome for associations with the "BRCA1-like" morphology. Genome-wide methylation in blood-derived DNA was measured using the Infinium HumanMethylation450K BeadChip assay for women under the age of 40?years participating in the Australian Breast Cancer Family Study (ABCFS) diagnosed with: i) BRCA1-like breast cancer (n?=?30); and ii) breast cancer without BRCA1-like morphological features (non BRCA1-like; n?=?30), and age-matched unaffected women (controls; n?=?30). Corresponding tumour-derived DNA from 43 of the affected women was also assessed. Methylation of blood-derived DNA was found to be elevated across 17 consecutive marks in the BRCA1 promoter region and decreased at several other genomic regions (including TWIST2 and CTBP1) for 7 women (23%) diagnosed with BRCA1-like breast cancer compared with women in the other groups. Corresponding tumour-derived DNA available from 5 of these 7 women had elevated methylation within the BRCA1 and SPHK2 promoter region and decreased methylation within the ADAP1, IGF2BP3 and SPATA13 promoter region when compared with the other breast tumours. These methylation marks could be biomarkers of risk for BRCA1-like breast cancer, and could be responsible in part for their distinctive morphological features and biology. As such, they may assist with prevention and targeted therapies for this cancer subtype.
Project description:Germline pathogenic mutations in BRCA1 increase risk of developing breast cancer. Screening for mutations in BRCA1 frequently identifies sequence variants of unknown pathogenicity and recent work has aimed to develop methods for determining pathogenicity. We previously observed that tumor DNA methylation can differentiate BRCA1-mutated from BRCA1-wild type tumors. We hypothesized that we could predict pathogenicity of variants based on DNA methylation profiles of tumors that had arisen in carriers of unclassified variants. We selected 150 FFPE breast tumor DNA samples [47 BRCA1 pathogenic mutation carriers, 65 BRCAx (BRCA1-wild type), 38 BRCA1 test variants] and analyzed a subset (n=54) using the Illumina 450K methylation platform, using the remaining samples for bisulphite pyrosequencing validation. Three validated markers (BACH2, C8orf31, and LOC654342) were combined with sequence bioinformatics in a model to predict pathogenicity of 27 variants (independent test set). Predictions were compared with standard multifactorial likelihood analysis. Prediction was consistent for c.5194-12G>A (IVS 19-12 G>A) (P>0.99); 13 variants were considered not pathogenic or likely not pathogenic using both approaches. We conclude that tumor DNA methylation data alone has potential to be used in prediction of BRCA1 variant pathogenicity but is not independent of estrogen receptor status and grade, which are used in current multifactorial models to predict pathogenicity.
Project description:Aging is characterized by a profound remodeling of the epigenetic architecture in terms of DNA methylation patterns. To date the most effective tool to study genome wide DNA methylation changes is Infinium HumanMethylation450 BeadChip (Infinium 450k). Despite the wealth of tools for Infinium 450k analysis, the identification of the most biologically relevant DNA methylation changes is still challenging. Here we propose an analytical pipeline to select differentially methylated regions (DMRs), tailored on microarray architecture, which is highly effective in highlighting biologically relevant results. The pipeline groups microarray probes on the basis of their localization respect to CpG islands and genic sequences and, depending on probes density, identifies DMRs through a single-probe or a region-centric approach that considers the concomitant variation of multiple adjacent CpG probes. We successfully applied this analytical pipeline on 3 independent Infinium 450k datasets that investigated age-associated changes in blood DNA methylation. We provide a consensus list of genes that systematically vary in DNA methylation levels from 0 to 100 years and that have a potentially relevant role in the aging process.
Project description:The Infinium Human Methylation450 BeadChip Array (Infinium 450K) is a robust and cost-efficient survey of genome-wide DNA methylation patterns. Macaca fascicularis (Cynomolgus macaque) is an important disease model; however, its genome sequence is only recently published, and few tools exist to interrogate the molecular state of Cynomolgus macaque tissues. Although the Infinium 450K is a hybridization array designed to the human genome, the relative conservation between the macaque and human genomes makes its use in macaques feasible. Here, we used the Infinium 450K array to assay DNA methylation in 11 macaque muscle biopsies. We showed that probe hybridization efficiency was related to the degree of sequence identity between the human probes and the macaque genome sequence. Approximately 61% of the Human Infinium 450K probes could be reliably mapped to the Cynomolgus macaque genome and contain a CpG site of interest. We also compared the Infinium 450K data to reduced representation bisulfite sequencing data generated on the same samples and found a high level of concordance between the two independent methodologies, which can be further improved by filtering for probe sequence identity and mismatch location. We conclude that the Infinium 450K array can be used to measure the DNA methylome of Cynomolgus macaque tissues using the provided filters. We also provide a pipeline for validation of the array in other species using a simple BLAST-based sequence identify filter.
Project description:We performed a two-stage molecular epidemiological study to explore DNA methylation profiles for potential biomarkers of esophageal squamous cell carcinoma (ESCC) in a Chinese population. Infinium Methylation 450K BeadChip was used to identify genes with differentially methylated CpG sites. Sixteen candidate genes were validated by sequencing 1160 CpG sites in their promoter regions using the Illumina MiSeq platform. When excluding sites with negative changes, 10 genes (BNIP3, BRCA1, CCND1, CDKN2A, HTATIP2, ITGAV, NFKB1, PIK3R1, PRDM16 and PTX3) showed significantly different methylation levels among cancer lesions, remote normal-appearing tissues, and healthy controls. PRDM16 had the highest diagnostic value with the AUC (95% CI) of 0.988 (0.965-1.000), followed by PIK3R1, with the AUC (95% CI) of 0.969 (0.928-1.000). In addition, the methylation status was higher in patients with advanced cancer stages. These results indicate that aberrant DNA methylation may be a potential biomarker for the diagnosis of ESCC.
Project description:DNA methylation can mimic the effects of both germline and somatic mutations for cancer predisposition genes such as BRCA1 and p16INK4a. Constitutional DNA methylation of the BRCA1 promoter has been well described and is associated with an increased risk of early-onset breast cancers that have BRCA1-mutation associated histological features. The role of methylation in the context of other breast cancer predisposition genes has been less well studied and often with conflicting or ambiguous outcomes. We examined the role of methylation in known breast cancer susceptibility genes in breast cancer predisposition and tumor development. We applied the Infinium HumanMethylation450 Beadchip (HM450K) array to blood and tumor-derived DNA from 43 women diagnosed with breast cancer before the age of 40 years and measured the methylation profiles across promoter regions of BRCA1, BRCA2, ATM, PALB2, CDH1, TP53, FANCM, CHEK2, MLH1, MSH2, MSH6 and PMS2. Prior genetic testing had demonstrated that these women did not carry a germline mutation in BRCA1, ATM, CHEK2, PALB2, TP53, BRCA2, CDH1 or FANCM. In addition to the BRCA1 promoter region, this work identified regions with variable methylation at multiple breast cancer susceptibility genes including PALB2 and MLH1. Methylation at the region of MLH1 in these breast cancers was not associated with microsatellite instability. This work informs future studies of the role of methylation in breast cancer susceptibility gene silencing.
Project description:We examined concordance of methylation levels across the Illumina Infinium HumanMethylation450 BeadChip and the Infinium MethylationEPIC BeadChip.We computed the correlation for 145 whole blood DNA samples at each of the 422,524 CpG sites measured by both chips.The correlation at some sites was high (up to r = 0.95), but many sites had low correlation (55% had r < 0.20). The low correspondence between 450K and EPIC measured methylation values at many loci was largely due to the low variability in methylation values for the majority of the CpG sites in blood.Filtering out probes based on the observed correlation or low variability may increase reproducibility of BeadChip-based epidemiological studies.
Project description:DNA methylation analysis has become an integral part of biomedical research. For high-throughput applications such as epigenome-wide association studies, the Infinium HumanMethylation450 (450K) BeadChip is currently the platform of choice. However, BeadChip processing relies on traditional bisulfite (BS) based protocols which cannot discriminate between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Here, we report the adaptation of the recently developed oxidative bisulfite (oxBS) chemistry to specifically detect both 5mC and 5hmC in a single workflow using 450K BeadChips, termed oxBS-450K. Supported by validation using mass spectrometry and pyrosequencing, we demonstrate reproducible (R(2)>0.99) detection of 5hmC in human brain tissue using the optimised oxBS-450K protocol described here.
Project description:The Infinium Human Methylation450 BeadChip Array (TM) (Infinium 450K) is an important tool for studying epigenetic patterns associated with disease. This array offers a high-throughput, low cost alternative to more comprehensive sequencing-based methodologies. Here we compare data generated by interrogation of the same seven clinical samples by Infinium 450K and reduced representation bisulfite sequencing (RRBS). This is the largest data set comparing Infinium 450K array to the comprehensive RRBS methodology reported so far. We show good agreement between the two methodologies. A read depth of four or more reads in the RRBS data was sufficient to achieve good agreement with Infinium 450K. However, we observe that intermediate methylation values (20-80%) are more variable between technologies than values at the extremes of the bimodal methylation distribution. We describe careful processing of Infinium 450K data to correct for known limitations and batch effects. Using methodologies proposed by others and newly implemented and combined in this report, agreement of Infinium 450K data with independent techniques can be vastly improved.