sodium-bisulfite and Colonic-Neoplasms

Whole-genome bisulfite sequencing (WGBS) is an approach of growing importance. It is the only approach that provides a comprehensive picture of the genome-wide DNA methylation profile. However, obtaining a sufficient amount of genome and read coverage typically requires high sequencing costs. Bioinformatics tools can reduce this cost burden by improving the quality of sequencing data. We have developed a statistical method Ajusted Local Kernel Smoother (AKSmooth) that can accurately and efficiently reconstruct the single CpG methylation estimate across the entire methylome using low-coverage bisulfite sequencing (Bi-Seq) data. We demonstrate the AKSmooth performance on the low-coverage (~ 4 ×) DNA methylation profiles of three human colon cancer samples and matched controls. Under the best set of parameters, AKSmooth-curated data showed high concordance with the gold standard high-coverage sample (Pearson 0.90), outperforming the popular analogous method. In addition, AKSmooth showed computational efficiency with runtime benchmark over 4.5 times better than the reference tool. To summarize, AKSmooth is a simple and efficient tool that can provide an accurate human colon methylome estimation profile from low-coverage WGBS data. The proposed method is implemented in R and is available at https://github.com/Junfang/AKSmooth.

Topics: Algorithms; Base Sequence; Chromosome Mapping; Colonic Neoplasms; CpG Islands; DNA Methylation; DNA, Neoplasm; Genome, Human; Humans; Molecular Sequence Data; Sequence Analysis, DNA; Software; Sulfites

Deleted in liver cancer‑1 (DLC‑1), a candidate tumor suppressor gene which is inactive in liver carcinogenesis, is located at 8p21.3, where deletions are frequently found in several types of human cancer. Promoter hypermethylation is an epigenetic mechanism leading to silencing of the gene expression, which may be the primary cause for the absence of DLC‑1. We investigated the expression of the DLC‑1 gene and the methylation of the DLC‑1 gene in colon cancer cell lines (Caco‑2, LoVo and HT‑29). The data showed that reduced or undetectable levels of DLC‑1 mRNA were found in HT‑29 by reverse transcription-polymerase chain reaction (RT‑PCR). By contrast, the DLC‑1 gene was significantly expressed in Caco‑2 and LoVo cells. These findings were in agreement with the data obtained from western blot analysis. To further determine whether aberrant methylation is a contributing factor to transcriptional inactivation of DLC‑1 in HT‑29, the methylation of promoter was examined using methylation‑specific PCR and sodium bisulfite genomic sequencing in LoVo and HT‑29 cells, which suggests that promoter hypermethylation accounts for silencing of the DLC‑1 gene in HT‑29 cells. Since DLC‑1 is a candidate tumor suppressor gene, we sought to determine whether DLC‑1 expression is associated with cell proliferation in colon cancer cell lines. RNA interference techniques were adopted to inhibit DLC‑1 expression in the LoVo cell line and resulted in inhibition of cell growth and reduced colony formation. Collectively, our observations suggest that hypermethylation is responsible for abrogating the function of the DLC‑1 gene in colon cancer and indicate a role of DLC‑1 in colon carcinogenesis.

Topics: Antioxidants; Blotting, Western; Cell Proliferation; Colonic Neoplasms; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; GTPase-Activating Proteins; Humans; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Sulfites; Tumor Cells, Cultured; Tumor Stem Cell Assay; Tumor Suppressor Proteins

Cytosine methylation provides an epigenetic level of cellular plasticity that is important for development, differentiation and cancerogenesis. We adopted microdroplet PCR to bisulfite treated target DNA in combination with second generation sequencing to simultaneously assess DNA sequence and methylation. We show measurement of methylation status in a wide range of target sequences (total 34 kb) with an average coverage of 95% (median 100%) and good correlation to the opposite strand (rho = 0.96) and to pyrosequencing (rho = 0.87). Data from lymphoma and colorectal cancer samples for SNRPN (imprinted gene), FGF6 (demethylated in the cancer samples) and HS3ST2 (methylated in the cancer samples) serve as a proof of principle showing the integration of SNP data and phased DNA-methylation information into "hepitypes" and thus the analysis of DNA methylation phylogeny in the somatic evolution of cancer.

Topics: Aged; Colonic Neoplasms; DNA Methylation; DNA, Neoplasm; Female; Fibroblast Growth Factor 6; Genetic Predisposition to Disease; Genome-Wide Association Study; Genome, Human; Haplotypes; Humans; Lymphoma, Follicular; Male; Middle Aged; Neoplasms; Phylogeny; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Sequence Analysis, DNA; snRNP Core Proteins; Sulfites; Sulfotransferases

Genome-wide DNA hypomethylation plays an important role in epigenomic and genomic instability and colorectal carcinogenesis. DNA methylation in the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of global DNA methylation level. In addition, LINE-1 hypomethylation in blood cells has been associated with colorectal adenoma risk, and LINE-1 hypomethylation in colorectal cancer is related with prognosis and linearly predicts shorter patient survival. However, no study has comprehensively evaluated the precision of sodium bisulfite conversion and PCR-pyrosequencing to measure LINE-1 methylation. Using 10 paraffin-embedded colon cancers, 5 matched normal colon mucosa, and 5 unrelated peripheral blood buffy coat leukocyte specimens, we enriched tumor DNA by macrodissection and laser capture microdissection. LINE-1 methylation was calculated as an average of 100 * C/(C + T) at 4 CpG sites after bisulfite-PCR-pyrosequencing. The LINE-1 methylation value in colon cancers varied, ranging approximately from 30 to 80. To measure assay precision, we performed bisulfite conversion on seven different DNA specimen aliquots and repeated PCR-pyrosequencing seven times. Run-to-run (between-run) SD ranged from 1.3 to 4.4 (median, 3.0) in macrodissected colon cancers; 1.1 to 10.5 (median, 3.8) in laser capture microdissection specimens; 1.3 to 2.5 (median, 1.9) in normal colon; and 1.5 to 3.4 (median, 1.9) in leukocyte DNA. In conclusion, bisulfite conversion and PCR-pyrosequencing assay can measure LINE-1 methylation in macrodissected colon cancer, normal colon, and blood DNA, and may be useful in clinical and research settings.

Topics: Blood Cells; Colon; Colonic Neoplasms; DNA Methylation; Genomic Instability; Humans; Intestinal Mucosa; Long Interspersed Nucleotide Elements; Risk Factors; Sensitivity and Specificity; Sequence Analysis, DNA; Sulfites

In this study, we adapted the well known uracil DNA glycosylase (UNG) carry-over prevention system for PCR, and applied it to the analysis of DNA methylation based on sodium bisulfite conversion. As sodium bisulfite treatment converts unmethylated cytosine bases into uracil residues, bisulfite treated DNA is sensitive to UNG treatment. Therefore, UNG cannot be used for carry-over prevention of PCR using bisulfite treated template DNA, as not only contaminating products of previous PCR, but also the actual template will be degraded. We modified the bisulfite treatment procedure and generated DNA containing sulfonated uracil residues. Surprisingly, and in contrast to uracil, 6-sulfonyl uracil containing DNA (SafeBis DNA) is resistant to UNG. We showed that the new procedure removes up to 10,000 copies of contaminating PCR product in a closed PCR vessel without significant loss of analytical or clinical sensitivity of the DNA methylation analysis.

Topics: Colonic Neoplasms; DNA; DNA Methylation; DNA, Neoplasm; Female; Humans; Male; Polymerase Chain Reaction; Sulfites; Uracil; Uracil-DNA Glycosidase

We report in silico identification and characterisation of a novel member of the ras association domain family 1 (RASSF1)/NORE1 family, namely, RASSF2, located at chromosomal region 20p13. It has three isoforms, all contain a ras association domain in the C-terminus. The longest isoform RASSF2A contains a 5' CpG island. RASSF2A was cloned from a brain cDNA library and directly sequenced, confirming the genomic gene structure. In previous reports, we and others have demonstrated that RASSF1A is epigenetically inactivated in a variety of cancers, including sporadic colorectal cancer (CRC). In the present report, we analysed the methylation status of RASSF2A promoter region CpG island in sporadic CRC and compared it to K-ras mutation status. RASSF2A promoter region CpG island was hypermethylated in a majority of colorectal tumour cell lines (89%) and in primary colorectal tumours (70%), while DNA from matched normal mucosa was found to be unmethylated (tumour-specific methylation). RASSF2A expression was reactivated in methylated tumour cell lines after treatment with 5-aza 2-deoxycytidine. RASSF2A methylation is an early event, detectable in 7/8 colon adenomas. Furthermore, 75% of colorectal tumours with RASSF2A methylation had no K-ras mutations (codons, 12 and 13) (P=0.048), Fisher's exact test). Our data demonstrate that RASSF2A is frequently inactivated in CRCs by CpG island promoter hypermethylation, and that epigenetic (RASSF2A) and genetic (K-ras) changes are mutually exclusive and provide alternative pathways for affecting Ras signalling.

Topics: Base Sequence; Brain; Cell Line, Tumor; Cloning, Molecular; Colonic Neoplasms; Colorectal Neoplasms; DNA Primers; Female; Gene Expression Regulation, Neoplastic; Gene Library; Humans; Male; Mutation; Neoplasm Staging; Promoter Regions, Genetic; Proteins; Sulfites; Tumor Suppressor Proteins

Aberrant DNA methylation at CpG dinucleotides can result in epigenetic silencing of tumour suppressor genes and represents one of the earliest events in tumourigenesis. To date, however, high-throughput tools that are capable of surveying the methylation status of multiple gene promoters have been restricted to a limited number of cytosines. Here, we present an oligonucleotide microarray that permits the parallel analysis of the methylation status of individual cytosines, thus combining high throughput and high resolution. The approach was used to study the CpG island in the promoter region of the tumour suppressor gene p16(INK4A). In total, 876 oligonucleotide probes of 21 nt in length were used to inspect the methylation status of 53 CpG dinucleotides, producing correct signals in colorectal cancer cell lines as well as control samples with a defined methylation status. The information was validated by established alternative methods. The overall methylation pattern was consistent for each cell line, while different between them. At the level of individual cytosines, however, significant variations between individual cells of the same type were found, but also consistencies across the panel of cancer cell lines were observed.

Topics: Base Sequence; Carcinoma; Cell Line, Tumor; Colonic Neoplasms; CpG Islands; DNA Methylation; Genes, p16; Genomics; Humans; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Oligonucleotide Probes; Polymerase Chain Reaction; Promoter Regions, Genetic; Sulfites