Number Of Methylated CpG Sites Research Articles

Aberrant DNA Methylation: Have We Entered the Era of More Than One Type of Colorectal Cancer?

It has been known for over two decades that DNA from colorectal cancer (CRC) is hypomethylated compared to that from normal colonic epithelia. 1,2 More recently it has also been recognized that specific CpG dinucleotide islands are often hypermethylated in this tumor type, 3 giving rise to the term CpG island methylator phenotype, or CIMP+. 4 Hypomethylation may contribute to the activation of oncogenes, whereas CpG-island specific hypermethylation has been linked to the transcriptional silencing of tumor suppressor genes. The relationship between these two epigenetic phenomena has not been examined, however, in CRC. Intuitively one might expect that tumors with severe global DNA hypomethylation might also show a lower frequency of CpG island hypermethylation due to a lower overall content of methyl groups. The meticulous study by Bariol et al 5 in this issue of The American Journal of Pathology reveals this not to be the case. No association was apparent between global 5-methyl cytosine content and the number of methylated CpG sites in a relatively large and representative series of 97 premalignant and malignant lesions. With only one exception, all of the 22 CRCs investigated showed a lower 5-methyl cytosine content compared to normal mucosa from the same patient. Moreover, approximately 30 to 40% of hyperplastic polyps, adenomas, and CRCs showed no methylation at any of the five CpG sites examined. This most recent study by the Ward laboratory follows on from their previous work in characterizing CIMP+ CRCs. 6 It is also relevant to the notion put forward by these and other workers that CIMP+ and the closely related tumor subgroup with microsatellite instability (MSI+) originate in large hyperplastic polyps and serrated adenomas. 7,8 Increasingly, it is becoming recognized that tumors with widespread DNA hypermethylation have distinctive clinicopathological and molecular features. 4,6,9 Although the study by Bariol et al 5 emphasizes and probably serves to justify the current interest in hypermethylation as opposed to hypomethylation, there is clearly much more to be learned about the general role of aberrant DNA methylation in the development and progression of CRC.

Heterogeneity in the methylation status of genomic DNA fragments demonstrating similar elution profiles in methyl-CpG binding domain column chromatography

Methyl-CpG binding domain column chromatography enables the separation of DNA fragments on the basis of the number of methylated CpG sites within the fragment. However, the exact elution profile of a specific DNA fragment is unpredictable. In order to address this problem, we have investigated the methylation status of genomic DNA fragments having similar elution profiles. We found that genomic DNA fragments that had high affinity to the column included those with high-density methylation, moderate methylation, and monoallelic methylation. Some DNA fragments associated with both bona fide CpG islands, which are methylation-free, and methylated Alu repeated sequence had high affinity to the column. These results suggest that methylation status of DNA fragments having high affinity to the column is heterogeneous and caution is required for the interpretation of the results.

DNA methylation patterns in the calcitonin gene region at first diagnosis and at relapse of acute lymphoblastic leukemia (ALL).

Aberrant DNA methylation can occur early in neoplastic transformation and may lead to the development of cancer. We describe the alterations of methylation patterns at the DNA sequence level which occurred in the 5' region of the calcitonin gene in lymphoblasts from 14 pediatric patients with acute lymphoblastic leukemia (ALL). The DNA methylation status of 25 CpG sites was determined by sequence analysis after bisulfite treatment of the DNA. This method showed that 13 out of 14 patients had increased numbers of methylated CpG sites in the calcitonin gene region at initial diagnosis when compared to control DNA from healthy individuals. The 5' region of the calcitonin gene appears to be methylated to a significantly higher degree in T lineage ALL compared to B lineage ALL (P < 0.01). Each of six ALL patients who were investigated at initial diagnosis and at relapse showed alterations in DNA methylation between the two stages. These six cases were also investigated by Southern blot analysis with methylcytosine-sensitive restriction enzymes and this method showed an increase in DNA methylation in only four of the six cases. The DNA sequencing method thus appears to be better suited to assess alterations of DNA methylation than Southern blot analysis. There are marked regional differences in the frequency of methylation of individual CpG sites and in the frequency of alterations between the two stages. Our results show that alterations in DNA methylation continue to occur from the initial stage to the relapse stage of ALL, suggesting that aberrant DNA methylation may play a role in tumor progression.

DNA methylation in early development.

Several lines of evidence strongly suggest that DNA methylation is involved in embryo development. Perhaps the most direct evidence comes from experiments with methyltransferase deficient mice. Embryos that express low levels of the maintenance methyltransferase do not develop to term and die at the 5 to 20 somite stage corresponding to the level of the enzyme. In the mouse, dramatic methylation changes have been observed during the early steps of embryo development. Most genes are subject to a process of active demethylation starting promptly after fertilization. Except for a small number of methylated CpG sites in imprinted genes the vast majority of the sites are unmethylated by the stage of cavitation (16 cell). Such genome-wide demethylation may signify an erasure of epigenetic information originating in the highly differentiated gametes. This erasure may be essential for the establishment of a pluripotent state, before specific cell lineages can be determined. The process of laying down a new developmental program involves, initially, global de novo methylation at the stage of pregastrulation followed by gene specific demethylations associated with the onset of activity of these genes. CpG islands characteristic of housekeeping genes, appear to be protected from the global de novo methylation. An exception to this rule is the hypermethylation of CpG islands in X-linked housekeeping genes on the inactive X chromosome and of specific differentially methylated CpG sites in imprinted genes. Primordial germ cells escape the global de novo methylation which takes place at the pregastrula stage and undergo a very similar de novo methylation process in the differentiated gonads (15.5-18.5 days post coitum), forming the methylation patterns which are specific to the gametes. Specific demethylations then form a terminal methylation pattern which is then clonaly inherited in the soma and erased after fertilization.