Novel Tools for Unbiased Dna Differential Methylation Screening

Abstract

DNA differential methylation screening approaches may be hypothesis driven (preselection of the loci to screen) or unbiased (screening precedes mapping of differentially methylated loci). The latter allow for the identification of sequences demonstrating 'nonclassical' methylation behavior in cancer and, thus, widen our concept of tumor biology. Extensive employment of unbiased screening methods is hampered by the troublesome procedures involved in physically mapping the identified differentially methylated DNA fragments. In this special report, we describe a positive experience of optimizing two screening methods, methylation-sensitive arbitrarily primed PCR and amplification of intermethylated sites, with a special focus on simplification, or even exclusion, of sequencing procedures. With our modifications, unbiased screening of DNA differential methylation acquires an easy-to-use workflow, including sophisticated experimental design, high-resolution analysis and simple genomic mapping of the fragments of interest.

Genome-Wide Methylotyping Resolves Breast Cancer Epigenetic Heterogeneity and Suggests Novel Therapeutic Perspectives

“…Abnormal methylation has been demonstrated for the genes involved in cell cycle regulation (CDKN2A, CDKN2B, p14/ARF, RB1, etc. ), apoptosis (TP53, CDKN1A, HOX5, MDM2, DAPK1, TWIST1, TMS1 and FHIT), invasion and metastasis (CDH1, CDH13 and CTNB), receptor mediated signaling (ESR1, PR and RARB) and many others [9][10][11][12][13][14][15].…”

Evolution of Cancer DNA Methylotyping

“…As far as pan-cancer abnormally methylated genes were unsuitable as markers that would potentially discriminate cancer types, and knowledge of cancer biology was still insufficient to confidently pick candidate regions, a cohort of methods naturally emerged to provide unbiased search for differentially methylated loci in cancer genomes [12]. Application of such methods produced results rather surprising for the time.…”

“…First, new functional classes of genes were discovered that were not expected to be specifically methylated in cancers based on the previous concepts, such as genes encoding ion channels, loosely characterized transmembrane proteins, nerve differentiation and growth regulators, as well as the genes of completely unknown functions and even deep intergenic regions containing functionally uncharacterized genomic elements [12,13]. Second, it became apparent that the exact positions of the regions abnormally methylated in cancer were not necessarily coincident with the promoters, readily positioning not only in 5 UTRs and around the TSSs, but in the gene bodies and 3 UTRs as well, and, again, in the intergenic regions thousands of base pairs from the nearest genes [12,14]. Despite the scientific importance of the findings achieved, extensive employment of unbiased screening methods was hampered by the troublesome physical mapping of the identified differentially methylated DNA fragments.…”

“…Demand for the unbiased genome-scale approach in differential DNA methylation analysis has become one of the key challenges proposed in the very first issues of the Epigenomics journal a decade ago [12,15]. It was the technological revolution that made it possible to widely screen multiple samples by providing the opportunity of genome-wide DNA methylation analysis with simultaneous mapping of the differentially methylated regions.…”