title: The stem cell signature of CHH/CHG methylation is not present in 271 cancer associated 5'UTR gene regions.
abstract: Non-CpG methylation is frequently present in stem cell DNA. We investigated the value of this epigenetic modification in cancerous DNA in order to establish the implications of CHH/CHG methylation for biomarker development. Therefore we used the restriction enzymes BstNI and PspGI within a combined multiplex PCR and targeted microarray approach for the elucidation of non-CpG (CCWGG) methylation. Targeting 544 CCWGG sites in 271 5' gene regions, the CHH/CHG methylation status of the MCF7 breast cancer cell line and blood from healthy volunteers and childhood ALL was analyzed. Statistical analysis of microarray data and subsequent SYBR green based qPCR on DNA digests was applied to confirm the results from the microarray screen.
  
  
  CONCLUSIONS
  The microarray experiments identified for the MCF7 cell line the genes MSH2 (p < 0.001), EREG (p < 0.001) and HSPA2 (p = 0.029) with CHH/CHG methylation, and in childhood ALL the genes HIST1H2AG (p = 0.003), PGF (p = 0.02), CPEB4 (p = 0.039) and TJP2 (p = 0.04). Validation using qPCR upon restriction digestion did not confirm the presence of CHH/CHG methylation in MCF7 DNA. In ALL samples only TJP2 was found harboring CHH/CHG methylation (p = 0.02). However, applying Bonferroni-correction for multiple testing that qPCR-result was not rated as statistically significant anymore. We concluded that non-CpG methylation in 544 CCWGG sites analyzed did not change in tumor cells. Thus any change of the CHH/CHG methylation pattern is a minor event in tumorigenesis, even if the stem cell markers OCT4, NANOG, STELLAR and GDF3 are expressed like in the MCF7 breast cancer cell line.
authors: AIT - Austrian Institute of Technology GmbH, Health & Environment Department, Molecular Diagnostics, Muthgasse 11/2, A-1190 Vienna, Austria. walter.pulverer@ait.ac.at
authors: Walter
authors: W
authors: Pulverer
authors: Matthias
authors: M
authors: Wielscher
authors: Renate
authors: R
authors: Panzer-Grümayer
authors: Tanja
authors: T
authors: Plessl
authors: Albert
authors: A
authors: Kriegner
authors: Klemens
authors: K
authors: Vierlinger
authors: Andreas
authors: A
authors: Weinhäusel
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics*; D014408:Biomarkers, Tumor / Q000235:genetics; D045744:Cell Line, Tumor; D002648:Child; D018899:CpG Islands / Q000235:genetics*; D004247:DNA / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology*; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis
chemical_list: D020121:5' Untranslated Regions; D014408:Biomarkers, Tumor; D004247:DNA