title: Methylation status of individual CpG sites within Alu elements in the human genome and Alu hypomethylation in gastric carcinomas.
abstract: BACKGROUND
  Alu methylation is correlated with the overall level of DNA methylation and recombination activity of the genome. However, the maintenance and methylation status of each CpG site within Alu elements (Alu) and its methylation status have not well characterized. This information is useful for understanding natural status of Alu in the genome and helpful for developing an optimal assay to quantify Alu hypomethylation.
  
  
  METHODS
  Bisulfite clone sequencing was carried out in 14 human gastric samples initially. A Cac8I COBRA-DHPLC assay was developed to detect methylated-Alu proportion in cell lines and 48 paired gastric carcinomas and 55 gastritis samples. DHPLC data were statistically interpreted using SPSS version 16.0.
  
  
  RESULTS
  From the results of 427 Alu bisulfite clone sequences, we found that only 27.2% of CpG sites within Alu elements were preserved (4.6 of 17 analyzed CpGs, A approximately Q) and that 86.6% of remaining-CpGs were methylated. Deamination was the main reason for low preservation of methylation targets. A high correlation coefficient of methylation was observed between Alu clones and CpG site J (0.963), A (0.950), H (0.946), D (0.945). Comethylation of the sites H and J were used as an indicator of the proportion of methylated-Alu in a Cac8I COBRA-DHPLC assay. Validation studies showed that hypermethylation or hypomethylation of Alu elements in human cell lines could be detected sensitively by the assay after treatment with 5-aza-dC and M.SssI, respectively. The proportion of methylated-Alu copies in gastric carcinomas (3.01%) was significantly lower than that in the corresponding normal samples (3.19%) and gastritis biopsies (3.23%).
  
  
  CONCLUSIONS
  Most Alu CpG sites are deaminated in the genome. 27% of Alu CpG sites represented in our amplification products. 87% of the remaining CpG sites are methylated. Alu hypomethylation in primary gastric carcinomas could be detected with the Cac8I COBRA-DHPLC assay quantitatively.
authors.affiliation: Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Beijing Cancer Hospital and Institute, Peking University School of Oncology, Haidian District, Beijing, 100142, China.
authors.forename: Shengyan
authors.initials: S
authors.lastname: Xiang
authors.forename: Zhaojun
authors.initials: Z
authors.lastname: Liu
authors.forename: Baozhen
authors.initials: B
authors.lastname: Zhang
authors.forename: Jing
authors.initials: J
authors.lastname: Zhou
authors.forename: Bu-Dong
authors.initials: BD
authors.lastname: Zhu
authors.forename: Jiafu
authors.initials: J
authors.lastname: Ji
authors.forename: Dajun
authors.initials: D
authors.lastname: Deng
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D020087:Alu Elements; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D018899:CpG Islands; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D005260:Female; D015894:Genome, Human; D006801:Humans; D008297:Male; D008875:Middle Aged; D013274:Stomach Neoplasms / Q000235:genetics*