DNA methylation of CpG islands from the critical region as measured with real-time PCR.

(A) Methylation of CpG islands localized at chromosomal band 13q14.3 and in the BCL2 promotor was assessed with quantitative real-time PCR by using primers specific for bisulfite-converted and nonmethylated DNA from peripheral blood lymphocytes (PBL; n = 7), CD-19⁺ peripheral blood lymphocytes (B cells; n = 11), B-CLL patients with tumors biallelic for the critical region in 13q14.3 (disomic; n = 9), loss of one allele (deleted; n = 14), or loss of both alleles (biallelically deleted; n = 1). Depicted are the ratios of 13q14.3- orBCL2-specific primer pairs and 2 methylation-insensitive primer pairs that are specific for bisulfite-converted DNA (Eads et al26). The color coding gives fold nonmethylated DNA (green = methylated) as compared with the average of sorted B cells; gray = not done. (B) A double-sided Student t test shows no significant differences between the patient groups (disomic or deleted) and sorted B cells.