Treatment of lymphoblastoid cells (LCs) with the demethylation drug 5-aza-2′-deoxycytidine (AzadC).
(A) Methylation analysis of CpG 85 by methylation-specific PCR (MS-PCR) and quantification of allelic expression imbalance of the RB1 gene. The top chart shows the methylation status of CpG 85 in blood, mock-treated and AzadC-treated LCs. The percentage of MS-PCR products specific for methylated and unmethylated alleles is indicated by black and grey bars, respectively. The bottom plot shows the ratio of allelic expression as determined by SNaPshot primer extension on RT–PCR products obtained from RNA. For each sample 3–8 independent experiments were performed. The top and bottom of the means diamonds represent the 95% confidence interval for the means. In family H, we could not investigate allelic RB1 expression in blood, because we did not have RNA from fresh blood. In this family, a male patient with unilateral retinoblastoma (HII-1) inherited the rare variant from his unaffected mother and transmitted it to his unaffected daughter (HIII-1). (B) Electropherograms of SNaPshot primer extension on RT–PCR products specific for the 2B-transcript. Black and red peaks correspond to C and T alleles, respectively. In A III-1, the C allele is known to be of paternal origin. Numbers next to peaks indicate peak areas. Numbers below electropherograms with two peaks show the ratios of peak areas (T-allele/C-allele).