The rs1024611 Regulatory Region Polymorphism Is Associated with CCL2 Allelic Expression Imbalance
Figure 2
Validation of a pyrosequencing assay to measure AEI at rs13900 polymorphism.
A. Schematic of the CCL2 gene structure and the LD between regulatory region polymorphism rs1024611 and the transcribed polymorphism rs13900. Numbered boxes are exons and the dashed lines connecting the exons are the introns. rs1024611 is located 2578 bp upstream of the CCL2 translational start site and rs13900 is located in the 3′UTR. B. Stacked bar graph represents the levels of the C (black) and T (grey) alleles in PCR products as determined by pyrosequencing. The PCRs were performed on the plasmid mixtures containing rs13900C and rs 13900T allele combined at the indicated ratios to simulate homozygous (10∶0 and 0∶10) and heterozygous samples (5∶5) as well as different allelic levels (other indicated ratios). Data shown is from one representative experiment from three independent experiments which gave similar results. C. Regression analysis of amplification products obtained from the rs13900 C- and rs13900 T-bearing plasmids combined at different proportions. The measured levels of the C allele (y-axis) were plotted against the expected levels (x-axis). There was a near linear relationship between these values (R2 = 0.9969) suggesting that the pyrosequencing can serve as a sensitive assay to measure the levels of rs13900C and rs13900T alleles in heterozygous individuals. Similar results were obtained with using genomic DNA mixtures from homozygous and heterozygous individuals (data not shown).