Evaluation of candidate regulatory SNPs <i>in vitro</i>.

<p>(A) Binding of nuclear factors from lymphoblastoid B-cells (PSC cells) and Jurkat cells to the 31-bp sequences around each SNP was evaluated by EMSA. Unlabeled probes in 200-fold excess as compared to the labeled probes were used for the competition experiment. The densities of the bands were quantified and normalized to that of the risk allele. rs2233424 in <i>NFKBIE</i> (C(NR)/T(R)) (left), rs12248974 (A(NR)/G(R)) (middle) and rs61852964 (G(NR)/T(R)) (right) in <i>RTKN2</i>. (B) Transcriptional activities were evaluated by luciferase assays. Each 31-bp oligonucleotide was inserted into the pGL4.24[<i>Luc2P</i>/minP] vector. Luc, luciferase; minP, minimal promoter. Transcfection was performed with HEK293A (for all the SNPs), PSC cells (for rs2233424), and Jurkat cells (for rs12248974 and rs61852964). rs2233424 (left), rs12248974 (middle), and rs61852964 (right). Data represent the mean ± s.d. Each experiment was performed in sextuplicate and independently repeated three times. *<i>P</i><0.05 by Student's <i>t</i>-test. n.s.: not significant. (C) Liner regression analysis of the relationship between SNP genotype and gene expression level. <i>NFKBIE</i> expression data in lymphoblastoid B-cell lines of HapMap individuals (JPT+CHB, CEU and YRI; <i>n</i> = 151), and <i>RTKN2</i> expression data in primary T cells from umbilical cords of Western European individuals (<i>n</i> = 85) were used. The x-axis shows the SNP genotypes and the y-axis represents the log₂-transformed gene expression level. R: correlation coefficient between SNP genotype and gene expression. Rs2233424 genotypes and <i>NFKBIE</i> expression level (left). The genotype classification by population: JPT+CHB, CC = 52, CT = 1; CEU, CC = 35, CT = 2; YRI, CC = 32, CT = 2, TT = 4. Rs1432411 genotypes and <i>RTKN2</i> expression level (right). Rs1432411 was used as a proxy SNP of rs12248974 (<i>r²</i> = 0.97).