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Functional characterization of human variants of NFKBIA : a key regulator of immune responsiveness implicated in susceptibility to infectious and inflammatory disease Ali, Salman


IkBα is an important regulator of inflammation. Single nucleotide polymorphisms (SNPs) rs3138053, rs2233406 and rs2233409 in the promoter of the gene NFKBIA, which encodes for IkBα, have been shown to be associated with a variety of infectious and inflammatory conditions. In this study, we investigated the functional impact of the promoter variants of NFKBIA on human immune responsiveness. Using a coding SNP that was in strong linkage disequilibrium (LD) with NFKBIA SNPs rs3138053/rs2233406/rs2233409, we designed and validated an allele-specific PCR assay that could detect subtle differences in allele ratios between the major (ACC) and minor (GTT) promoter variants of SNPs rs3138053/rs2233406/rs2233409. Peripheral blood mononuclear cells (PBMCs) of homozygous (ACC/ACC) and heterozygous (ACC/GTT) individuals were stimulated with 100ng/ml LPS and live cultures of Streptococcus pneumoniae (moi 7.8-30) serotype 14 for 3 and 4 hours. PBMCs of neonatal NFKBIA homozygotes and heterozygotes were stimulated with various Toll-like-receptor (TLR) ligands of the innate immunity cascade to assay for differences in the innate immune response. NFKBIA heterozygotes of European descent displayed 1.21 (1.14-1.27 95% CI)-1.26 (1.18-1.34 95% CI) fold higher expression of the major allele transcript (ACC) relative to the minor allele transcript (GTT). For the same ethnicity, at 3 hours stimulation, NFKBIA homozygotes (ACC/ACC) produced higher levels of NFKBIA mRNA than heterozygotes following stimulation with LPS (1.4 fold. p=0.0095) and S. pneumoniae (1.51 fold, p=0.024). Higher TNFα secretion was seen from PBMCs of heterozygotes as compared to homozygotes (of European descent) in the presence of LPS (1.57 fold , p<0.05 at a dose of 100ng/ml), Pam3CSK4 (2.29 fold, p<0.01 at a dose of 100ng/ml and 1.91 fold, p<0.05 at a dose of 1000 ng/ml), 3M003 (1.79 fold, p<0.001 at a dose 10μM) and 3M002 (3.30 fold, p<0.001 at a dose of 10μM). The results presented here provide preliminary functional evidence behind the observed associations of these SNPs with infectious and inflammatory conditions. A global understanding of the functional consequences of regulatory polymorphisms of NFKBIA will be provided with subsequent experiments that examine differences between all NFKBIA genotypes (including minor allele homozygotes) for IκBα protein expression and NF-κB translocation in all major ethnic groups.

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