Abstract 421: The NLRC4 Variant rs385076 Affects PU.1 Transcription Factor Binding, NLRC4 Isoform Expression and Circulating Interleukin-18 Concentrations
Circulating levels of the pro-inflammatory cytokine Interleukin-18 (IL-18) associate with cardiovascular events. IL-18 GWAS identified the NLRC4 locus, with rs385076 in the 5’UTR exon 2 of NLRC4 as lead SNPs. Data from the ENCODE project indicates accessible DNA and PU.1 transcription factor binding around rs385076.
We assessed the hypothesis that rs385076 alleles regulate NLRC4 gene expression by modulating binding of PU.1.
To investigate the influence of rs385076 alleles on PU.1 binding, a luciferase reporter gene assay with T or C allele containing plasmids was transfected into human embryonic kidney cells. The cells were treated with 0 / 12.5 / 25 or 50 ng PU.1. Gene expression of 1) total NLRC4, 2) NLRC4 isoforms containing 5’UTR exon 2 and 3) isoforms without exon 2 was quantified by qPCR in 1,500 individuals of the Gutenberg Health Study. Effects of rs385076 T/C allele on NLRC4 isoform levels were calculated using a linear mixed model.
In PU.1 treated cells, luciferase activity of the rs385076 C allele was 6-fold (±1.3) higher compared to controls, whereas the T allele showed no significant increase. Total NLRC4 gene expression was decreased in T allele carriers (p=7x10-3). Comparing isoforms containing the exon 2 (decreased, p=2x10-10) to those without (increased, p=7x10-4), expression of NLRC4 isoforms was regulated by T alleles in opposite directions, indicating a switched isoform usage (see figure).
In conclusion, our analyses indicate that the rs385076 T allele leads to decreased gene expression of NLRC4 and a switched isoform usage, putatively affected by decreased PU.1 binding. This is consistent with the finding that the T allele associated with lower IL-18 levels.
Author Disclosures: C. Mueller: None. T. Haase: None. S. Zeller: None. J. Krause: None. P.S. Wild: None. T. Münzel: None. A. Ziegler: None. K.J. Lackner: None. R.B. Schnabel: None. S. Blankenberg: None. T. Zeller: None.
- © 2015 by American Heart Association, Inc.