Clinical relevance of single nucleotide polymorphisms within the entire NLRP3 gene in patients with major blunt trauma

Abstract

Introduction: The nucleotide-binding oligomerization domain-like receptor (NLR) family has been recognized as comprising intracellular pattern recognition receptors in which NLRP3 (NLR family, pyrin domain containing 3) plays an important role in the initiation of host immune inflammatory responses. The genetic variants have been recognized to be critical determinants of interindividual differences in both inflammatory responses and clinical outcomes in critical illness. However, little is known about the clinical relevance of NLRP3 gene polymorphisms in critical illness.

Methods: A total of 718 patients with major blunt trauma were included in this study. Six tag SNPs (tSNPs) were selected from the entire NLRP3 gene through construction of haplotype bins, and they were genotyped using a pyrosequencing method. They were analyzed in relation to sepsis morbidity rate, multiple organ dysfunction (MOD) scores and IL-1β production. Moreover, the functionality of the rs2027432 polymorphism was assessed by the observation of its effect on transcriptional activities.

Results: Among the six tSNPs genotyped in this study, two of them (rs2027432 and rs12048215) were significantly associated with sepsis morbidity rate and MOD scores. A significant association was also observed between these two polymorphisms and IL-1β production by peripheral leukocytes in response to ex vivo lipopolysaccharide stimulation. However, no combined effects were found between these two polymorphisms. In addition, the rs2027432 polymorphism could significantly enhance the promoter activities of the NLRP3 gene.

Conclusions: rs2027432 and rs12048215 polymorphisms might be used as relevant risk estimates for the development of sepsis and MOD syndrome in patients with major trauma, in which rs2027432 might be a functional SNP.

Figures

Figure 1

Overview of selected tag SNPs within the entire NLRP3 gene. (A) The pairwise analysis of linkage disequilibrium (LD), based on r2, among the 35 SNPs with a minor allele frequency (MAF) ≥ 5% within the NLRP3 gene and the 5-kb up- and downstream regions. The selected six tag SNPs (tSNPs) are indicated by trigones. A LD plot of the 35 SNPs in the 37.953-kb region is displayed by using an r2 black-and-white color scheme. Black represents very high LD correlation between SNPs (r2 = 0.8 to 1), and white indicates the absence of correlation between SNPs (r2 = 0 to 0.2). (B) The six tSNPs and SNPs that are indirectly measured by tSNPs are listed with corresponding r2 values. Major and minor alleles of the selected tSNPs are given with their frequencies, based on the HapMap data for the Chinese Han Beijing (CHB) population. (C) The exon-intron structure of the NLRP3 gene and the locations of the tSNPs are shown. rs2027432 (-1,017 G/A) and rs3738448 (-23 G/T) map to within the 5'-flanking region. rs12048215 (5,134 A/G), rs4612666 (19,613 C/T) and rs1539019 (20,844 C/A) are located in introns 3, 7 and 8, and rs10754558 (32,579 C/G) is located in the 3'-untranslated region (3'-UTR).

Figure 2

Effect of the rs2027432 and rs12048215 polymorphisms of the NLRP3 gene on IL-1β production. The whole-blood samples collected from trauma patients immediately after admission were mixed at a 1:1 ratio (vol/vol) with RPMI 1640 culture medium and incubated with 100 ng/ml bacterial lipopolysaccharide at 37°C for 4 hours. The levels of IL-1β in the supernatants were assayed by performing a sandwich ELISA. The data are presented as means ± SD. One-way analysis of covariance (ANCOVA) was used to assess statistical significance. *Dominant effect (variant homozygotes + heterozygotes vs wild homozygotes) assessed by ANCOVA: *1P = 0.003, *2P = 0.018. #Recessive effect (variant homozygotes vs heterozygotes + wild-type homozygotes) assessed by ANCOVA: #1P < 0.0001, #2P = 0.00016.

Figure 3

Effect of the rs2027432 polymorphism on the transcription activity of the NLRP3 promoter. Relative luciferase activity (RLA) was measured in human U937 cells transfected with rs2027432G or rs2027432A plasmid constructs as described in Methods. Luciferase activity was normalized for transfection efficiency by using a control plasmid, pRL-CMV. The results are expressed as fold increases in RLA of the NLRP3 promoter construct vector as compared with pGL3-Basic (means ± SD). LPS = lipopolysaccharide.