Enhancer polymorphism rs10865710 associated with traumatic sepsis is a regulator of PPARG gene expression

Abstract

Background: Peroxisome proliferator-activated receptor gamma (PPARγ) is a major regulator in sepsis. Our previous study identified the enhancer polymorphism rs10865710C/G to be associated with susceptibility to sepsis in trauma patients. We performed two-stage cohort studies integrating biological experiments of potential functional variants that modify susceptibility to traumatic sepsis.

Methods: Improved multiplex ligation detection reaction (iMLDR) was used to genotype rs10865710 in 797 Han Chinese trauma patients in Chongqing. Clinical relevance was validated in 334 patients in Guizhou. The potential function of rs10865710 in transcriptional regulation was explored through a dual luciferase reporter assay and electrophoretic mobility shift assay (EMSA). Expression of PPARγ was assessed by expression quantitative trait locus (e-QTL) and western blot analyses.

Results: The association results confirmed rs10865710 to be significantly strongly associated with sepsis risk in trauma patients of the Chongqing and Guizhou cohorts (OR = 1.41 (1.11-1.79), P = 0.004 and OR = 1.45 (1.01-2.09), P = 0.046, both for allele-dose effect, respectively). A meta-analysis of both cohorts and a previous study indicated strong evidence for this association (OR = 1.41 (1.17-1.71), P = 0.0004 for the dominant model, OR = 1.78 (1.34-2.36), P < 0.0001 for the recessive model and OR = 1.38 (1.20-1.58), P < 0.0001 for the allelic model). Functional experiments verified that rs10865710 was a causative variant influencing enhancer activity (G vs. C, 0.068 ± 0.004 vs. 0.096 ± 0.002, P = 0.0005) and CREB2 binding. Expression analysis also indicatevd rs10865710 genotypes to be associated with levels of PPARγ expression (P = 9.2 × 10-5 for dominant effect and P = 0.005 for recessive effect).

Conclusions: Our study provides evidence that the enhancer-region polymorphism rs10865710 might influence transcription factor binding and regulate PPARγ expression, thus conferring susceptibility to traumatic sepsis.

Trial registration: ClinicalTrials.gov, NCT01713205. Registered 18 October 2012, retrospectively registered.

Keywords: Peroxisome proliferator-activated receptor gamma; Sepsis; Transcriptional regulation; Trauma; rs10865710.

Conflict of interest statement

All authors declare that they have no competing interests.

Figures

Fig. 1

Rs10865710 is associated with lower LPS-induced PPARG protein expression. Whole blood samples were collected from 30 trauma patients (CC: n = 10, CG: n = 10, GG: n = 10, respectively). PPARG expression by peripheral leukocytes was detected using western blotting and is presented as a gray value. *P = 9.2 × 10−5 for a dominant association (CG + GG vs. CC), #P = 0.005 for a recessive association (GG vs. CG + CC). Student’s t test was used to assess statistical significance

Fig. 2

Rs10865710 decreased PPARG transcriptional enhancer activity. a Plasmid constructs used for transfection. b Transcriptional enhancer activities of rs10865710 measured by luciferase (luc) activity 48 h after transfection. Values of relative luciferase activity are shown as means ± SDs from three independent experiments. *P = 0.005, Student t test. C, C allele; G, G allele

Fig. 3

The rs10865710 risk allele disrupts transcription factor CREB2 binding. a EMSA with biotin-labeled probes containing either the C or the G allele of rs10865710, incubated with THP-1 cell nuclear extract. The arrow indicates an allele-specific band that interacts with nuclear protein. 10× and 200× indicate 10- and 200-fold unlabeled probes excess over labeled probes. “+” and “−” mean added and unadded, respectively. b Super-shift EMSA of rs10865710. Two independent experiments were performed with similar results. Ab, antibody; C, C allele; G, G allele