inflammation is receiving an increasing amount of attention for its potential role in the pathogenesis of a variety of disorders , from insulin resistance and type 2 diabetes mellitus ( t2 dm ) to fatty liver and cardiovascular disease ( cvd ) .
current evidence shows that c - reactive protein ( crp ) level is a plasma protein and a sensitive and dynamic systemic marker of inflammation   could be influenced by both clinical and genetic factors .
ppars are orphan nuclear receptors belonging to the steroid , retinoid , and thyroid hormone receptor superfamily of ligand - activated transcription factors [ 4 , 5 ] .
three distinct receptor types have been cloned and characterized : ppar , ppar/ , and ppar .
ppar was less studied in previous study , compared with ppar and ppar. several evidences suggested biological roles for ppar in many genotypes .
deletion of ppar from foam cells increased the availability of inflammatory suppressors , which in turn reduced atherosclerotic lesions . on the other hand , activation of ppar
ppar agonists could promote cholesterol accumulation in human macrophages and increase serum high density lipoprotein ( hdl ) while lowering triglyceride levels in obese animal models [ 9 , 10 ] .
however , the associations between variants of the ppar , corresponding gene - gene interactions with crp level , were rarely studied .
so in this study , we sought to examine the association between 4 ppar polymorphisms with crp level and the additional interaction among the 4 single nucleotide polymorphisms ( snps ) .
a total of 1096 subjects were included in investigated population , excluded subjects with abnormal increasing of crp level ( 10  mg
/ l ) because of any reasons , such as infection ( n = 35 ) , trauma ( n = 15 ) , and any others factor ( n = 8) , and crp were missing ( n = 10 ) , a total of 1028 subjects ( 538 men , 490 women ) , with a mean age of 45.3  13.8 years , were included in the study , including the genotyping of polymorphisms .
the selected subjects were similar to those who were not selected in terms of age , sex , smoking status , and alcohol consumption .
data on demographic information and lifestyle risk factors for all participants were obtained using a standard questionnaire administered by trained staffs .
body weight and height were measured , and body mass index ( bmi ) was calculated as weight in kilograms divided by the square of the height in meters .
cigarette smokers were those who self - reported smoking cigarettes at least once a day for 1 year or more .
alcohol consumption was expressed as the sum of milliliters of alcohol per week from wine , beer , and spirits .
physical activity was divided into three levels , including light , moderate , and heavy .
we selected snps within the ppar gene using the following methods : ( 1 ) previously reported associations with metabolic abnormalities , ( 2 ) known heterozygosity and a minor allele frequency ( maf ) greater than 1%
. 4 snps of ppar were selected for genotyping in the study : rs2016520 , rs9794 , rs1053046 , and rs1053049 .
genomic dna from participants was extracted from edta - treated whole blood , using the dna blood mini kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions .
abi prism7000 software and allelic discrimination procedure were used for genotyping of aforementioned 4 snps . a 25  l reaction mixture including 1.25  l snp genotyping assays ( 20x ) , 12.5  l genotyping master mix ( 2x ) , and 20  ng dna and the conditions were as follows : initial denaturation for 10  min and 95c , denaturation for 15  s and 92c , annealing and extension for 90  s and 60c , and 50 cycles .
the mean and standard deviation ( sd ) for normally distributed continuous variables and percentages for categorical variable were calculated and compared .
the categorical data were analyzed using  test or the fisher exact test if necessary .
further , continuous variables were analyzed using student 's t - test or one - way analysis of variance , followed by the least significant difference multiple - range tests for comparison between groups .
hardy - weinberg equilibrium ( hwe ) was performed by using snpstats ( available online at http://bioinfo.iconcologia.net/snpstats ) .
line regression analysis was performed to verify polymorphism association between snp with crp levels using gender , age , smoking and alcohol status , and physical activity as covariates in the model .
generalized multifactor dimensionality reduction ( gmdr )   was employed to analyze the interaction among 4 snps ; some parameters was calculated , including cross - validation consistency , the testing balanced accuracy , and the sign test , to assess each selected interaction .
the cross - validation consistency score is a measure of the degree of consistency with which the selected interaction is identified as the best model among all possibilities considered .
the testing balanced accuracy is a measure of the degree to which the interaction accurately predicts case - control status with scores between 0.50 ( indicating that the model predicts no better than chance ) and 1.00 ( indicating perfect prediction ) .
finally , a sign test or a permutation test ( providing empirical p values ) for prediction accuracy can be used to measure the significance of an identified model .
a total of 1028 subjects ( 538 men , 490 women ) , with a mean age of 45.3  13.8 years , were selected .
minor allele frequencies of rs2016520 , rs9794 , rs1053046 , and rs1053049 were shown in table 2 .
all genotypes were distributed according to hardy - weinberg equilibrium ( all p values more than 0.05 ) .
line regression analysis on association between 4 snps and crp level was conducted ( table 3 ) .
we found that the crp level was lower in the subjects with minor alleles of rs2016520 and rs9794 , compared to subjects with wild genotype .
compared to the carriers of the common genotype ( rs2016520-tt ) , the carriers of the c allele ( tc + cc ) of rs2016520 were associated with a significant decreased level of crp , regression coefficients were 0.338 , and standard error was 0.104 ( p = 0.001 ) . compared to the carriers of the common genotype ( rs9794-cc ) , the carriers of the g allele ( cg + gg ) of rs9794 were also significantly associated with decreased level of crp , regression coefficients were 0.219 , and standard error was 0.114 ( p = 0.012 ) . however , the other 2 snps in ppar were not significantly associated with crp level after covariate adjustment .
we employed the gmdr analysis to assess the impact of the interaction among 4 snps , after adjustment for covariates including gender , age , smoke and alcohol status , and physical activity .   table 4 summarizes the results obtained from gmdr analysis for two- to four - locus models with covariates adjustment .
there was a significant two - locus model ( p = 0.0107 ) involving rs2016520 and rs9794 , indicating a potential gene - gene interaction between rs2016520 and rs9794 .
overall , the two - locus models had a cross - validation consistency of 10 and had the testing accuracy of 62.17% . to obtain ors and 95% cis for the joint effects of candidate snps ( rs2016520 and rs9794 ) on crp , we conducted interaction analysis among snps in the 2-locus models .
subjects with rs2016520-tc or cc and rs9794-cg or gg genotypes have lowest crp level , difference ( 95% ci ) = 0.50 ( 0.69 to 0.21 ) ( p < 0.001 ) , compared to subjects with rs2016520-tt and rs9794-cc genotypes , after adjustment for gender , age , smoke and alcohol status , and physical activity ( table 5 ) .
the frequency of the c allele was 24.9% in the present population , which is lower than the proportion in the han population of dalian reported by yu et al .
c - reactive protein ( crp ) is a well - established biochemical marker of inflammation and has been used to predict future cardiovascular disease , metabolic syndrome , and future development of type 2 diabetes mellitus [ 3 , 1921 ] .
current evidence shows that crp level is a complex trait , influenced by both clinical and genetic factors .
family and twin studies have found that additive genetic factors account for 2740% of the variance in crp level , suggesting a role for genetic variation in determining serum levels .
the results of this study indicated that minor alleles of rs2016520 and rs9794 were associated with lower crp level , compared to subjects with wild genotype . compared to the carriers of the common genotype ( rs2016520-tt ) ,
the carriers of the c allele ( tc + cc ) of rs2016520 were associated with a significant decreased level of crp .
compared to the carriers of the common genotype ( rs9794-cc ) , the carriers of the g allele ( cg + gg ) of rs9794 were also significantly associated with decreased level of crp .
however , the other 2 snps in ppar were not significantly associated with crp level after covariate adjustment .
in contrast to ppar and ppar , the role of ppar in crp level is less well defined .
gu et al .   indicated that rs2016520 in ppar was not associated with crp level both in normal weight and obese subjects in a chinese population .
liang et al .   suggested that ppar attenuated crp - induced proinflammatory effects through cd32 and nf-b pathway .
ppar may serve as a more potent therapeutic target than ppar in atherosclerosis or inflammatory therapy , and the potency of 1  m ppar is similar to that of 10  m ppar in anti - inflammatory effect . as we have known that a multiple genotype was influenced by both genetic and environment factors and the genetic factors including many gene mutations , so it is necessary to investigate the synergistic effect of multiple snp on crp level .
we employed the gmdr analysis to assess the impact of the interaction among the 4 snps on crp level with covariates adjustment .
subjects with rs2016520-tc or cc and rs9794-cg or gg genotypes have lowest crp level , difference ( 95% ci ) = 0.50 ( 0.69 to 0.21 ) ( p < 0.001 ) , compared to subjects with rs2016520-tt and rs9794-cc genotypes .
in addition , rs9794 was not associated with crp , but it can significantly affect level when accompanied with rs2016520 .
these findings indicate that a minor gene ( even when its main effects are close to nil ) can have a strong effect on level , due to the presence of gene - gene interaction .
several mechanisms of association between activation of ppar and decreased crp level have been suggested .
liang et al .   indicated that untreated endothelial cells express a basal level of ppar and ppar agonist exerts an anti - inflammatory effect in crp - treated endothelial cells .
we also demonstrated that less concentration of ppar than ppar agonist is needed to attenuate both crp - induced adhesion molecules and the ability of monocyte to attach to endothelial cells .
ppar could attenuate crp - induced nf-b activation and vcam-1 and mcp-1 expression in endothelial cells .
moreover , it was found that the decreased adhesion molecule expression would result in significant fewer monocytes attaching on the crp - treated endothelial cells .
similar to one of the mechanisms in endothelial cells , activation of ppar has been shown to promote the binding of inflammatory suppressor bcl-6 to the promoter of group iia secretory phospholipase a2 , an inflammatory marker for atherosclerosis , and therefore inhibit the inflammation induced by il-1b in vsmcs . limitations of this study should be considered .
the selected snps were not sufficient to capture most genetic information of the ppar. further studies should include more snps , even the other ppar isoforms , such as ppar or ppar. secondly , there was a relatively small sample size in the study , though the number of study participants met the requirement for analysis ; future studies should be conducted in different races .
thirdly , the interaction between gene - environment factors on level should be studied in the future papers . in conclusion
, the results of this study indicated that minor alleles of rs2016520 and rs9794 were associated with lower crp level , compared to subjects with wild genotype , and we also found a potential gene - gene interaction between rs2016520 and rs9794 on decreased crp level .
 
 aims . to examine the association between 4 single nucleotide polymorphisms ( snps ) of peroxisome proliferator - activated receptors  ( ppar ) polymorphisms and c - reactive protein ( crp ) level and additional gene - gene interaction . 
 methods . 
 line regression analysis was performed to verify polymorphism association between snp and crp levels . 
 generalized multifactor dimensionality reduction ( gmdr ) was employed to analyze the interaction . 
 results . a total of 1028 subjects ( 538 men , 490 women ) were selected . 
 the carriers of the c allele ( tc or cc ) of rs2016520 were associated with a significant decreased level of crp , regression coefficients was 0.338 , and standard error was 0.104 ( p = 0.001 ) . 
 the carriers of the g allele ( cg or gg ) of rs9794 were also significantly associated with decreased level of crp , regression coefficients was 0.219 , and standard error was 0.114 ( p = 0.012 ) . 
 we also found a potential gene - gene interaction between rs2016520 and rs9794 . 
 subjects with rs2016520-tc or cc , rs9794-cg or gg genotypes have lowest crp level , difference ( 95% ci ) = 0.50 ( 0.69 to 0.21 ) ( p < 0.001 ) , compared to subjects with rs2016520-tt and rs9794-cc genotypes . conclusions . 
 rs2016520 and rs9794 minor allele of ppar and combined effect between the two snp were associated with decreased crp level . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion