Abstract
The aim of the present study was to investigate the association between seven functional SNPs in NALP3 gene and the susceptibility to primary gout. A total of 247 patients with primary gout and 247 controls were selected in this study. Genotyping of NALP3 rs4612666, rs3806268, rs12239046, rs10754558, rs7512998, rs12137901 and rs12565738 was performed using the Sequenom MassARRAY platform. Comparison analysis showed that primary gout patients were more likely to have a higher body mass index, DBP, SBP, TG, urea nitrogen and uric acid (P < 0.05). According to logistic regression analysis, individuals carrying with the GG genotype of rs3806268 were associated with increased risk of primary gout when compared with the AA genotype (OR=1.83, 95% CI=1.03-3.26). However, no significant associations were identified for the remaining SNPs. In conclusion, we found a significant association between rs3806268 in NLRP3 gene and the risk of primary gout in a Chinese population. Further clinical and genetic studies are required to investigate the mechanisms underlying the association between NALP3 polymorphisms and the development of primary gout.
Keywords: NLRP3, polymorphism, primary gout
Introduction
Gout is the most common form of autoinflammatory arthritis among men, and this disease is characterized by higher urate in serum and monosodium urate crystal deposition in tissues, and thus to cause acute arthritis in and around the joints, nephrolithiasis and urate nephropathy [1,2]. It is reported that the morbidity is showing an increasing trend recently worldwide, and gout has affected about 6% of adults [3]. A recent epidemiology study has reported that the prevalence of gout in eastern China is about 2% in men and 0.5% in women, while it is less than 0.1% about 10 years ago [4]. It is well known that gout is a long-term chronic disease, and the pathogenesis of this disease is involved in a complex and multifactorial process. Many environmental factors and immune dysregulation may play an important role in the development of gout, such as hyperuricemia. However, not all the individuals who expose to the similar risk factors would develop gout, which suggest that genetic factors could contribute to the underlying pathogenesis of this disease.
The accumulation of monosodium urate in tissues plays a critical role in the attack of gouty arthritis [5]. Blood monocytes and monocytes and macrophages in synovial fluid could release a mass of IL-1β stimulated by monosodium urate, and mediate inflammatory response. Caspase-1 catalyzes inactive proIL-1β, and thus causes the production of active interleukin IL-1β. The activation of caspase-1 depends on active NALP3 inflammasome [6]. NLRP3 is a member of the nucleotide-binding domain and one of best-described members of nod like receptor (NLR) family [7]. NLRP3 could active caspase-1 and subsequent secretion of proinflammatory cytokines such as IL-1β and IL-18 [8,9]. The variants of NLRP3 could influence the mRNA stability and expression of NLRP3. Recent studies have reported the association of NLRP3 polymorphisms with the pathogenesis of autoinflammatory diseases, such as type-2 diabetes and inflammatory bowel disease [10,11]. Currently, only one study reported the role of NLRP3 polymorphisms in the development of gout [12]. Therefore, we hypothesize that NLRP3 polymorphisms could contribute to the development of gout, and we conducted a case-control study to investigate the association between seven functional SNPs in NALP3 and the susceptibility to primary gout.
Material and methods
Patients
Between March 2013 and January 2015, a total of 247 patients with primary gout were collected from the Love & Health Hospital of Huangshi. The diagnosis of primary gout was based on preliminary criteria for the classification of gout of the American Rheumatism Association published by the American College of Rheumatology in 1997 (Wallace et al., 1977). Additionally, a total of 247 adult healthy subjects without primary gout were collected from individuals who received health check-up examinations in the Love & Health Hospital of Huangshi during the same period of collecting the patients with primary gout. The exclusion criteria for control subjects were those who had no history of primary gout, coronary atherosclerosis heart disease, diabetes, hyperlipidemia, cancer, hepatic disease, or renal disease.
The demographic characteristics of patients with primary gout and control subjects were investigated by trained nurses with a self-designed questionnaire, such as BMI index, tobacco smoking and alcohol drinking. The clinical characteristics were collected from medical records, such as blood pressure, urea nitrogen, creatinine, uric acid and etc. All individuals voluntarily participated in the study and gave their informed consent. The protocol of this study was approved by the ethics committee of the Love & Health Hospital of Huangshi.
DNA extraction and genotyping
2 ml peripheral blood sample was drawn from each patient with primary gout and control subject, which was stored at -20°C until use. DNA was extracted from peripheral blood by using a commercially available Qiagen kit (Hilden, Germany). All patients and health controls were asked to provide 5 mL venous blood, which was stored at -20°C until use and included 0.5 mg/mL EDTA as an anticoagulant. DNA was extracted from peripheral blood leukocytes using a commercially available Qiagen kit (Hilden, Germany). Genotyping of NALP3 rs4612666, rs3806268, rs12239046, rs10754558, rs7512998, rs12137901 and rs12565738 was performed using the Sequenom MassARRAY platform (San Diego, CA, USA). Multiplex PCR mixture contained 1× HotStar Taq buffer, 2.8 mM MgCl2, 0.1 U of HotStar Taq polymerase, 2 ng of genomic DNA, 0.5 pmol of each primer, and 0.5 mmol of dNTPs. Reaction was performed at 94°C for 15 min, followed by 45 cycles at 94°C for 20 s, 56°C for 30 s, and 72°C for 1 min, with a final incubation at 72°C for 3 min. Unincorporated dNTPs were deactivated using 0.3 U of shrimp alkaline phosphatase (SAP) followed by primer extension using 5.4 pmol of each extension probe, 50 mmol of iPlex Termination Mix, and 0.5 U of iPLEX enzyme (Sequenom; San Diego, CA). The extension reactions were carried out with an initial denaturation step of 8 min at 94°C, followed by 30 cycles at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min. Purified extension reaction products with cation exchange resin were spotted onto SpectroCHIPs and measured by MALDI-TOF mass spectrometry.
Statistical analysis
Frequencies were used to describe the distribution of categorical variables and median and interquartile range was used for continuous variables. Deviations from Hardy-Weinberg equilibrium of NALP3 rs4612666, rs3806268, rs12239046, rs10754558, rs7512998, rs12137901 and rs12565738 were assessed by χ2-test with one degree of freedom. Multiple conditional logistic regression models were established to estimate the association between NALP3 rs4612666, rs3806268, rs12239046, rs10754558, rs7512998, and rs12137901 and risk of primary gout, and the results were evaluated by the odds ratio (OR) and 95% confidence intervals (CIs). Additional stratification analysis was designed to analyze the gene-environmental interaction on the risk of primary gout. These values were adjusted for potential confounding factors, and the wild-type genotype was used as the reference group. Differences for 2-sided P values < 0.05 were considered statistically significant.
Results
The baseline information of patients with primary gout and control subjects were shown in Table 1. The mean ages of the primary gout patients and control subjects were 51.60±9.15 and 51.95±8.52 years, respectively. As expected, no significantly difference was found between patients with primary gout and control subjects in terms of age and gender (P > 0.05). When compared with control subjects, the patients with primary gout had a higher BMI, DBP, SBP, TG, urea nitrogen and uric acid by χ2-test (All P value < 0.05) (Table 1). Additionally, no significant differences were found between the 2 groups in terms of tobacco smoking, alcohol drinking, TC and creatinine (All P value > 0.05).
Table 1.
Characteristics of patients with primary gout and control subjects
| Variables | Patients N=247 | % | Controls N=247 | % | t or χ2 value | P value |
|---|---|---|---|---|---|---|
| Age, years | ||||||
| Mean age, years | 51.60±9.15 | 51.95±8.52 | 0.44 | 0.33 | ||
| < 50 | 144 | 58.30 | 147 | 59.51 | ||
| ≥ 50 | 103 | 41.70 | 129 | 52.23 | 1.34 | 0.25 |
| Gender | ||||||
| Female | 31 | 12.55 | 31 | 12.55 | ||
| Male | 216 | 87.45 | 216 | 87.45 | 0.00 | 1.00 |
| Tobacco smoking | ||||||
| Never | 159 | 64.37 | 146 | 59.11 | ||
| Ever | 88 | 35.63 | 101 | 40.89 | 1.45 | 0.23 |
| Alcohol drinking | ||||||
| Never | 132 | 53.44 | 124 | 50.20 | ||
| Ever | 115 | 46.56 | 123 | 49.80 | 0.52 | 0.47 |
| BMI, kg/m2 | 25.13±1.15 | 24.20±2.05 | 6.22 | < 0.001 | ||
| DBP, mmHg | 139.40±13.75 | 131.55±15.60 | 5.93 | < 0.001 | ||
| SBP, mm Hg | 89.42±11.30 | 85.43±10.53 | 4.06 | < 0.001 | ||
| TG, mmol/L | 2.47±2.10 | 1.53±0.93 | 6.43 | < 0.001 | ||
| TC, mmol/L | 5.16±1.26 | 5.22±2.10 | 0.39 | 0.35 | ||
| Urea nitrogen, mmol/L | 6.15±2.40 | 5.81±1.55 | 1.87 | 0.03 | ||
| Creatinine, μmol/L | 92.50±35.35 | 95.65±16.73 | 1.27 | 0.10 | ||
| Uric acid, μmol/L | 513.50±19.40 | 335.50±48.65 | 53.41 | < 0.001 |
BMI: body mass index; DBP: Diastolic blood pressure; SBP: Systolic blood pressure; TC: Total cholesterol; TG: Triglyceride; Continuous variables analyzed using independent sample t-test; categorical variables analyzed using χ2-test.
By χ2-test with one degree of freedom, the genotype distributions of rs4612666, rs3806268, rs12239046, rs7512998 and rs12137901 confirmed with Hardy-Weinberg equilibrium (P value < 0.05), while the rs10754558 and rs12565738 deviated from Hardy-Weinberg equilibrium (P value > 0.05) (Table 2). The minor allele frequencies of the seven SNPs in controls were similar with those in the general population, as found in the NCBI dbSNP databases.
Table 2.
Genotype characteristics of the eight nucleotide polymorphisms of NALP3
| SNP | Alleles | MAF From dbSNP | MAF | P for HWE in controls | |
|---|---|---|---|---|---|
|
| |||||
| Patients | Controls | ||||
| rs4612666 | A/G | 0.4109 | 0.3988 | 0.4271 | 0.07 |
| rs3806268 | A/G | 0.3874 | 0.4919 | 0.4089 | 0.08 |
| rs12239046 | C/T | 0.4067 | 0.4453 | 0.4494 | 0.42 |
| rs10754558 | C/G | 0.3558 | 0.3704 | 0.3522 | 0.001 |
| rs7512998 | T/C | 0.1359 | 0.1498 | 0.1336 | 0.16 |
| rs12137901 | T/C | 0.2869 | 0.3117 | 0.2834 | 0.06 |
| rs12565738 | C/T | 0.1661 | 0.1660 | 0.1457 | < 0.001 |
MAF: Minor allele frequency.
The association between the seven SNPs of NALP3 and risk of primary gout was shown in Table 3. By χ2-test, there were no significant differences between patients with primary gout and control subjects in terms of genotype distributions of the seven SNPs (All P values < 0.05). According to logistic regression analysis, individuals carrying with the GG genotype of rs3806268 were associated with increased risk of primary gout when compared with the AA genotype (OR=1.83, 95% CI=1.03-3.26). However, we did not find significant association of rs4612666, rs12239046, rs10754558, rs7512998, rs12137901 and rs12565738 with the development of primary gout (All P values > 0.05).
Table 3.
Association between the seven SNPs of NALP3 and risk of primary gout
| SNP | Patients | % | Controls | % | x2 value | P value | OR (95% CI)1 | P value |
|---|---|---|---|---|---|---|---|---|
| rs4612666 | ||||||||
| AA | 82 | 33.20 | 74 | 29.96 | Ref. (1.0) | - | ||
| AG | 131 | 53.04 | 135 | 54.66 | 0.87 (0.58-1.22) | 0.51 | ||
| GG | 33 | 13.36 | 38 | 15.38 | 0.82 | 0.66 | 0.78 (0.43-1.43) | 0.40 |
| rs3806268 | ||||||||
| AA | 56 | 22.67 | 77 | 31.17 | Ref. (1.0) | - | ||
| AG | 139 | 56.28 | 138 | 55.87 | 1.38 (0.89-2.15) | 0.12 | ||
| GG | 52 | 21.05 | 39 | 12.96 | 4.79 | 0.09 | 1.83 (1.03-3.26) | < 0.03 |
| rs12239046 | ||||||||
| CC | 72 | 29.15 | 78 | 31.58 | Ref. (1.0) | - | ||
| CT | 130 | 52.63 | 116 | 46.96 | 1.21 (0.79-1.86) | 0.35 | ||
| TT | 45 | 18.22 | 53 | 21.46 | 1.69 | 0.43 | 0.92 (0.53-1.58) | 0.75 |
| rs10754558 | ||||||||
| CC | 110 | 44.53 | 116 | 46.96 | Ref. (1.0) | - | ||
| CG | 91 | 36.84 | 88 | 35.63 | 1.09 (0.72-1.65) | 0.67 | ||
| GG | 46 | 18.62 | 43 | 17.41 | 0.31 | 0.86 | 1.13 (0.67-1.90) | 0.63 |
| rs7512998 | ||||||||
| TT | 182 | 73.68 | 188 | 76.11 | Ref. (1.0) | - | ||
| TC | 56 | 22.67 | 52 | 21.05 | 1.11 (0.71-1.75) | 0.63 | ||
| CC | 9 | 3.64 | 7 | 2.83 | 0.50 | 0.78 | 1.33 (0.43-4.29) | 0.58 |
| rs12137901 | ||||||||
| TT | 128 | 51.82 | 136 | 55.06 | Ref. (1.0) | - | ||
| TC | 84 | 34.01 | 82 | 33.20 | 1.09 (0.72-1.64) | 0.67 | ||
| CC | 35 | 14.17 | 29 | 11.74 | 5.87 | 0.05 | 1.28 (0.71-2.31) | 0.37 |
| rs12565738 | ||||||||
| CC | 185 | 74.90 | 191 | 77.33 | Ref. (1.0) | - | ||
| CT | 42 | 17.00 | 40 | 16.19 | 1.08 (0.65-1.80) | 0.74 | ||
| TT | 20 | 8.10 | 16 | 6.48 | 0.59 | 0.75 | 1.29 (0.61-2.75) | 0.47 |
Adjusted for BMI, DBP, SBP, TG, urea nitrogen and uric acid.
We further analyzed the association between rs3806268 and risk of primary gout stratified by BMI, DBP, SBP, TG, TC, urea nitrogen, creatinine and uric acid, and no significant interaction was found between rs3806268 and demographic and clinical characteristics (P values > 0.05).
Discussion
It is well known that polymorphisms have an effect on the regulation of gene expression, which could contribute to the differences between individuals in the susceptibility to a disease and its severity. Gout is a polygenic heterogeneity disease, and the pathogenesis of gout showed individualization in human. The distributions of SNP loci in different races showed genetic heterogeneity. More than 30 different single nucleotide polymorphisms (SNPs) located on the exon3 of NLRP3 gene have been reported, which encodes the nucleotide binding site domain and boundary regions [13]. In our study, we found that the GG genotype of rs3806268 in NLRP3 gene was associated with an increased risk of primary gout in a Chinese population.
NLRP3 are known to be responsible for the recognition of several molecular patterns inducing the assembling of inflammasome, the activation of caspase-1 and finally the secretion of the pro-inflammatory cytokine IL-1β, and consequently innate immune response [14]. Polymorphisms in NLRP3 were previously associated with inflammation-related diseases, such as ulcerative colitis, inflammatory bowel disease, juvenile idiopathic arthritis, neonatal-onset multisystem inflammatory disease and type 2 diabetes [15-20]. Zhang et al. conducted a study in a Chinese population, and have reported that rs10754558 and rs10925019 of NLRP3 contribute to the development of ulcerative colitis [15]. Varghese et al. conducted a study in Caucasian, and they have reported that no association was found between NLRP3 polymorphisms and development of inflammatory bowel disease [16]. Yang et al. have reported that rs4353135 polymorphism may contribute to the pathophysiology of juvenile idiopathic arthritis in a Taiwanese population [17]. Dehghan et al. reported that SNPs in NLRP3 may play a critical role in regulation of inflammation underlying cardiovascular disease [19]. These above mentioned studies have suggested that NLRP3 polymorphisms were associated to susceptibility to inflammation-related diseases. Therefore, we hypothesized that functional SNPs in NALP3 inflammasome may involve in the development of primary gout.
Only one previous study reported the association of NALP3 polymorphisms with the susceptibility to primary gout in human [12]. Meng et al. have conducted a study including 480 cases with primary gout and 480 control subjects to investigate the association between genetic variants in 17 SNPs of NALP3 and the susceptibility to the development of primary gout in a Chinese population. However, they did not find any association between them [12]. In our study, we also conducted a study in a Chinese population, and we found that the GG genotype of rs3806268 in NLRP3 was associated with the development of primary gout, whereas no significant association was identified for the remaining six SNPs. The discrepancies of our results may be related to variation in origin of population, sample size, genotyping method and also by chance. Further clinical genetic studies are greatly needed to be performed to confirm our results.
Several limitations should be considered in our study. First, subject selection bias may overestimate the true size of effect or lead to spurious findings. Although there may exist selection bias for hospital-based study design, both cases and controls were matched on age and sex, which may have minimized the bias. Second, we investigated the association between selected seven SNPs in NLRP3 and development of primary gout, but the associations of the functional SNPs in NLRP3 with risk factors of gouty arthritis were not assessed. Therefore, a more detailed data are needed to analyze their association. Third, the rs10754558 and rs12565738 deviated from Hardy-Weinberg equilibrium which suggests that the sample size may not better represent the general population. Four, the sample size of the present study was small and all were selected from Chinese population, and thus the results could not representative of other ethnicities. Therefore, the results of our study should be repeated in other populations with more sample sizes.
In conclusion, we found a significant association between rs3806268 in NLRP3 gene and the risk of primary gout in a Chinese population. Further clinical and genetic studies are required to investigate the mechanisms underlying the association between NALP3 polymorphisms and the development of primary gout.
Acknowledgements
We thanks for the funding from Natural Science Foundation of Hubei Province (Grant No.2014CFC1039), and Health & Family Planning Research Program of Huangshi (Grant No.[2014]72).
Disclosure of conflict of interest
None.
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