| Aims:Primary gout is a group of heterogeneous diseases caused by long-term purine metabolic disorders(increased uric acid synthesis and / or reduced uric acid excretion)that causes elevated uric acid and consequently tissue damage.Epidemiological studies have shown that,due to the improvement of economic level,the incidence of hyperuricemia and gout in China increased year by year,especially in China's coastal areas.In addition,hyperuricemia and gout are also associated with obesity,diabetes,high blood pressure,dyslipidemia,cardiovascular and cerebrovascular diseases.Gout is closely related to serious harm to human health and has been a public health problem to be solved.Gout is a polygene genetic disease,but its exact pathogenesis is not yet clear.It is known that genetic factors are the major contributors to gout.The latest Genome-Wide Association(GWAS)study revealed that the single nucleotide polymorphisms of the rs179785 locus located in the intron region of the KCNQ1 gene were associated with gout in the Chinese Han population.The KCNQ1 gene encodes a potassium channel protein,but the association with gout is unknown.A preliminary study of my study group found that KCNQ1 regulates the secretion of IL-1? in monocytes.We speculated that KCNQ1 participate in the onset of gout through mononuclear cells.The aim of this study was to investigate the effect of rs179785 single nucleotide polymorphism on the expression of KCNQ1 gene in peripheral blood mononuclear cells of gout patients,so as to further reveal the relationship between KCNQ1 and gout and contribute to the study of new mechanism of gout.Methods:A total of 326 subjects from March 2015 to November 2015 at the Affiliated Hospital of Qingdao University were enrolled in this case-control study(159 gout cases vs 167 controls).All subjects were male.All subjects in this study excluded hypertension,heart disease,diabetes,dyslipidemia,malignancy,renal insufficiency,blood system diseases,infections,asthma,inflammatory bowel disease,autoimmune diseases and other serious system diseases.All subjects were carefully recorded clinical data,height,weight,blood pressure,and a total of 10 ml venous blood was collected in fasting(including ordinary biochemical tube and sodium citrate anticoagulant 5ml).Alanine aminotransferase(ALT),aspartate transaminase(AST),triglyceride(TG),total cholesterol(CH),glucose(GLU),uric acid(UA),urea nitrogen(BUN)and creatinine(CR)were measured by automatic biochemical analyzer;anticoagulant blood was used to extract genomic DNA,and all the subjectswere genotyped by Taqman? fluorescence quantitative PCR.The peripheral blood mononuclear cells were isolated from anticoagulant blood,the total RNA and the total protein were extracted from the mononuclear cells.The expression level of KCNQ1 gene mRNA was detected by reverse transcription-polymerase chain reaction(RT-PCR),and the expression of KCNQ1 protein was detected by Western blot.The level of IL-1? was detected by ELISA.Statistical analysis was performed using the SPSS 16.0 statistical software package.The genetic balance of the gout group and the control group was tested by Hardy-Weinberg genetic balance principle.Genotype and allele frequency distribution were expressed as frequencies and percentages,the comparison between groups using ? 2test.Continuous data were expressed as mean ± standard deviation,the comparison between two groups was analysed using independent sample t-test,while multiple groups comparison using one-way ANOVA and LSD method.All the statistical tests were two-sided at a significance of 0.05.Results:(1)Comparison of clinical data and biochemical indexes between gout group and control group: Compared with the control group,there was no significant difference between the two groups in ages(45.37 ± 11.56 vs 44.19 ± 9.83 years,P> 0.05);The BMI in the gout group(27.06 ± 3.34)was significantly higher than that in the control group(25.21 ± 3.10)(P <0.001);The systolic blood pressure(137.07 ± 15.36)and diastolic blood pressure(87.75 ± 12.19)in the gout group were significantly higher than those in the control group(systolic blood pressure 132.31 ± 18.53;diastolic blood pressure 84.15± 9.84)(P <0.001).The levels of serum alanine aminotransferase,blood glucose,triglyceride,total cholesterol and serum uric acid in the gout group were significantly higher than those in the control group(P <0.001),and the serum creatinine and urea nitrogen were significantly lower than those in the control group(P <0.001).There was no significant difference in the level of aspartate transaminase between the two groups(P> 0.05).(2)Genotype frequency and allele frequency comparison: SNP rs179785 genotype in the gout group and control group were distributed to the genetic balance,with the representative of the population.The frequencies of AA,AG and GG genotypes of rs179785 in the gout group were 30.19%,53.46% and 16.35% respectively,there was no significant difference compared with the control group(29.34%,46.11% and 24.55%,P =0.308).There was no significant difference in the frequency of A and G allele between the two groups(P = 0.327).(3)Comparison of KCNQ1 expression and IL-1? levels between gout group and control group: The expression of KCNQ1 mRNA was detected by real-time PCR.The expression of KCNQ1 mRNA in gout group was significantly higher than that in thecontrol group(1.12 ± 0.09 vs 1.08 ± 0.08),the difference was statistically significant(P =0.013).The expression of KCNQ1 protein was detected by western blot.We confirmed that KCNQ1 expressed in peripheral blood mononuclear cells of normal controls and gout patients.The expression of KCNQ1 protein in gout group(0.32 ± 0.07)was significantly higher than that in the control group(0.18 ± 0.04)(P <0.001),and the result was consistent with the mRNA test.The level of IL-1? in gout group is higher than that in normal control group(17.14 ± 1.06 vs 2.67 ± 0.07,P <0.001).(4)Comparison of biochemical indexes,KCNQ1 mRNA expression and IL-1? levels in different genotypes of gout group and control group:Biochemical indexes : The gout group and control group were divided into AA,AG and GG genotypes according to rs179785 genotype.The levels of serum alanine aminotransferase,blood glucose,triglyceride,total cholesterol,creatinine,urea nitrogen and uric acid were compared between the three subtypes of the gout group and the control group respectively,but there was no significant difference both in the two groups(P>0.05).KCNQ1 mRNA expression level: Compared with AA(1.10 ± 0.07)or AG(1.08 ±0.08)genotype,the KCNQ1 mRNA expression level of GG genotype(1.04 ± 0.08)was significantly decreased in the control group(P = 0.002).In the gout group,the KCNQ1 m RNA expression level of GG genotype was lower than that of the other two genotypes too,but there was no significant difference between the three subgroups(P> 0.05).IL-1? levels: There was no significant difference in serum IL-1? levels between the three genotypes both in gout and normal control group(P> 0.05).Conclusions:KCNQ1 was associated with gout in Han Chinese.The single nucleotide polymorphisms of rs179785 may be related to the onset of gout by affecting the expression of KCNQ1 gene in monocytes.Study of greater sample size and data from different regions and different races is needed in the future to validate the findings of this study.There is still a need to conduct further functional studies to raise awareness of the pathogenesis of gout. |
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