| Objective 1)To explore the association of KOA,TCM constitution and syndromes with SNPs in ADAMTS-4,-5 gene;2)To research the association of KOA,TCM constitution and syndromes,as well as the genotypes of ADAMTS-4,-5 SNPs with serum level of ADAMTS-4,-5.Methods 1)288 people were randomly selected for this study(188 KOA cases and 100 control),all patients were told to sign informed consent to study.General information such as gender,age,BMI and occupational category were recorded,as well,according to the questionnaire of TCM constitution judgment standard and the diagnostic criteria of TCM syndromes,the TCM constitution and TCM syndromes were judged.Meanwhile,tag SNPs which will be studied in this experiment were chosen from SNP database,such as NCBI db SNP and 1000 Genomes database.Importantly,Haplo View software was used to choose tag SNPs and analyze the experimental data.Whole blood and serum which was donated by participants was collected and stored.All of the SNPs in this experiment were genotyped by SNPs Mass Spectrometry,and the serum ADAMTS-4 and ADAMTS-5 concentration were detected by ELISA kit.2)Experimental information were summarized for statistical analysis.When the association of KOA,TCM constitutions and syndromes with general risk factors were analyzed,chi-square test was performed first,and then the Logistic regression analysis was carried out.Hardy-Weinberg balance test were performed by Haplo View software.SNPs analysis includes alleles,genotypes,genetic models,linkage disequilibrium(LD)analysis,and haplotype analysis.Association of KOA,TCM constitutions and syndromes were analyzed firstly by chi-square test,and secondly Logistic regression analysis.LD analysis were performed by Haplo View software.Comparison of serum ADAMTS-4 and ADAMTS-5 levels between groups were analyzed by ANOVA combined with Logistic regression analysis.When multiple independent pairwise comparisons were made between groups in the same dataset,the more rigorous Bonfferoni correction P value(0.05 / comparison times)was used as the statistical significance level.Results 1)There were significant differences in general risk factors(sex,age,weight type,occupation)between KOA and control group(P<0.05).In addition,the regression analysis showed that,except for age intervals,sex(female,OR=4.581),BMI(obesity,OR=3.493),occupation category(physical strength,OR=2.334)were all related to KOA susceptibility(P<0.01).2)In KOA group,constitutions of yang asthenia(29.3%),qi asthenia(22.3%),yin-yang harmony(18.6%)and yin asthenia(14.9%)were the main TCM constitutions;there was significant difference in the overall distribution of TCM constitutions between KOA and control group(P=0.001<0.05).In addition,the biased constitutions of KOA group were more than that of control group.In KOA group,the biased constitutions were mainly mixed biased constitutions,in which constitutions of yang asthenia(11.76%),qi asthenia(8.50%),yang and qi asthenia(8.50%),yin asthenia(7.84%),qi and yin asthenia(5.88%),yang asthenia and qi stagnation(4.58%)were the most common TCM biased constitution.Additionally,chi-square test and Logistic regression analysis showed that there was no correlation between TCM constitutions and general factors,as well as K-L radiographic grade(P>0.05).3)In KOA group,the liver and kidney deficiency syndrome was the most common TCM syndrome,followed by cold-dampness obstruction syndrome(34.6%),deficiency of both qi and blood syndrome(11.7%),stagnation of qi and blood stasis syndrome(7.4%).Symptoms of most of KOA patients were complicated and often combined with some symptoms of other syndromes.Chi-square test did not find correlation of TCM syndromes and general factors as well as TCM constitutions(P>0.05),but interestingly,Logistic regression analysis found that KOA TCM syndromes were associated with general factors and TCM constitutions.The results showed that liver and kidney deficiency syndrome is related to female(P=0.000,OR=4.906),obesity(P=0.048,OR=2.490),manual occupation(P=0.040,OR=2.176),and three common TCM constitutions-yang asthenia(P=0.003,OR=4.296),qi asthenia(P=0.009,OR=3.826)and yin asthenia(P=0.009,OR=4.637).In addition,cold-dampness obstruction syndrome was correlated with female(P=0.001,OR=3.762),overweight(P=0.036,OR=2.423)and obesity(P=0.000,OR=5.876),manual occupation(P=0.009,R=3.046),and TCM constitution of qi asthenia(P=0.005,OR=4.355).What's more,deficiency of both qi and blood syndrome is associated with female(P=0.009,OR=5.041)and three common TCM constitutions-yang asthenia(P=0.030,OR=5.349),qi asthenia(P=0.041,OR=5.369)and yin asthenia(P=0.007,OR=9.916).Moreover,stagnation of qi and blood stasis syndrome was also connected with female(P=0.022,OR=6.500)and TCM constitution of yang asthenia(P=0.031,OR=4.885).4)In this study,16 tag SNPs were selected,of which 13 were in accordance with the HardyWeinberg equilibrium and entered the follow-up study.The distribution of A allele at rs2249350(A<C)of ADAMTS-5 gene was less in KOA group(P=0.016,OR=0.761)than that in control group.Similarly,the AA genotype of KOA group was less than that of CA+CC(P=0.031).In addition,Logistic regression analysis showed that AA genotype decreased the risk of KOA(P=0.004,OR=0.288)compared with CC.The recessive gene model regression analysis also found that the genotype of AA compared with CA+CC was negatively correlated with the susceptibility of KOA.However,the correlation between other loci and KOA was not significant(P>0.05).5)LD and haplotypes analysis showed that there were 2 haplotype blocks and 8 haplotypes in ADAMTS-5 SNPs included in this study.The frequency of haplotype GC(rs229054,rs2249350)in KOA group was significantly higher than that in control group(P<0.019).In addition,the haplotype GA(rs229054,rs2249350)in the KOA group was significantly lower than that in the control group(P>0.05).There was no significant difference in the distribution of other haplotypes between the two groups(P>0.05).6)In this study,chi-square test and Logistic regression analysis showed that there was no correlation between KOA TCM constitutions and SNPs(P>0.05),however,there was a negative correlation between TCM syndrome of liver and kidney deficiency and AA genotype at rs2249350(A<C)locus of ADAMTS-5 gene in KOA patients.7)Comparison of serum ADAMTS-4 and ADMATS-5 levels showed that the two proteins in the KOA group were significantly higher than those in the control group(P<0.05).In addition,Logistic regression analysis showed that the serum ADAMTS-4(P=0.000,OR=1.055)and ADAMTS-5(P=0.000,OR=1.055)was positively correlated with the susceptibility to KOA.Moreover,general stratification comparison showed that the serum ADAMTS-4 level in older patients with KOA was higher than that in middle-aged KOA patients(P<0.05).There was no significant difference between the two protein levels in different TCM physique types,but the ADAMTS-5 level in KOA patients with TCM constitution of blood stasis was significantly lower than that in KOA patients with non-blood stasis constitution(P=0.001<0.05).Additionally,regression analysis showed that there was a negative correlation between TCM constitution of blood stasis and the level of serum ADAMTS-5.No positive results were found in serum protein levels of KOA TCM syndromes or related SNPs genotypes,including Logistic regression analysis.Conclusion 1)Female,obesity,physical work increased susceptibility to KOA,while age may not be related to KOA;2)The main type of TCM constitutions in KOA patients was biased constitution,in which most of them were mixed biased constitutions.Yang asthenia,qi asthenia and yin asthenia were the main TCM constitutions.Compared with constitution of yin-yang harmony,yang asthenia,qi asthenia and yin asthenia increased the susceptibility to KOA.TCM constitutions in KOA patients may not be related to gender,age,occupational category,K-L radiographic grade.3)In KOA patients,the liver and kidney deficiency syndrome was the most common TCM syndrome,followed by cold-dampness obstruction syndrome,deficiency of both qi and blood syndrome and stagnation of qi and blood stasis syndrome.Symptoms of most of KOA patients were complicated and often combined with some symptoms of other syndromes.The TCM syndromes of KOA may be related to the gender,BMI,occupational category,as well as TCM constitutions.Specifically,female,overweight and obesity,manual occupation and TCM constitution of qi asthenia may increase the susceptibility of KOA with cold-dampness obstruction syndrome;in addiction,female,overweight and obesity,manual occupation and TCM constitution of yang asthenia,qi asthenia and yin asthenia may increase the susceptibility of KOA with liver and kidney deficiency syndrome;meanwhile,female and TCM constitutions of yang asthenia,qi asthenia and yin asthenia may increase the susceptibility to KOA with deficiency of both qi and blood syndrome;female and TCM constitution of yang asthenia may increase the susceptibility of KOA with stagnation of qi and blood stasis syndrome.4)The rs2249350(A<C)locus of the ADAMTS-5 gene may be associated with KOA.The A allele of the locus may be a protective recessive gene,while C may be a pathogenic dominant gene.In addition,the AA genotype may be a protective genotype,and reduce the risk of KOA,while CC+CA genotypes may increase the susceptibility of KOA.The genetic pattern of AA genotype may be recessive,and the other SNPs loci may not be related to KOA.What's more,there were 2 LD regions in SNPs of ADAMTS-5 gene and 8 haplotypes in ADAMTS-5 SNPs included in this study,in which GC(rs229054rs2249350)haplotypes may be negatively correlated with susceptibility to KOA,while GA may be negatively correlated with KOA,and other haplotypes may not be associated with KOA.5)TCM constitutions of KOA patients may not be related to the 13 SNPs loci in this study.The AA genotype at rs2249350(A<C)locus of ADAMTS-5 gene may be negatively correlated with susceptibility to KOA with liver and kidney deficiency syndrome,while other KOA syndrome types may not be associated with SNPs in this study.6)The expression of ADAMTS-4 and ADAMTS-5 protein may be increased in KOA population.In addition,the level of serum ADAMTS-4 in older KOA patients may be higher than that in middle-aged people.The serum ADAMTS-5 level of KOA patients with TCM constitution of blood stasis may be lower than that of other types of KOA.The TCM syndromes of KOA and 13 SNPs locus genotypes may not be associated with serum ADAMTS-4 and ADAMTS-5 protein levels in KOA patients. |
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