The Clinical Correlations Of Single Nucleotide Polymorphism Of Vascular Endothelial Growth Factor And Rheumatoid Arthritis

Background:Rheumatoid arthritis(RA)is a chronic,systemic and progressive inflammatory disorder primarily characterized by persistent chronic synovitis,progressive erosions,and cartilage destruction,which may cause deformed and painful joints,even resulting in loss of function.RA affects 0.5-1%adults in the developed world,with 5-50 per 100,000 people being newly diagnosed with the condition,annually.Furthermore,the onset of RA is most frequent in females and the elderly.RA,without intervention,may eventually cause joint damage,disability,reduced quality of life,cardiovascular events and other comorbidities.Although a variety of environmental and behavioral factors confer a high risk of developing RA,genetic factors are suspected to account for up to 50%of the risk for developing RA.Vascular endothelial growth factor(VEGF)is an important signaling protein and a secreted ligand released by cells to stimulate vasculogenesis and angiogenesis.VEGF plays an important role in regulating angiogenesis through promoting vascular endothelial cell growth,migration,and lumen formation.In addition,VEGF is also capable of inducing proinflammatory change,seen in chronic inflammation,which involves leukocyte accumulation,collagen deposition,and blood vessel remodeling.VEGF is also released by inflammatory cells and,in turn may represent the inflammatory component in many disease processes.In this context,elevated serum VEGF levels are associated with the duration and severity in many disorders,such as RA.The gene vegf of human is located on chromosome 6p21.3,containsing 8 exons.The gene vegf,is also an independent risk factor for RA severity,and correlates with multiple disease parameters,such as disease activity,joint damage,and functional disability.Two common single-nucleotide polymorphisms(SNPs),rs833070(G>A)located in intron 2 and rs3025030(G>C)located in intron 5,are suspected to result in altered protein expression of VEGF and have strong links to the onset of RA,although some studies dispute such a link.Our present study evaluates the common SNPs in the VEGFA gene[rs833070(G>A)and rs3025030(G>C)]for their influences on the circulating levels of VEGF protein and their effects on disease activity and synovial lesions in RA.Therefore,we attempted to investigate 2 common single-nucleotide polymorphisms(SNPs)of VEGF for their influences on,VEGF levels in serum,disease activity,and synovial lesions in rheumatoid arthritis(RA).Methods:The study was approved by the Ethics Committee of the Zhujiang Hospital.The written informed consent was proved by each eligible patient and the study conformed to the Declaration of Helsinki.Patients:This study was conducted between October 2010 and May 2012 on a population of RA patients(n=98)from the Zhujiang Hospital of Southern Medical University.Patients with RA who satisfied all aspects of the 2010 American College of Rheumatology(ACR)and European League Against Rheumatism(EULAR)classification criteria for RA were recruited to the study.There were 32 male and 66 female RA patients,with an age range of 18-80 years(mean age,50.82±12.53 years)and mean disease duration of 3.0 years(range,0.8-8.0 years).A group of 100 healthy volunteers(male,35;female,65;age range,15-85 years;mean age,48.67±13.41 years)were enrolled as the control group from the Medical Examination Center of Zhujiang Hospital.No statistical difference in age or sex existed between the RA group and the control group.Patients with systemic lupus erythematosus(SLE),Sjogren syndrome(SS),juvenile idiopathic arthritis(JIA),ankylosing spondylitis(AS),polymyositis(PM),dermatomyositis(DM),other genetic diseases,hereditary diseases,severe heart,lung,liver,or kidney dysfunction,benign or malignant tumors,or other related diseases were excluded from this study.Clinical data collection:General clinical data of all the study subjects such as age,sex,disease duration,present and past medical history,and the history of hereditary disease were collected and recorded.Common clinical laboratory parameters for RA including routine blood test,erythrocyte sedimentation rate(ESR),rheumatoid factor(RF)and acute C-reactive protein(CRP)were collected.Detection of VEGF polymorphisms:Morning fasting venous blood samples(4 ml)were collected from all subjects.EDTA-K2 was used as an anticoagulant for blood collection.Genomic DNA was extracted from the white blood cells collected from venous blood by using a DNA Extraction Kit according to the manufacturer's protocol.The SNP genotyping was conducted using the matrix-assisted laser desorption ionization time of flight mass spectrometry.The purification of PCR products was performed by demineralization with resin.The DNA samples were transferred from the 384-well plate onto the MassARRAY SpectroCHIP covered with matrix,and detected with the mass spectrometer.The genotyping analysis was performed by using software.Enzyme-linked immunosorbent assay:The serum concentrations of VEGF were measured by the ELISA method using an ELISA kit.Disease activity score(DAS):A total of 28 joints,including shoulders,elbows,wrists,metacarpophalangeal joints,proximal interphalangeal joints of hands,and knee joints,were scored by DAS scoring system.The number of swollen and tender joints in each RA patient was examined and recorded,and the DAS28 score was calculated by combining the ESR and self-evaluation in patients with RA.Ultrasonography:The ultrasonic examination was performed.The 28 joints of each RA patient were examined using direct contact method.All patients adopted the supine or sitting position,adjusted based on the examined joints,with the examined joint fully exposed.An experienced physician with ultrasound expertise,who did not know the disease status and treatment status of the patients,monitored the ultrasound examinations.Ultrasonic examination:Check when using Siemens S2000 color doppler ultrasonic diagnostic instrument testing.Power Doppler evaluation:The semi-quantitative signal of each point of synovium in the 28 joints was observed and classified.(0-3 grade)Recording items:Recording items consisted of the followings:(1)US joint count SH:the number of joints with thickened synovium;(2)US joint count SF:the number of joints with effusion;(3)US joint count PD:the number of joints with energy signals;(4)US index SH:total synovial thickening score,which was the sum of the synovial thickening score of the 28 joints;(5)US index SF:total joint effusion score,which was the sum of joint effusion score of the 28 joints;(6)total sharp scores(TSS):the sum of the highest power Doppler score of each joint.Statistical analysis:Statistical analysis was conducted using SPSS 19.0 software(SPSS Inc.,Chicago,IL,USA),and the data are represented as means±standard deviation(SD),median,or percentage.The statistical comparison between 2 groups was conducted using the t-test or the analysis of variance(ANOVA).Genotype distribution in the control group was tested by Hardy-Weinberg equilibrium(HWE).The differences in genotype and allele distribution between the RA group and the control group are represented as odds ratio(OR)and 95%confidence interval(CI).P values for all tests are 2-tailed,and<0.05 was considered as statistically significant.ResultsCharacteristics of RA patients:The demographic and clinical characteristics of subjects,consisting of 98 RA patients and 100 controls,from a hospital based population at the time of recruitment.Comparisons between the RA group and the control group demonstrated that ESR,RF,acute CRP,and serum VEGF levels were significantly higher in patients with RA than in controls(all P<0.05).No statistical differences were seen in age,gender,WBC,red blood cell(RBC),hemoglobin(HGB)and platelet(PLT)count(all P>0.05).Distributions of VEGF SNPs:The genotype and allele frequencies of the VEGF genetic polymorphisms,rs833070(G>A)and rs3025030(G>C),Genotypes of VEGF rs833070(G>A)and rs3025030(G>C)in controls were distributed in accordance with HWE(all P>0.05).The distributions of allele and genotype frequencies of the VEGF rs833070(G>A)in the RA group were significantly different from the control group(all P<0.05).The VEGF rs833070 AA genotype and A allele frequency were significant higher in the RA group than in the control group(AA genotype frequency:59.2%vs.43.0%,P<0.05;A allele frequency:78.1%vs.67.0%,P<0.05).We detected that the polymorphisms located in rs833070 of the VEGF gene were strongly linked with an increased risk of RA(AA vs.GG+AG,OR=1.78,95%CI=0.621-5.103,P=0.007;A vs.G,OR=1.812,95%CI=1.156-2.840,P=0.009).Similarly,statistical differences in the distribution of genotypic and allele frequencies of VEGF rs3025030(G>C)were observed between the RA group and the control group(all P<0.05).VEGF rs3025030 CC genotype frequency was 50.0%in RA patients vs.67.0%in controls(P<0.05).In addition,VEGF rs3025030 C allele frequency was 69.4%in RA patients and 81.0%in controls(P<0.01).VEGF rs3025030 GG genotype might be related to a reduced risk of RA(GG vs.GC+GG,OR=0.488,95%CI=0.387-0.785,P=0.019).Comparison of serum VEGF levels:The comparison of VEGF serum levels between different genotypes of rs833070 and rs3025030 in RA patients.The comparisons of serum levels of VEGF based on genotypes of rs833070 showed that the serum level of VEGF was significantly higher in RA patients with AA genotype(1737.5±250.3 pg/mL)compared to RA patients with GG genotype(623.7±183.6 pg/mL)and AG genotype(1030.2± 107.9 pg/mL)(P<0.01)(Figure 1).Additionally,serum VEGF levels in RA patients with rs3025030 CC genotype were lower than in patients carrying the rs3025030 GC genotype and GG genotype(1232.94±358.39 vs.1586.67±398.39 vs.1812.37±309.07,P<0.01).Association of VEGF SNPs with disease severity in RA and DAS28 stratified by the genotypes of the VEGF rs833070 and rs3025030 polymorphisms in all patients with RA.The mean DAS28 of all RA patients was 4.68±0.76,and RA patients were classified into low-activity RA patients(DAS28?5.1)and high-activity RA patients(DAS28>5.1)according to DAS28 score system.Statistical difference was observed in DAS28 between different genotypes of VEGF rs833070 in RA patients(P<0.05).RA patients carrying rs833070 AA genotype had a higher proportion of DAS28>5.1 compared with patients carrying rs833070 GG and AG genotype(100%vs.33.3%vs.56.8%).No statistical difference was detected in DAS28 between different genotypes of VEGF rs3025030 in RA patients(P>0.05).Synovial lesions in RA:Among the 98 RA patients,synovial thickening was detected in 82 patients and in a total of 1,065 joints,with a positive rate of 38.8%(1065/2744).Joint effusion was observed in 90 patients and 747 joints,with a positive rate of 27.2%(747/2744).Doppler signal in 63 patients and 661 joints,showed a positive rate of 24.1%(661/2744).Association of VEGF SNPs with synovial lesions in RA:As illustrated in Table 6,there was statistical significance in the positive rate of synovial thickening,joint effusion and synovial angiogenesis between different genotypes of VEGF rs833070 and rs3025030(P<0.05).The positive rate of synovial thickening,joint effusion and synovial angiogenesis in RA patients carrying rs833070 AG and AA genotype was evidently higher than patients carrying rs833070 GG genotype(P<0.05).Similarly,the positive rate of synovial thickening,joint effusion and synovial angiogenesis in RA patients with rs3025030 GC and GG genotype was higher compared to RA patients with rs3025030 CC genotype(P<0.05).Conclusions:Our study provides evidence that VEGF polymorphisms might be important indicators of disease activity and synovial lesions,and prognostic factors in evaluating the treatment effectiveness in RA.