Value Of Single Nucleotide Polymorphism Of RANKL And OPG Gene, Expression Of Protein On Assessing Disease Activity,bone And Joint Damage In Patients With Rheumatoid Arthritis

BackgroundReceptor or Activator of NF-KB Ligand(RANKL) / Receptor or Activator of NF-KB(RANK) / Osteoprotegerin(OPG) system is proved to closely correlate with bone matablism and bone injury. They are all considered as members of tumor necrosis factor gene family. This system, which becomes the hot topic in osteoimmunology recently, is also tightly associated with immune system. Some researches indicated certain single-nuclear polymorphism(SNP) of RANKL /RANK/OPG system may play an important role on susceptibility of osteoporosis(OP), any autoimmune disease and their bone injury. Rheumatoid arthritis(RA) is a systemic autoimmune disease characterized by chronic sonovitis in joints, which may lead to invasion of synovial tissue into the adjacent cartilage matrix with damage of bone and joint as a consequence. Local bone erosion and general bone loss including osteoporosis are the main mainifests of bone and joint injury in RA, deformity of joint, dyfunctiong and disability are the inevitable outcomes with recurrent attacks over time. Cumulative studies indicated that many medications could not control the occurrence and progress of bone and joint damage in RA, although they could effectively surpress the synovitis. It is emphasis and difficult point for us to explore the mechanism of bone and joint injury and probe new therapic agents for inhibiting its happening.ObjectiveTo explore the SNP in Receptor of Activator of Nuclear Factor kappa B Ligand(RANKL)(TNFRSF11) 401A>G(rs2277438) and Osteoprotegerin(OPG) gene(TNFRSF11B) 1181G>C(rs2073618), 163A>G(rs3102735), measure the peripheral serum level of above genetic expression product: OPG and s RANKL(soluble RANKL), assess the value of SNP in RANKL and OPG gene and the levels of expression protein on disease susceptibility, disease activity, bone and joint injury – general bone loss(BMD) and local bone erosion(Sharp score in hands) in patients with rheumatoid arthritis(RA).MethodsTwo hundred RA patients(28 males and 172 females) and 201 matched controls(30 males and 101 females) were analyzed from Han Chinese populations by case–control design, and their samples were genotyped using a panel of two SNP markers(rs2073618 +1181G/C, rs3102735 +163A/G) within the OPG gene(TNFRSF11B) and one SNP marker(rs2277438 +401A/G) within the RANKL gene(TNFRSF11) by ligase detection reactions(LDRs). Peripheral serum levels of s RANKL and OPG were detected with ELISA. Radiographic changes in both hands of all RA patients were assessed by Sharp’ method(Sharp score). Bone mineral density(BMD) values at femur and lumbar spine were assessed using dual-energy X-ray absorptiometry in 113 patients and 120 contrlos. Joint counts for tenderness and swelling, Health Assessment Questionnaire(HAQ) score, Visual Assessment Score(VAS) of overall pain, erythrocyte sedimentation rate(ESR), C-reactive protein(CRP), rheumatic factor(RF) values,anti-cyclic citrulline polypeptide(anti-CCP) and disease activity score(DAS28) were measured in detail by rhumatologues simultaneously.Results1. No significant differences in the distribution frequency of the alleles and genotypes of TNFRSF11B(rs2073618 and rs3102735) and TNFRSF11(rs2277438) were observedbetween RA and control(P>0.05).2. The haplotype analyses for TNFRSF11 B and TNFRSF11 SNPs showed the rs2073618/rs2277438/rs3102735 GGG haplotype reduced the risk of RA(1.5% vs 6.0%)(P=0.008, OR 0.216, 95% CI: 0.081 to 0.575) and the rs2073618/rs2277438/rs3102735 GAG haplotype increased the risk of RA(14.5% vs 8.4%)(P=0.007; OR 1.862, 95% CI: 1.179 to 2.943).3. The levels of s RANKL [79.69(43.59) vs 70.13(30.61),z=2.368,P=0.018] and the ratio of s RANKL/OPG [0.41(0.20) vs 0.27(0.26),z=5.259,P<0.0001] in RA increased significantly, while serum OPG [202.86(123.44) vs 285.14(318.61), z=4.029,P<0.0001] decreased obviously, compared with normal controls.4. In comparison with normal controls, BMDs of all detected regions such as femur neck, Ward area, greater trochanter, total femur, L2, L3, L4 and L2-4 in RA decreased obviously(P<0.01-0.0001). The incidence of osteoporosis in RA(31.9%) was higher than that in normal controls(13.3%)(x2=11.520,P=0.001). Patients with osteoporosis had elder age(59.03±12.67 vs 51.75±11.62,t=3.021,P=0.003), more numbers of swollen joints[10.5(7) vs 7(6),z=2.138,P=0.032], higher scores of space narrowing [54.5(65) vs 11.5(30),z=3.210,P=0.001], more severe bone erosion of joint [47(82) vs 10(31),z=3.263,P=0.001] and higher scores of total Sharp scores [103(146.5) vs 22(62.75),z=3.355,P=0.001] by X-ray’sharp method than those of patients without osteoporosis. There were few differences in the changes of other clinical and laboratory parameters including serum s RANKL and OPG levels between the two groups(P>0.05).5. Patients with TNFRSF11(rs2277438) AA or GG genotypes(n=62) had significantlyhigher BMD values compared to those with AG genotypes(n=39) at lumbar spine 3(1.05±0.22 vs 0.93±0.26, t=2.314,P=0.023), lumbar spine 4(1.06±0.24 vs 0.94±0.28, t=2.27,P=0.030), lumbar spine 2-4(1.04±0.21 vs 0.89±0.28, t=2.788,P=0.007) and clear lower Sharp score in both hands [15.50(46.75) vs 43.00(108.75),z=2.419,P=0.016],obviously lower peripheral serum s RANKL level [66.18(29.05) vs 84.51(73.09),z=2.491,P=0.013],respectively. There existed no obvious discrepancies in BMD at all detected positions, Sharp score and peripheral serum OPG level among different genotype groups of TNFRSF11B(rs2073618 or rs3102735)(P>0.05).6. The tender joint counts(12.69±6.36 vs 9.04±5.22,t=2.352,P=0.022), tender joint index(17.19±8.09 vs 11.88±6.32,t=2.732,P=0.008),morning stiffness time [30(180) vs 5(30),z=2.161,P=0.031], VAS score [6(2) vs 4(2),z=2.962,P=0.003] and DAS28 score(6.20±0.97 vs 5.30±1.61,t=2.388,P=0.023)differed significantly between patients with TNFRSF11B(rs2073618) AA or GG genotypes(n=60) and AG genotypes(n=40). There were no apparent dissimilarities on clinical and laboratorial disease activity factors among different genotype groups of TNFRSF11B(rs3102735) or TNFRSF11(rs2277438)(P>0.05).7. The level of s RANKL in peripheral blood of RA patients correlated with the level of OPG which presented as a positive linear correlation(r=0.568,P<0.0001). BMD at all deteted region negatively correlated with age(P<0.0001). BMD exclusive of L2-4 negatively correlated with the X-ray sharp scores in RA(P<0.05-0.0001). There was a negative line corelations between BMD of L2-4 and the ratio of s RANKL/OPG(P<0.05-0.0001). Negative linear correlations were also found between BMD of femoral neck, ward area, G.T., total hip and disease duration, numbers of swollen joints in RA(P<0.05-0.0001). There were also negative linear correlations between BMD of L2, L3, L4 and CRP(P<0.05-0.001). There were no any correlations between serum s RANKL,OPG or ratio of s RANKL/OPG levels and any parameters which could reflect disease activity(P>0.05).8. Defining age, disease duration, level of s RANKL and OPG in peripheral blood, ratio of s RANKL/OPG, rheumatoid factor, DAS28, anti-CCP antibody as independent variable, and the Sharp score as the dependent variable, analysis of multivariate linear regression showed that and age(β=-0.179, t=2.274, P=0.026), disease duration(β=0.725, t=9.078, P<0.0001) and ratio of s RANKL/OPG in peripheral blood(β=0.166,t=2.104, P=0.039) were the contrubutors for Sharp score(R2=0.570,F=33.137,P<0.0001). Neither serum s RANKL, OPG levels nor ratio of s RANKL/OPG levels were found as the contributors for DAS28 by analysis of multivariate linear regression(P>0.05).9. Analysis of binary logistic regression(backward: LR) was executed to investigate the potential risk factor for RA-induced osteoporosis. A new target, accounting for whether one patient with RA accompanied with OP or not, was defined as a dependent variable(0=No, 1=Yes). Age, sex, BMI, duration of disease, use of glucocoticoid or not, DAS28, Sharp score were defined as independent variables, results showed that age(OR=1.058,P=0.014, 95%CI:1.012-1.106) and Sharp score(OR=1.021,P=0.001,95%CI:1.008-1.033) were the risk factors for the occurrence of osteoporosis in patients with RA(R2=0.262,x2=17.692,P=0.001). When RANKL(rs2277438) SNP(1=AA or GG,2=AG), OPG(rs2073618) SNP(1=CC or GG, 2=CG),OPG(rs3102735) SNP(1=AA or GG, 2=AG) were added as independent variables, analysis of logistic regression showed genotypes of TNFRSF11(rs2277438) was the unique risk factor for the occurrence of osteoporosis in RA(OR=2.932,P=0.048,95% CI:1.008-8.529).Conclusion1. SNPs of OPG gene(rs2073618 and rs3102735) and RANKL gene(rs2277438) have no correlations with susceptibility of Anhui Han RA population. The rs2073618/rs2277438/rs3102735 GGG haplotype may be a protective factor against RA, while GAG haplotype probably increases susceptibility to RA.2. SNP of RANKL and OPG serve differently in RA. SNP of TNFRSF11(rs2277438) may affect serum s RANKL level in patients with RA, further lead to local bone erosion and influence BMD value at lumbar spine. SNP of TNFRSF11B(rs2073618) may have an influence on disease activity of RA.3. General bone loss and local bone erosion in RA are in clear association with each other. approximate 2.40 times that of the normal subjects. RANKL/OPG system in the linkage.