A Study On Methotrexate Treatment Response In Patients With Rheumatoid Arthritis: Genetic Contributions Of RFC-1 G80A And ABCB-1 C3435T Polymorphism To Methotrexate Polyglutamates In Erythrocytes

BackgroundRFC-1 and ABCB1 are the transport proteins for Methotrexate(MTX) flowing into or out of cells. Recently, researchers have found that RFC-1 G80 A and ABCB1 C 3435 T genes polymorphism is associated with treatment effect and adverse reactions of MTX. We hypothesized that RFC-1 G80 A and ABCB1 C 3435 T may cause the adverse effect of MTX by influencing methotrexate polyglutamates(MTX-PGs). Using real-time quantitative polymerase chain reaction(real-time fluorescent quantitative polymerase chain reaction, FQ-PCR) method for detection of RA patients in this region, the association between RFC-1 G80 A and ABCB1 C 3435 T genes polymorphism with concentrations of MTX-PGs in erythrocytes of RA patients was investigated. The underlying mechanism that RFC-1 G80 A and ABCB1 C 3435 T genes polymorphism cause MTX treatment response and adverse reaction was explored.Objectives:To establish a novel enzyme-linked immunosorbent assay(ELISA) method to determine the concentration of MTX-PGs in erythrocytes of patients with rheumatoid arthritis. To investigate the correlation between RFC-1 G80 A and ABCB1 C 3435 T polymorphism and MTX-PGs concentration for exploring the possible mechanism that RFC-1 G80 A and ABCB1 C 3435 T polymorphism affects MTX treatment response and adverse reaction in rheumatoid arthritis, which provide a theoretical foundation for individualized treatment of rheumatoid arthritis.Methods:1. Inpatients and outpatients with treatment of MTX, fulfilling the American College of Rheumatology/European League Against Rheumatism criteria for RA, were enrolled, from the department of rheumatology, Southwest Hospital, during from Oct. 2012 to Oct. 2012. The demographic information and MTX dose were registered and 4mL venous blood per patient was collected 72 hours after patients took MTX. In total, 95 patients were assessed in the present study, including 18 males and 77 female.2. MTX-PGs concentration in red blood cells of rheumatoid arthritis was determined by ELISA. The DNA of whole blood in RA patients was extracted by the method of phenol chloroform. Using real-time quantitative polymerase chain(FQ-PCR) reaction method RFC-1 gene in patients with RA G80 A and ABCB1 gene C 3435 T 2 SNP loci were genotyped3. Multiple linear regression analysis of body weight, dosage, body mass index, body surface area per unit weight of oral dosage unit body surface area affected and the age of the oral dose MTX-PGs concentration of red blood cells, using ANOVA gene SNPs correlated with drug concentration analysis.Results 1、1. A novel method of ELISA for quantifying the MTX-PGs in red blood cells has been established. MTX-PGs interference from plasma folate, between 0.1ng/ ml ~ 10ng/ ml had a good linear relationship(R2 = 0.9962), the regression equations are y =-0.4772X-0.1472(R2 = 0.9902), the detection sensitivity of up to 0.087ng/ mL, the detection limit is: 0.2ng/ mL, the experiment recoveries: 94.2% ~ 112.8%, the average recovery: 103.0%; RSD ranges: 7.2% ~ 17.7% average RSD: RSD 13.5%, less than 15%, indicating that the present method has higher accuracy. Precision experiments: The intra RSD is: 3.3% to 8.7%, the average grant in the RSD as follows: 6.5%, inter-assay RSD is: 7.6% to 9.9%, the average grant is between RSD: 8.3%, less than 10%.2、MTX-PGs within the concentration range of 95 cases of red blood cells in patients with RA is: MTX-PGs concentration range of 59.9 ~ 149ng/ ml, the mean concentration was 88.09 ± 19.29ng/ ml. Age, sex, weight, body mass index and body surface area of the MTX-PGs no significant effect on the concentration, dose and dose per body weight and MTX-PGs concentrations were positively correlated(P = 0.000, P = 0.049)3、95 cases of methotrexate in patients with rheumatoid arthritis, RFC-1 G80 A genotype distributions(GG/ AG/ AA) to(/47.4% 32.5%/ 20.0%), genotype ABCB1 C 3435 T’s(TT/ CT/ CC) was(18.9%/46.3%/34.7%), genotypes are in line with Hardy-weiberg equilibrium(χ2 = 1.309, P = 0.595; χ2 = 0.654, P = 0.721), with a group of samples were representative.4、No MTX-PGs concentration RFC-1 G80 A and ABCB1 C 3435 T statistically significant difference between the different genotypes, all P values were greater than 0.05, indicating that RFC-1 G80 A and ABCB1 C 3435 T polymorphism of these two sites in this study MTX-PGs concentration does not affect the red blood cells.Conclusion1. the method for measuring MTX-PGs by ELSIA developed in this study is simple, accurate, strong application, meet the clinical blood concentration monitoring.2. RA patients in Chongqing RFC-1 G80 A distribution of the gene(GG/ AG/ AA) :( 32.5%/47.4%/ 20.0%), with no significant difference in the distribution of Chinese Han population3. RA patients in Chongqing distribution of gene ABCB1 C 3435 T(TT/ CT/ CC) :( 18.9%/46.3%/34.7%) with no significant difference in the distribution of Chinese Han population4. MTX-PGs concentrations in erythrocyte of RA patients ranged from 59.9 ~ 149ng/ ml, and the mean concentration was 88.09 ± 19.29 ng / ml5. MTX-PGs concentrations in erythrocyte of RA patients were not relevant with age, sex, height, body mass index.6. MTX-PGs concentrations in erythrocyte of RA patients were relevant with MTX treatment doses, dose-related weight unit.7. the RFC-G80 A and ABCB1 C 3435 T genes polymorphism of RA patients in Chongqing was not correlated with MTX-PGs concentrations in erythrocyte.