Association Of UGT1A9、UGT1A8、UGT2B7、ABCC2、ABCG2�'�SLC01B3 Polymorphisms With Adverse Reactions Caused By Mycophenolate Mofetil(MMF) In Kidney Transplant Recipients

BackgroundsOrgan transplantation has become an effective clinical therapy for end-stage renal disease. The rejection of organ transplantation is an important factor to affect the transplant recipients’ postoperative long-term survival rate and their quality of life. Even worse, the serious immunological rejection can course the failure of organ transplantation. The immunosuppressive drugs are used to inhibit the rejection. There is individual variability of the effect and toxicity of transplant recipients. In order to improve the prognosis of organ transplant recipients, the individualization of immunosuppressive drugs is the major subject of organ transplant research.Mycophenolate mofetil(Mycophenolate mofetil, MMF) is a rapamycin target molecule inhibitors, which is widely used in clinic for its good immunosuppressive effect and low incidence of adverse reactions. But mycophenolate mofetil has significant individual metabolic differences and narrow therapeutic window. Therapeutic drug monitoring(Therapeutic drug monitoring, TDM) can guide the use of drugs in a narrow range in order to blance the treatment efficacy between side effects. However TDM is insufficient to provide a comprehensive solution of using the mycophenolate mofetil for the existence of its side effects even with low doses. TDM also has a certain hysteresis, some patients had appeared adverse reactions before the blood drug concentration monitoring.As the immunosuppressive drugs need to be taken for a long-term or even all lifetime. In terms of patients, they not only need a good curative effect but also need to consider the economic bear ability of themselves’. Therefore, if we can create an individualized medication of mycophenolate mofetil based on the genetic analysis, we can forecast the initial and daily doses before the patients taking the medicine. In this way, we can reduce adverse reactions in a certain degree, and we will make mycophenolate mofetil more safe, effective and economic.Individual genetic difference is one of the most representative factors that affect absorption, distribution and metabolism of drugs. Studies have shown that the differences of 20% to 95% of drugs disposition and effection are related to genetic factors. Therefore, pharmacogenomics research, which is the study of the role of genetics in pharmacokinetics of medicine, is necessary for guiding the clinic medicine. Recently, in the domestic and foreign, there are some research about the association between the gene polymorphisms and the mycophenolate mofetil. The gene polymorphisms of genes coding mainly concentrate in uridine diphosphate glucuronic acid transferase(UGT), multi-resistant binding protein- 2(MRP2) and organic anion transfer peptide 1B3(OATP1B3). There are some gene that are closely related to the blood drug concentration of mycophenolate mofetil, such as UGT1A9-440 C > T, UGT1A9 I399 C > T, UGT1A8 * 2, UGT2B7 G211 T, UGT2B7 A268 G ABCC2-24 C > T, ABCC2 1249 G > A, ABCG2 C421 A, SLCO1B3 T334 G.New studies have showed that OATP1B3 can affect transcription of pregnane X receptor(PXR) and constitutive androstane receptor(CAR) by acting on the transfer of PXR and CAR, and then it regulates the expression of drug metabolism enzymes such as cytochrome P450(CYP450), of which the gene polymorphism have a very important influence on the pharmacokinetic of mycophenolate mofetil. Therefore, the investigation of the gene locus mutations which effects on the pharmacokinetic of mycophenolate mofetil is an important way to individualize of this medicine.At present, there are many researches about the relationship between related gene polymorphism and MMF blood valley oncentrations, but no more research about the association of gene polymorphism and the MMF related adverse reactions these days. ObjectiveFor the personalizing of the mycophenolate mofetil, we determine the polymorphisms of gene UGT1A9, UGT1A8, UGT2B7, ABCC2, ABCG2 and SLCO1B3 to investigate the association between the gene polymorphisms of these proteins and the adverse reactions of mycophenolate mofetil in kidney transplant patients. MethodsWe have determined single nucleotide polymorphisms genotypes of these genes using the PCR-RFLP(polymerase chain reaction-restriction fragment length polymorphism): UGT1A9-440 C>T, UGT1A9 I399 C>T, UGT1A8 * 2, UGT2B7 G211 T, UGT2B7 A268 G, ABCC2-24C>T, ABCC2 1249G>A, ABCG2 C421 A and SLCO1B3 T334 G. Then sending some parts of PCR products sequencing directly to verify the accuracy of the agarose gel electrophoresis. Using HPLC-fluorescence detector method to determine the blood drug concentration of MPA. We have consulted the clinical data of the 236 kidney transplant patients, including the age, gender, height, cadaveric kidney or living relative kidney, weight, BMI, dialysis time, transplanting time, history of diabetes or hypertension and history of hepatitis b or hepatitis c. We also have recorded the oral daily doses of MMF, FK506 and prednisone at 3, 6, 12, 24, 36 months, as well as the plasma glucose(FPG), blood routine, blood lipids and the function of liver and kidney.All data were processed using SPSS20.0 software and P<0.05 indicate a statistically significant difference. The measurement data results were using the mean ± standard deviation. The t-test, ANOVA analysis and repeated measures analysis of variance were used to compare the clinical data to get the statistical difference between the groups. The χ2 test was used to detect genotypes and genetic allele differences between groups. The logistic statistical analysis was used to analyze risk factors for related adverse reactions caused by MMF in post-transplant patients. ResultsThere are 77 cases of adverse reactions, 96 person-times totally. Among the 236 patients, including 36 cases of bone marrow suppression, 15 cases of gastrointestinal reaction, 26 cases of infection. The incidence of adverse reactions was 40.68%. According the general data contrast, there are significant difference among the BMI,dialysis and medication time of the patients who occurred MMF related adverse reactions. Logistic stepwise regression analysis showed that medication time and the trough blood concentrations of MMF after 3 months postoperative are the risk factors of bone marrow suppression coursed by MMF.The results of gel electrophoresis were consistent with that from the direct sequencing.The mutation frequency of UGT1A9-440C>T was 6.10%. But no TT type gene mutation was detected in our study. There was no significant difference among the different genotypes and alleles distribution. The general the clinical dates from the different genotypes showed no significant difference. The trough blood concentrations of MMF in CT group were significantly higher than that in CC group after 3 months postoperative.The mutation frequency of UGT1A9 I399C>T was 53.20%. The frequency of CC genotype in the group of bone marrow suppression was remarkably lower than that in the control group. There was no statistical significance of the allele distribution differences among groups. The doses of 24 months postoperatively were Dose CC>Dose CT>Dose TT and the the doses of TT genotype was remarkably lower than that of the CC genotype. No significant difference of the trough blood concentrations of MMF was found among the different genotypes in each time point.The mutation frequency of UGT2B7 G211 T was 16.01%. Genotypes and alleles distribution had no significant difference in each group. The dialysis time in TT genotype was significantly higher than that in GG type group, but the dose of 12 months postoperatively was remarkably lower than that of GG group. No significant difference of the trough blood concentrations of MMF was found among the different genotypes in each time point.The mutation frequency of UGT2B7 A268 G was 63.72%. The frequencies of AA genotypes in bone marrow suppression and infection group were both remarkably higher than that in control group. The distribution of A allele in control group was remarkably lower than that of infection group. The dose of 3 months postoperatively in GG group was remarkably higher than that in AA and AG group. The trough blood concentrations of MMF in AA group were higher than that in GG type group after 3 months postoperative.The mutation frequency of UGT1A8 * 2 was 56.40%. The frequency of GG genotypes in bone marrow suppression was remarkably higher than that in control group. There was no statistical significance of the allele distribution differences among groups. The dose of 3 months postoperatively in GG group was remarkably lower than that in CG group. The trough blood concentrations of MMF in GG group were higher than that in CC type group after 3 months postoperative.The mutation frequency of ABCC2-24C/T was 21.34%. The frequency of CC genotypes in bone marrow suppression was remarkably higher than that in control group. There was no statistical significance of the allele distribution differences among groups. Between CT genotypes and CC genotype groups, there was a significant difference in age. No significant difference of the trough blood concentrations of MMF was found among the different genotypes group in each time point.The mutation frequency of ABCC2 1249G>A was 11.59%. There was no significant difference among the different genotypes and alleles distribution. The values of BMI were significant difference between GG and GA group. The trough blood concentrations of MMF in GG group were significantly higher than GA group after 12 months postoperative.The mutation frequency of ABCG2 C421 A was 33.84%. The frequency of AA genotypes in distribution of gastrointestinal reaction group was higher than that in control group. There was no statistical significance difference of allele distribution between each group. The distribution in AA group was significance different from that in CT and CC groups in gender. The trough blood concentrations of MMF in AA group were significantly higher than CC group after 6 months postoperative.The mutation frequency of SLCO1B3 T334 G was 71.65%. The frequency of GG genotypes in distribution of gastrointestinal reaction group was higher than that in control group. G allele in the distribution of gastrointestinal reaction group was remarkably higher than that of control group. The general the clinical dates from the different genotypes showed no significant difference. The trough blood concentrations of MMF in GG group were significantly higher than TT group after 24 months postoperative.The risk of toxicity of bone marrow suppression in UGT1A9-440 CT genotypes was 3.514 fold higher than that in CC genotypes. The risk of toxicity of bone marrow suppression in UGT1A9 I399 TT genotypes was 4.326 fold higher than that in CC genotypes. There is no significant association between UGT2B7 G211 T genotypes and the adverse reactions caused by MMF. The risk of toxicity of infections in UGT2B7 268 GG genotypes was 3.254 fold higher than that in AA genotypes. The risk of toxicity of bone marrow suppression in UGT1A8*2 GG genotypes was 4.527 fold higher than that in CC genotypes. The risk of toxicity of bone marrow suppression in ABCC2-24 CC genotypes was 3.461 fold higher than that in TT genotypes. There is no significant association between the ABCC2 1249G>A genotypes and the adverse reactions caused by MMF. The risk of toxicity of gastrointestinal reaction in ABCG2 421 AA genotypes was 4.410 fold higher than that in CC genotypes. The risk of toxicity of gastrointestinal reaction in SLCO1B3 334 GG genotypes was 5.624 fold higher than that in TT genotypes. ConclusionThe polymorphisms of UGT1A9-440C>T 、 UGT1A9 I399C>T 、UGT1A8*2 、 UGT2B7 A268 G 、 ABCC2-24C>T 、 ABCG2 C421 A and SLCO1B3 T334 G were associated with adverse reaction caused by MMF after renal transplantation. The gene locus, such as UGT1A9-440CT、UGT1A9 I399TT、UGT1A8*2 GG and ABCC2-24 CC, were the independent risk factors for the bone marrow inhibition. UGT2B7 268 GG were the independent risk factor for the opportunistic infections. ABCG2 421 AA and SLCO1B3 334 GG were the independent risk factors for the gastrointestinal reaction.