Systematic Review For Association Between TPMT SNP And Thiopurine-induced Adverse Drug Reactions And Construction Of Detection For TPMT SNP

BackgroundThiopurine drugs,including 6-mercaptopurine,6-thioguanine and its pro-drug(azathioprine),were first invented by G.B.Elion and G.H.Hitchings in 1950 s.6-mercaptopurine and 6-thioguanine are one of the well-established treatments in the management of inflammatory bowel disease,as well as in the treatment of childhood acute lymphoblastic leukemia.Azathioprineis is widely used as an immunosuppressive drug in autoimmune diseases and transplant rejection.The variable response to,and efficacy of,thiopurine drugs still remains.Hence,safe,effective,individualized,and rational thiopurine drugs dosing is of huge clinical importance.Thiopurine S-methyltransferase(TPMT)is an important and key cytoplasmic enzyme in the metabolism of thiopurine drugs,whose activity can directly determine the amount of thiopurine drugs metabolized to cytotoxic 6-thioguanine nucleotides and consequently influence clinical efficacy and adverse drug reactions of thiopurine drugs.The gene encoding for TPMT is subject to genetic polymorphisms and ethnic regional differences that have been studied extensively.There is no comprehensive compilation of TPMT genotype distributions in Asia,which is the Earth's largest and most populous continent.Besides,China officially recognizes 56 distinct ethnic groups,whose genetic background,environment,living habits and living area differ to each other.An all-round and detailed compilation of TPMT genotype distributions in China is also absent.Until now,lots of studies have investigated the association between TPMT polymorphisms and thiopurine-induced adverse drug reactions in inflammatory bowel disease and autoimmune diseases patients,the results are inconsistent.Namely,controversy still exists about the association.The available detection methods for TPMT polymorphisms or single nucleotide polymorphism have some limitations and cannot meet the complexity and diversity of TPMT polymorphisms.Based on the above background,we intended to compile TPMT genotype distributions in Asian and Chinese populations through a comprehensive search of relevant published literatures.Therefore we could have a good knowledge of TPMT genotype distributions,which can provide a theoretical basis for individualized dosing of thiopurine drugs and also provide data support for the next step of our research.On this basis,we performed systematic review and meta-analysis in order to resolve the dispute of association between TPMT polymorphisms and thiopurine induced adverse drug reactions(ADRs)in both inflammatory bowel disease and autoimmune diseases patients,which can further indicate the importance of TPMT genotyping prior to commencing thiopurine drug treatments.Lastly,we would build a novel,convenient,high throughput,and single tubed detection method based on ligation-during-polymerization probe for TPMT single nucleotide polymorphism.Objectives(1)To compile TPMT genotype distributions in Asian and Chinese populations,which can provide a theoretical basis for individualized dosing of thiopurine drugs and also provide data support for the next step of our research.(2)To resolve the dispute of association between TPMT polymorphisms and thiopurine induced adverse drug reactions in both inflammatory bowel disease and autoimmune diseases patients through performing systematic review and meta-analysis,which can further indicate importance of TPMT genotyping prior to commencing thiopurine drug treatments.(3)To build a novel,convenient,high throughput and single tubed detection method based on ligation-during-polymerization probe for TPMT single nucleotide polymorphism.Methods(1)We compiled TPMT distributions in Asian and Chinese populations through exploring Pub Med,Excerpta Medica Database and Chinese Biomedical Database for relevant articles using different key words like ?TPMT?,?Thiopurine S-methyltranferase?,?Polymporphism?,?Hepalotype? et al.(2)We explored Pub Med,Web of Science and Embase for articles on TPMT polymorphisms and thiopurine-induced ADRs.Studies that compared TPMT polymorphisms with-ADRs and without-ADRs in patients with inflammatory bowel disease and autoimmune diseases were included.Relevant outcome data from all the included articles were extracted and the pooled odds ratios(ORs)with corresponding ninety-five percentages confidence intervals(95% CIs)were calculated using Revman 5.3 software.(3)We designed and synthesized ligation-during-polymerization probes targeted TPMT single nucleotide polymorphism;optimized the preferred general primers,polymerase,ligase,concentration of d NTPs.Results(1)To date,data from 15 countries and 1 region for TPMT genotypes could be retrieved and 68 articles concentrated on the distribution of TPMT genotype with a total number of 18,234 individuals were included in this compilation according to the preset inclusion and exclusion criteria.The overall frequency of mutant TPMT genotype in Asian populations was 3.54%(664/18,234).TPMT*1/ *3C and TPMT*1/ *3A are the most common mutant genotypes,which account for 69.72% and 16.15% of the overall mutant genotypes,respectively.Homozygous mutant genotypes are not common in Asian populations,when all the studies were considered,including a total of 18,234 patients,one compound heterozygous and eleven homozygous mutant genotypes were detected.The overall frequency of mutant TPMT allele in Asian populations was 1.80%(657/36,468),TPMT*3C accounts for 70.93%(66/657)of the overall mutant alleles,which were followed by TPMT*3A.Our analysis further indicated that TPMT*3C is the predominant mutant allele only in East Asia and Southeast Asia,not in all regions of Asia.(2)A total of 44 articles(30 articles writen in English and 14 articles writen in Chinese,respectively)concentrated on the distribution of TPMT genotype with a total number of 10,458 individuals were included in this compilation according to the preset inclusion and exclusion criteria.The overall frequency of mutant TPMT genotype in Chinese populations was 2.91%(304/10,458).TPMT*1/ *3C and TPMT*1/ *3A are the most common mutant genotypes in China.TPMT*3C was the most common mutant allele,which accounts for 97.05%(296/305)of the overall mutant alleles.TPMT*3A was mainly detected in Kazakhstan and the Uygur populations,which was not detected in Han population.Until now there were a small number of researches on TPMT distributions in Ethnic Minorities,which needs more researches to confirm the results.(3)Fourteen published studies,with a total of 2,206 IBD patients,which investigated associations between TPMT polymorphisms and thiopurine-induced ADRs were included in this meta-analysis.Our meta-analysis demonstrated that TPMT polymorphisms were significantly associated with thiopurine-induced overall ADRs and bone marrow toxicity;pooled ORs were 3.36(95%CI: 1.82-6.19)and 6.67(95%CI: 3.88-11.47),respectively.TPMT polymorphisms were not associated with the development of other ADRs including hepatotoxicity,pancreatitis,gastric intolerance,flu-like symptoms and skin reactions;the corresponding pooled ORs were 1.27(95%CI: 0.60-2.71),0.97(95%CI: 0.38-2.48),1.82(95%CI: 0.93-3.53),1.28(95%CI: 0.47-3.46)and 2.32(95%CI: 0.86-6.25),respectively.(4)Eleven published studies,with a total of 651 patients with autoimmune diseases,which investigated associations between TPMT polymorphisms and azathioprine-induced ADRs,were included in this meta-analysis.Our meta-analysis demonstrated that TPMT polymorphisms were significantly associated with azathioprine-induced overall ADRs,bone marrow toxicity and gastric intolerance;pooled ORs(95% CI)were 3.12(1.48–6.56),3.76(1.97–7.17)and 6.43(2.04–20.25),respectively.TPMT polymorphisms were not associated with the development of hepatotoxicity;the corresponding pooled OR was 2.86(95%CI: 0.32–25.86).(5)Completed the design and synthesis of ligation-during-polymerization probes targeted TPMT* 2 and TPMT*3A~*3C.Optimization was defined as follows: general primers were derived from Phage M13 promoter sequence;polymerase was Exo(-)pfu DNA polymerase without exonuclease activity;concentration of d NTP was 2 m M;POP4 gel and 36 cm capillary were used in ABI 3500 for fragment analysis.Conclusions(1)TPMT genotype distributions differed to each other,which further indicates the importance of TPMT genotype based thiopurine dosing in each country.Since many countries in Asia are still underrepresented in pharmacogenomic researches,additional data from these Asian countries will be useful for safe thiopurine dosing in patients in those countries.(2)China officially recognizes 56 distinct ethnic groups,whose genetic background,environment,living habits and living area differ to each other.Until now there were a small number of researches on TPMT distributions in Ethnic Minorities,which needs more researches to confirm the results.The selective detection of TPMT According to the different regions and ethnic groups in our country not only guide the individualized thiopurine drug dosing,but also save the medical cost and reduce the economic burden of the patients.(3)TPMT polymorphisms were significantly associated with thiopurine-induced overall ADRs and bone marrow toxicity,but not with the development of other ADRs including hepatotoxicity,pancreatitis,gastric intolerance,flu-like symptoms and skin reactions in inflammatory bowel disease.Our research also clearly demonstrated that TPMT polymorphisms were significantly associated with azathioprine-induced overall ADRs,bone marrow toxicity and gastric intolerance but not with the development of hepatotoxicity in autoimmune disease patients.The results may translate to improved clinical outcomes in the management of both inflammatory bowel disease and autoimmune disease patients.Nowadays several clinical guidelines recommend TPMT genotyping prior to commencing thiopurine drug therapies;our results indirectly confirmed the importance and necessity of TPMT genotyping and can provide evidence based medicine basis.But there is one thing worth noting: TPMT genotyping prior to commencing thiopurine therapies cannot replace,may augment,the current practice of regular monitoring of the white blood cell.The combination of the two can maximize the efficacy while minimizing the toxicity of thiopurine drugs.(4)The method based on ligation-during-polymerization probe in our study could specifically TPMT single nucleotide polymorphisms and point out mutant type.