Establishment And Preliminary Application Of The Analysis Method Of TPTM Gene Polymorphism

Background and purpose:The large-scale studies carried out by the World Health Organization(WHO), Europe and USA showed that the drug safety issues have become one of the most important reasons leading to death of hospitalized patients[1, 2]. It ranks the fifth place in the reasons which caused death. Thiopurine drugs, including 6-mercaptopurine(6-MP), 6-thioguanine(6-TG) and azathiopurine(AZA), are all purine antagonist. They can interfere the nucleotide metabolism and play an important role in anti-proliferation and immunosuppression. These drugs are mainly used for inflammatory bowel disease(IBD), or as immune inhibitors which are widely used in organ transplantation and the treatment of autoimmune diseases. Thiopurine drugs are inactive or of low activity. They need to be transformed to 6-thioguanine nucleotides(6-TGNs) by enzymes. Thiopurine S-methyltransferase(TPMT) is a kind of key enzymes in metabolism of thiopurine drugs in vivo. Single nucleotide polymorphisms(SNPs) of coding gene of TPMT may decide the activity of TPMT and directly influence the clinical efficacy and toxicity of the thiopurine drugs[2-4]. Phenotyping and genotyping are the two main ways used for the detection of the TPMT. Phenotyping are affected by blood transfusion or drug related action. On the contrary,genotyping won’t be affected by the factors mentioned above, while it is also more rapid and convenient. Phenotyping is gradually replaced by the genotyping, which is becoming the mainstream method for detecting the activity of TPMT. How to detect the genotype of TPMT fastly, comprehensively, systematically, accurately is the hot issue now.This study explored and optimized PCR reaction system of TPMT gene. A total of 37 kinds of TPMT gene of SNP locus have been reported. We found the incision enzyme which suited for each kind of SNP locus and then confirmed the fragment size after digestion. Finally, We determined the appropriate combinations of enzymes according to the fragment size after digestion, time of incubation and temperature.Methods:1. Searching the complete sequence of TPMT in Gen Bank. Then we determined the sequence of exon3-10 in the complete sequence of TPMT. We synthesized the specific primers of TPMT exon 3-10, established the polymerase chain reaction(PCR) amplification reaction system. Authenticating specificity and the size of the fragments of amplification primer by agarose gel electrophoresis(AGE).2. We amplified the exon 3-10 of TPMT gene using the optimized PCR system and done the cloning sequencing. Compared the results of cloning sequencing and the template region by BLAST and confirmed the accuracy of the new sequence. And then, made the point mutations in the sequence according to the different SNP locus. The location of the base mutations is: Exon2(TPMT*03E: rs3931660 T140+114A, TPMT*13E: rs72552742 A83 T, TPMT*14: rs9333569 A1 G, TPMT*17: ND C124 G, TPMT*29: rs267607275 T2 C, TPMT*30: ND G106A) Exon3( TPMT*03E: rs12529220 A141-101 T, TPMT*5: rs72552740 T146 C, TPMT*18: ND 211G>A, TPMT*21: rs200591577 C205G); Exon4(TPMT*02: rs1800462 G238 C, TPMT*03D: rs72552739 G292 T, TPMT*03E: rs2518463 T366+58C, TPMT*09: rs151149760 A356 C, TPMT*19: ND A356 C, TPMT*27: ND T319 G, TPMT*28: ND G349 C, TPMT*32: rs115106679 G340 A, TPMT*34: rs111901354 C244T); Exon5(TPMT*11: rs72552738 G395 A, TPMT*12: ND C374T); Exon6(TPMT*03B: rs1800460 G460 A, TPMT*03E: rs2842934 C474 T, TPMT*10: rs72552737 G430 C, TPMT*16: rs144041067 G488 A, TPMT*22: ND G488 C, TPMT*33: rs112339338 C487T); Exon7( TPMT*15: rs9333570 G495-1A, TPMT*06: rs75543815 A539 T, TPMT*23: rs74423290 C500 G, TPMT*24: rs6921269 G537T); Exon8( TPMT*26: rs72556347 T622 C, TPMT*31: rs79901429 T611C); Exon9( TPMT*04: rs1800584 G626-1A, TPMT*03C: rs1142345 A719 G, TPMT*07: rs72552736 T681 G, TPMT*08: rs56161402 G644 A, TPMT*20: rs150900439 A712 G, TPMT*25: ND T634C). The p UCm-T plasmid vector which was designed to simplify the PCR products clone was used for Wild-type sequence. The p UC57 plasmid vector was used for mutation sequence. 37 kinds of SNP locus were established.3.We sought the appropriate incision enzyme for each SNP locus using the DNAssist. However, there was no appropriate incision enzyme for 10 SNP locus. So we explored the appropriate incision enzyme by artificially modified base. After enzyme digestion, we recorded the fragment size. We combined different incision enzyme by fragment size, time of incubation and temperature.Results:1. The optimal PCR reaction conditions were: primer concentration is 0.4 mM, probe concentration is 0.1 mM, annealing temperature is 55℃.2. We amplified 8 exon(3-10) of TPMT gene and then done cloning sequencing. By comparing with standard sequence, we determined the correctness of all sequence of the exons. Then 37 kinds of SNPs locus were established including wild-type and mutant.3. Using different enzymes to identify 37 kinds of SNP locus of TPMT gene. The fragment size after digestion is determined.Conclusions:1. This study successfully established PCR reaction amplification system of TPMT gene.2. We built 37 kinds of SNP locus of TPMT gene by different plasmid vector.3. The fragment size of each SNP locus after digestion has been determined. We could combine the different enzymes by fragment size, time of incubation and temperature for future product development.