Application Research Of HRM Technology In Detection Of TPMT Gene Polymorphism In Inflammatory Bowel Disease Patients

Background:Azathioprine(AZA) is a common drug which is used to treat inflammatory bowel disease(IBD),however it may cause the myelotoxicity in some patients.Recent studies indicated that the drug adverse reaction had some relationship with the mutation of thiopurine methyltransferase (TPMT).Objective To discuss the relationship between the genotype of TPMT and AZA induced myelotoxicity in the patients with IBD.Methods Peripheral blood was collected from 82 IBD patients and 53 healthy Chinese people. Genomic DNA was isolated from these blood samples with a QIAamp DNA Blood Mini Kit(Qiagen),and three commom mutation alleles of TPMT [TPMT*3A(G460A/A719G),TPMT*3B(G460A),TPMT*3C(A719G)]were detected by polymerase chain reaction-high resolution melting and sequencing.Result Clinical data:There were 82 IBD patients(UC 38/CD 42/IC 2),who were divided into group A and B based on whether they used AZA or not.Group A was composed by 31 patients who received AZA(50-100mg/d)(male 20/female 11) whose average age was 39.6(18-68) years old.The healthy control group was group C(male28/female 25),average age 41.05(23-68).There was no significant difference between the age of either group A and C or group B and C.Six cases of heterozygote TPMT*1/*3C were detected,which accounted for 4.44%of the test population.Four cases of heterozygote TPMT*1/*3C were found in IBD patients,making genotypic frequency of the heterozygote TPMT*1/*3C 4.88%,and mutation allele frequency in these patients 2.44%. While two cases of heterozygote TPMT*1/*3C were found in healthy people, so genotypic frequency of the heterozygote TPMT*1/*3C was3.77%.Mutation allele frequency in these people was 1.89%.No TPMT*3A or TPMT*3B was found. Bone marrow suppression occurred in four of the patients who received AZA, but only one of them has the TPMT*1/*3C mutation,the others are wild-type homozygote.What's more,TPMT*1S(T474C) was also detected.In IBD patients,genotype frequency of TPMT*IS homozygote was 1.96%, heterozygote was 35.85%,and the mutant allele frequency was 19.81%.The genotype frequency of TPMT*1S homozygote was 2.44%,heterozygote was 39.02%, and the mutant allele frequency was 21.95%in the healthy people.Conclusion: Patient who has the TPMT mutation is very likely to have the bone marrow suppression.But mutantion of TPMT gene could just partially explain the AZA induced myelotoxicity.Except the gene mutation,there are many other risks could cause bone marrow expression.