title: Genotyping of thiopurine methyltransferase using pyrosequencing.
abstract: Thiopurine methyltransferase (TPMT) metabolizes thiopurine drugs which are used in the treatment of leukemia and some autoimmune diseases. Previously, 11 mutant alleles of TPMT gene (TPMT*1S, *2, *3A, *3B, *3C, *3D, *4, *5, *6, *7, and *8) have been reported. These mutant alleles may cause life-threatening toxicity in patients exposed to thiopurine drugs, 6-mercaptopurine and azathioprine. We have developed a rapid and accurate protocol for TPMT genotype determination using Pyrosequencing(TM) technology in 96 Japanese subjects. Five fragments of the TPMT gene (exon 4, 5, 7, 8, 10) were amplified by PCR, and the 10 single-nucleotide polymorphisms (SNPs) for TPMT*1S, *2, *3A, *3B, *3C, *3D, *4, *5, *6, *7, and *8 were sequenced. The results of this pyrosequencing method corresponded exactly with those of the DNA sequencing method using BigDye terminator chemistry. We have demonstrated that typing of 10 SNPs can be performed within 30 min. Pyrosequencing has a wide application in the large-scale identification of individual TPMT genotypes.
authors.affiliation: Department of Clinical Pharmacology, Gunma University Graduate School of Medicine, Maebashi, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Okada
authors.forename: Katsunori
authors.initials: K
authors.lastname: Nakamura
authors.forename: Masanori
authors.initials: M
authors.lastname: Wada
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Nakamura
authors.forename: Norifumi
authors.initials: N
authors.lastname: Tsukamoto
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Nojima
authors.forename: Ryuya
authors.initials: R
authors.lastname: Horiuchi
authors.forename: Koujirou
authors.initials: K
authors.lastname: Yamamoto
mesh_terms: D000328:Adult; D000368:Aged; D001483:Base Sequence; D005260:Female; D005838:Genotype; D006801:Humans; D008297:Male; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D008780:Methyltransferases; C022745:thiopurine methyltransferase