Yoichi Kakuta1, Yasuhiro Izumiyama2, Daisuke Okamoto2, Takeru Nakano2, Ryo Ichikawa2, Takeo Naito2, Rintaro Moroi2, Masatake Kuroha2, Yoshitake Kanazawa2, Tomoya Kimura2, Hisashi Shiga2, Hisaaki Kudo3, Naoko Minegishi3, Yosuke Kawai4, Katsushi Tokunaga4, Masao Nagasaki5, Yoshitaka Kinouchi6, Yasuo Suzuki7, Atsushi Masasmune2. 1. Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo, Aoba, Sendai, 980-8574, Japan. ykakuta@med.tohoku.ac.jp. 2. Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo, Aoba, Sendai, 980-8574, Japan. 3. Department of Biobank Life Science, Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan. 4. Genome Medical Science Project, National Center for Global Health and Medicine, Tokyo, Japan. 5. Human Biosciences Unit for the Top Global Course Center for the Promotion of Interdisciplinary Education and Research (CPIER), Kyoto University, Kyoto, Japan. 6. Institute for Excellence in Higher Education, Student Health Care Center, Tohoku University, Sendai, Japan. 7. Inflammatory Bowel Disease Center, Toho University Sakura Medical Center, Sakura, Japan.
Abstract
BACKGROUND: The genetic variants of NUDT15 have been verified to induce adverse events (AEs) of thiopurines. Codon 139 variants are frequently observed in Asians, while multiple variants are seen in codon 18 which also cause AEs including the European ancestry. The purpose of this study is to establish a technique capable of the simple genotyping of NUDT15 codon 18 and to evaluate its efficacy. METHODS: A high-resolution melt (HRM) technique is performed to simply determine genotypes. The accuracy of HRM analysis was evaluated with DNAs from 1245 Japanese patients with inflammatory bowel diseases. Subsequently, another group of 572 patients was analyzed to verify the method. The diplotypes and the frequency of their AEs were estimated on the basis of codon 18 and 139 genotypes. RESULTS: The HRM analysis enabled the correct identification of the three main genotypes, ref/ref, ref/ins, and ref/V18I, in 1236 of 1241 cases. All rare genotypes including ref/del were identified as the impossible-to-determine group, the proper diagnosis rate was 99.6%. In the verification test using other samples, the diagnosis rate was 99.7%. By estimating diplotypes using both codon 18 and 139 genotypes, 2.74% and 2.13% of Japanese patients with Arg/Arg and Arg/Cys of codon 139 have a lower enzymatic activity of NUDT15 and a higher risk for adverse responses than those estimated by codon 139 genotypes alone. CONCLUSIONS: Our study showed that HRM method enables simple genotyping of complicated codon 18 variants essential to haplotype estimation of the NUDT15.
BACKGROUND: The genetic variants of NUDT15 have been verified to induce adverse events (AEs) of thiopurines. Codon 139 variants are frequently observed in Asians, while multiple variants are seen in codon 18 which also cause AEs including the European ancestry. The purpose of this study is to establish a technique capable of the simple genotyping of NUDT15 codon 18 and to evaluate its efficacy. METHODS: A high-resolution melt (HRM) technique is performed to simply determine genotypes. The accuracy of HRM analysis was evaluated with DNAs from 1245 Japanese patients with inflammatory bowel diseases. Subsequently, another group of 572 patients was analyzed to verify the method. The diplotypes and the frequency of their AEs were estimated on the basis of codon 18 and 139 genotypes. RESULTS: The HRM analysis enabled the correct identification of the three main genotypes, ref/ref, ref/ins, and ref/V18I, in 1236 of 1241 cases. All rare genotypes including ref/del were identified as the impossible-to-determine group, the proper diagnosis rate was 99.6%. In the verification test using other samples, the diagnosis rate was 99.7%. By estimating diplotypes using both codon 18 and 139 genotypes, 2.74% and 2.13% of Japanese patients with Arg/Arg and Arg/Cys of codon 139 have a lower enzymatic activity of NUDT15 and a higher risk for adverse responses than those estimated by codon 139 genotypes alone. CONCLUSIONS: Our study showed that HRM method enables simple genotyping of complicated codon 18 variants essential to haplotype estimation of the NUDT15.
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