Department of Laboratory Medicine, Ilsan Paik Hospital, Inje University College of Medicine, Joowha-ro 170, Ilsanseo-gu, Goyang, Gyeonggi, Republic of Korea.
Biomed Res Int. 2013;2013:305704. doi: 10.1155/2013/305704. Epub 2013 Feb 28.
Thiopurine drugs are metabolized via S-methylation and catalyzed by thiopurine S-methyltransferase (TPMT). Patients with very low TPMT activity are at high risk of fatal bone marrow toxicity when standard doses of thiopurine drugs are administered. TPMT genotyping can predict TPMT activity and is not affected by transfusion or red blood cell defects. Here, we report a new allele-specific real-time polymerase chain reaction (PCR) system for thiopurine methyltransferase genotyping that is validated in Korean population.
Three major TPMT single-nucleotide polymorphisms (TPMT 2, 3B, and 3C) were genotyped using real-time PCR with the allele-specific primers and probes. Internal positive controls were included in each well, and an automatic interpretative algorithm was applied. This system was validated using 244 clinical samples and 2 commercial DNA samples that had been previously genotyped using PCR-direct sequencing. Results. All of the obtained results are concordant with those of the reference method. All of the internal positive control reactions were successful. The allele frequency of TPMT 3C was 2.05% (10 of 488 alleles). All of the patients with variant alleles were heterozygotes, and no homozygotes were detected. No TPMT 2, 3A, or 3B alleles were observed in this Korean population.
This rapid, accurate, and user-friendly genotyping system can be readily used to improve the efficacy and safety of thiopurine treatments in clinical practice.
硫嘌呤药物通过 S-甲基化代谢，由硫嘌呤 S-甲基转移酶（TPMT）催化。当给予标准剂量的硫嘌呤药物时，TPMT 活性非常低的患者有发生致命骨髓毒性的高风险。TPMT 基因分型可预测 TPMT 活性，不受输血或红细胞缺陷的影响。在此，我们报告了一种新的用于硫嘌呤甲基转移酶基因分型的等位基因特异性实时聚合酶链反应（PCR）系统，该系统在韩国人群中得到验证。
使用实时 PCR 与等位基因特异性引物和探针对三个主要的 TPMT 单核苷酸多态性（TPMT 2、3B 和 3C）进行基因分型。每个孔中均包含内部阳性对照，并应用自动解释算法。该系统使用 244 个临床样本和 2 个先前使用 PCR-直接测序进行基因分型的商业 DNA 样本进行验证。结果：所有获得的结果均与参考方法一致。所有内部阳性对照反应均成功。TPMT 3C 的等位基因频率为 2.05%（488 个等位基因中的 10 个）。所有具有变异等位基因的患者均为杂合子，未检测到纯合子。在本韩国人群中未观察到 TPMT 2、3A 或 3B 等位基因。
这种快速、准确且易于使用的基因分型系统可用于提高临床实践中硫嘌呤治疗的疗效和安全性。
