Beránek M, Drastíková M, Bureš J, Palička V
Institute of Clinical Biochemistry and Diagnostics, Faculty of Medicine in Hradec Králové, Charles University in Prague and University Hospital Hradec Králové, Czech Republic.
2nd Department of Internal Medicine, Faculty of Medicine in Hradec Králové, Charles University in Prague and University Hospital Hradec Králové, Czech Republic.
Folia Biol (Praha). 2015;61(3):91-6. doi: 10.14712/fb2015061030091.
The aim of the study is to present a novel approach for preparing triple-compound heterozygous reference material (TCH-RM) for thiopurine S-methyltransferase (TPMT) genotyping by using the gene synthesis technology. The polynucleotide chain we prepared consisted of three wild-type and three mutant segments corresponding to the TPMT 238G>C, 460G>A, and 719A>G polymorphic sites. TCH-RM characteristics were assessed via four methods: reverse hybridization, real-time PCR with hydrolysis probes, real-time PCR followed by subsequent melting temperature analysis, and DNA sequencing. Consequently, we investigated the TPMT genotype of 371 patients suffering from autoimmune diseases requiring immunosuppressive therapy with thiopurine drugs, mostly inflammatory bowel disease. All methods confirmed the triple heterozygous character and commutability of TCH-RM. In evaluating its stability we obtained very comparable data before and after six months of storage at -80 °C. The determined genotypes were as follows: 352 wild-type subjects (94.8%), 17 TPMT3A heterozygotes (460G>A and 719A>G, 4.6%), one patient heterozygous for the TPMT2 allele (238G>C, 0.3%), and one TPMT3C heterozygote (719A>G, 0.3%). The frequencies of TPMT1, *3A, *3C, and *2 in the patients were 97.5%, 2.3%, 0.1%, and 0.1 %, respectively. Assembling segments of synthetic DNA into long polynucleotide chains is a universal way of obtaining compound heterozygous material for performing any simultaneous analysis of polymorphic sites in the human genome. The batches are manufactured with a perfect concentration match of wildtype and mutant fragments, and can be made in large quantities for most diagnostic techniques.
本研究的目的是提出一种利用基因合成技术制备用于硫嘌呤甲基转移酶（TPMT）基因分型的三联复合杂合参考物质（TCH-RM）的新方法。我们制备的多核苷酸链由对应于TPMT 238G>C、460G>A和719A>G多态性位点的三个野生型和三个突变片段组成。通过四种方法评估TCH-RM的特性：反向杂交、水解探针实时荧光定量PCR、实时荧光定量PCR随后进行熔解温度分析以及DNA测序。因此，我们对371例患有自身免疫性疾病且需要使用硫嘌呤类药物进行免疫抑制治疗（主要是炎症性肠病）的患者进行了TPMT基因分型研究。所有方法均证实了TCH-RM的三联杂合特性和互换性。在评估其稳定性时，我们在-80°C储存六个月前后获得了非常可比的数据。所确定的基因型如下：352例野生型受试者（94.8%）、17例TPMT3A杂合子（460G>A和719A>G，4.6%）、1例TPMT2等位基因杂合患者（238G>C，0.3%）和1例TPMT3C杂合子（719A>G，0.3%）。患者中TPMT1、*3A、3C和2的频率分别为97.5%、2.3%、0.1%和0.1%。将合成DNA片段组装成长多核苷酸链是获得复合杂合物质以对人类基因组中的多态性位点进行任何同时分析的通用方法。这些批次的产品在野生型和突变片段的浓度匹配方面非常完美，并且可以大量生产以用于大多数诊断技术。
