Kapoor Gauri, Maitra Arindam, Brahmachari Vani
Dr B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India.
Indian J Med Res. 2009 May;129(5):500-5.
BACKGROUND & OBJECTIVE: Mercaptopurine, azathioprine, and thioguanine, used as antineoplastic agents and immunosuppressants are catabolized by thiopurine methyltransferase (TPMT) enzyme, which exhibits genetic polymorphism. Genotyping patients and the population to which the patients belong, is important for effective treatment and reducing toxicity. There is a need for faster methods for genotyping. Hence the present study was planned to test the application of SNaPshot technique for analysis of the three common TPMT alleles: TPMT2, TPMT3A, and TPMT*3C in DNA from healthy Indian volunteers as well as to apply the method on cDNA samples obtained from children with acute lymphoblastic leukaemia (ALL).
A total of 120 healthy volunteers and 25 patients were analysed by multiplexed SNaPshot reaction. Genomic DNA was the template for most of the analyses, but additionally the cDNA synthesized for translocation detection was used as the template in case of patients with ALL. The results of SNaPshot reaction were confirmed by direct sequencing.
The TPMT genotype could be reliably identified by SNaPshot analysis in multiplex reactions both in genomic DNA samples and cDNA. The overall frequency of the three common polymorphisms was observed to be 4.9 per cent, arising from heterozygosity for TPMT3C (4.1%) and TPMT3A (0.8%).
INTERPRETATION & CONCLUSION: SNaPshot method for TPMT polymorphism analysis works faster with the potential for high throughput. By simultaneously interrogating the genotype at multiple sites, the method can provide future opportunity to multiplex, though multiplexing has not been done in the present analysis. Heterozygosity for TPMT3C (719 A>G) was detected in 4.1 per cent of the study population and no homozygosity was observed. Our results indicated that TPMT3C was the most common polymorphism in Indian population, while TPMT3*A, associated with the absence of catalytic activity of TPMT, was very rare.
巯嘌呤、硫唑嘌呤和硫鸟嘌呤用作抗肿瘤药和免疫抑制剂，可被具有遗传多态性的硫嘌呤甲基转移酶（TPMT）代谢。对患者及其所属人群进行基因分型，对于有效治疗和降低毒性至关重要。需要更快的基因分型方法。因此，本研究旨在测试SNaPshot技术在分析健康印度志愿者DNA中三种常见TPMT等位基因（TPMT2、TPMT3A和TPMT*3C）方面的应用，并将该方法应用于急性淋巴细胞白血病（ALL）患儿获得的cDNA样本。
通过多重SNaPshot反应对120名健康志愿者和25名患者进行分析。大多数分析以基因组DNA为模板，但对于ALL患者，另外将用于易位检测而合成的cDNA用作模板。SNaPshot反应的结果通过直接测序进行确认。
通过SNaPshot分析可在多重反应中可靠地鉴定基因组DNA样本和cDNA中的TPMT基因型。观察到三种常见多态性的总体频率为4.9%，源于TPMT3C（4.1%）和TPMT3A（0.8%）的杂合性。
用于TPMT多态性分析的SNaPshot方法速度更快，具有高通量潜力。通过同时检测多个位点的基因型，该方法未来有实现多重检测的机会，尽管本分析中未进行多重检测。在4.1%的研究人群中检测到TPMT3C（719 A>G）的杂合性，未观察到纯合性。我们的结果表明，TPMT3C是印度人群中最常见的多态性，而与TPMT催化活性缺失相关的TPMT3*A非常罕见。
