SULM – Schweizerische Union für Labormedizin | Union Suisse de Médecine de Laboratoire | Swiss Union of Laboratory Medicine

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M Stefanovic1 , E Topic1 , AM Simundic1

1Sestre milosrdnice University Hospital, Clinical Institute of Chemistry

Thiopurine S-methyltrasferase (TPMT) is a polymorphic drug metabolizing enzyme which catalyzes inactivation of mercaptopurine, azathioprine and thioguanine. 5-10% of patients are of intermediate TPMT activity (heterozygous genotype), and about 0.33% have almost no activity at all (homozygous genotype) which leads to excessive drug accumulation with possible severe myelosuppression or even fatal clinical outcome. Few TPMT genotyping methods were developed to date. They include expensive and laborious PCR RFLP or methods that require specific and expensive equipment, not everywhere available (Denaturing HPLC, or Horizontal Conformation Sensitive Gel Electrophoresis).
Aim of this study was to design simple PCR-SSCP method for genotyping 5 most common alleles -TPMT*1 (wild type), TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C which comprises for over 90-95% of TPMT genetic variation. By using the precast GMA(TM) (Gene Mutation Analysis) gels in the SEA 2000 apparatus (Elchrom Scientific, Switzerland), we genotyped 50 DNA samples from healthy subjects previously genotyped by PCR-RFLP. After PCR amplification of three TPMT gene regions containing allele specific point mutations, amplicons were denatured on 95°C, chilled on ice and run overnight under different SSCP electrophoresis conditions (10°C, 15°C and 5°C respectively). Gels were stained with SYBR Gold (Molecular probes, USA). We observed reproducible and uniform band patterns for different TPMT genotypes which were interpreted against positive control samples. Our findings were 100% consistent with samples previously genotyped by PCR RFLP. Among our samples, we found 97.8% TPMT*1 and 2.2% TPMT*3A alleles. Between them, 95.6% were wild type homozygotes and 4.4% TPMT*3A heterozygotes. These results are concordant with other authors’ findings. Our conclusion is that this method is reliable and more suitable than previously used PCR-RFLP for identification of known mutations, and it simultaneously screens for new mutations.


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