Determination of TPMT mutation status, allele *1, *2, *3A, *3B, *3C (mutations c.238G>C, c.460G>A and c.719A>G)

TPMT gene testing is performed by PCR and reverse hybridization strip assay to detect *1, *2, *3A, *3B, *3C alleles (c.238G>C, c.460G>A and c.719A>G mutations), which are responsible for the reduced activity of this enzyme catalyzing components of thiopurine drugs used in cancer therapy.

The TPMT gene encodes thiopurine S-methyltransferase, which is an essential enzyme for the biodegradation of thiopurines. Thiopurines include the anticancer drugs thioguanine and mercaptopurine, used mainly in haemato-oncology, and the immunosuppressant azathioprine, designed to treat autoimmune diseases and prevent transplant rejection. Adverse effects associated with the breakdown of thiopurines include neurotoxicity, hepatotoxicity, myelosuppression and inflammation of the mucous membranes. The reduced metabolic activity of the TPMT enzyme results from the presence of functional polymorphisms in the coding region of the TPMT gene, of which the most common deficiency alleles in the European population include TPMT2, TPMT3A and TPMT3C and the less common TPMT3B. Clinical manifestations of TPMT deficiency may be severe for patients who are heterozygous or homozygous for functionally variant alleles. 

TPMT gene testing is performed to detect alleles *1, *2, *3A, *3B, *3C (c.238G>C, c.460G>A and c.719A>G mutations). The reverse hybridization strip method works on the principle of assay to identify multiple gene polymorphisms in one experiment, and is based on a combination of polymerase chain reaction (PCR) and reverse hybridization. The process involves PCR amplification using biotinylated primers, and hybridization of the amplified products into test strips containing immobilised, allele-specific oligonucleotide probes. Visualisation of the hybridization reaction occurs due to the reaction of streptavidin-alkaline phosphatases and colour substrates. The advantage of this method is that the sample can be tested for many polymorphisms in one experiment using two multiplex PCR reactions.

Testing for TPMT gene mutation status is part of a pharmacogenetic algorithm for individualising drug doses prior to the administration of thiopurine drugs.