Determiniation of the Prevalence of Thiopurine Methyltransferase (TPMT) Phenotype Assay and Genotype in a Cohort of Rehumatology Patients at Heart of England NHS Trust

Oluwatobi Alabi, Haematology

The Thiopurine methyltransferase (TPMT)is a methylating enzyme which is involved in the metabolism of thiopurine drugs such as Azathioprine, which are used in the management of rheumatological diseases and other autoimmune diseases. The TPMT gene has been localised to chromosome 6p22.3 and encodes a 245 amino acid protein with a predicated molecule mass of 35 kD (Lee et al 1995).
Variation in TPMT activity has been observed to be largely the result of single nucleotide polymorphisms (SNPs). Four variant TPMT alleles, TPMT*2, TPMT*3A, TPMT*3B, TPMT*3C have been identified and account for low or intermediate TPMT activity in approximately 80% of Caucasians.

The aim of this project was to setup a working method to genotype TPMT and compare it with the phenotypic assay set up by Clinical chemistry.

50 patients were recruited from the Rheumatology clinic. DNA was extracted from peripheral blood mononuclear cells. A multiplex ARMS polymerase chain reaction (PCR) was set up simultaneously targeting the TPMT*2,*3A and *3C genotypes without the need for restriction endonuclease, this should detect between 60 and 95% of mutant alleles responsible for deficient TPMT activity (Tai et al 1996).

The results failed to detect any of the alternate TPMT genotypes in the patient cohort.
The reasons for this are unclear but may represent lack of sensitivity in the method used. Alternative methods have been described in the literature including a real time PCR assay for the detection of TPMT*2, *3, *4, *5, and *6 on the Light Cycler which allows rapid screening of patient samples for range of TPMT variants.

Further work required is required to optimise the PCR for TPMT and more sensitive methods including Real time PCR will be explored.