Date of Award


Document Type

Master Thesis

Degree Name

Masters of Science (Research)


Biological Sciences

First Advisor

Dr. Séamus Fanning

Second Advisor

Dr. Leslie Cotter


Thiopurine methyltransferase (TPMT) is a cytosolic enzyme that catalyses the S- methylation of aromatic and sulphydryl compounds known as the thiopurines. These therapeutic agents are used in the treatment of acute lymphoblastic leukemia, autoimmune disorders, inflammatory bowel disease and organ transplant recipients.

Erythrocyte TPMT activity is polymorphic and patients with intermediate or deficient TPMT are at significant risk for excessive toxicity after receiving ‘standard’ doses of thiopurine medications. The activity of TPMT is inherited as an autosomal co-dominant trait. Several mutations in the TPMT gene have been described that correlate with a low activity phenotype. Three mutations G238C, G460A and A719G account for the majority (96%) of the genetic polymorphisms found in all human populations studied to date. Significant ethnic differences in the frequency of these mutant alleles have been reported.

Advances in DNA analysis have led to the development of molecular approaches to aid in the predictive diagnosis of TPMT deficiency and determine heterozygosity, a step that has immense benefit for the prognostic outcome. Using such assays a sample population (100) was assessed for the presence of the three most common TPMT mutations.

In this work, the design of allele specific oligonucleotides for G460A and A719G permitted the development of Amplification Refractory Mutation Detection System (ARMS) assays for these mutations.

A novel multiplex ARMS PCR assay was designed to simultaneously detect the three most prevalent mutations using two formats i.e. conventional agarose gel electrophoresis and GeneScan analysis. The results of these new assays were compared with those conventional molecular approaches and the results were found to be in exact correlation. Of the sample population, 93 were determined not to have any of the three point mutations. The most common polymorphism in this population was found to be at the A719G position and this was not as expected, however with changes in population demographics, shifts in mutation prevalence may occur.

The multiplex ARMS strategy designed in this work provides a fast, reliable, easy to interpret and cost effective system to test simultaneously for the most prevalent point mutations. Such improvements will be necessary if molecular screening for TPMT polymorphisms is to be introduced into the clinical laboratory.


A thesis presented to the Higher Education and Training Awards Council for the degree of Master of Science

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