The use of denaturing high-pressure liquid chromatography for the detection of mutations in thiopurine methyltransferase.
The level of expression of the enzyme thiopurine methyltransferase (TPMT) is an important determinant of the metabolism of drugs used both in the treatment of acute leukaemia (6-mercaptopurine and 6-thioguanine) and as an immunosuppressant in patients with autoimmune diseases or following organ transplantation (azathioprine). Studies of enzyme activity in red blood cells have shown that TPMT expression displays genetic polymorphism with 11% of individuals having intermediate and one in 300 undetectable levels. Patients with biallelic mutations and undetectable enzyme activity suffer life-threatening myelosuppression when treated with conventional doses of these drugs. Patients with intermediate activity have an increased risk of drug-associated toxicity. In the Caucasian populations studied to date, intermediate activity is associated with mutations at two sites of the TPMT gene, G460A and A719G (designated TPMT*3A), in 80% of cases. Detection of these mutations has, to date, been based on the analysis of restriction digests of PCR products. In order to simplify this process we have investigated the ability of denaturing high pressure liquid chromatography (DHPLC) to detect the A719G mutation. DHPLC of PCR products from 15 known heterozygotes (TPMT*3A/TPMT*1) and 18 known homozygotes (TPMT*1/TPMT*1) gave a clear pattern difference between the groups and 100% concordance with the results of restriction digests. These results suggest DHPLC represents a valuable technique for accurate and rapid detection of pharmacologically important mutations in the TPMT gene.
Hall
A G
AG
Leukaemia Research Fund Laboratory, University of Newcastle Upon Tyne, Newcastle Upon Tyne, UK. a.g.hall@ncl.ac.uk
Hamilton
P
P
Minto
L
L
Coulthard
S A
SA
D000970:Antineoplastic Agents / Q000009:adverse effects / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods*; D006801:Humans; D007166:Immunosuppressive Agents / Q000009:adverse effects / Q000378:metabolism; D008780:Methyltransferases / Q000097:blood / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D017354:Point Mutation; D016133:Polymerase Chain Reaction
D000970:Antineoplastic Agents; D007166:Immunosuppressive Agents; D008780:Methyltransferases; C022745:thiopurine methyltransferase