In vitro characterization of four novel non-functional variants of the thiopurine S-methyltransferase

Human thiopurine S-methyltransferase (TPMT) is an enzyme responsible for the detoxification of widely used thiopurine drugs such as azathioprine (Aza). Its activity is inversely related to the risk of developing severe hematopoietic toxicity in certain patients treated with standard doses of thiopurines. DNA samples from four leucopenic patients treated with Aza were screened by PCR-SSCP analysis for mutations in the 10 exons of the TPMT gene. Four missense mutations comprising two novel mutations, A83T (TPMT*13, Glu²⁸Val) and C374T (TPMT*12, Ser¹²⁵Leu), and two previously described mutations, G430C (TPMT*10, Gly¹⁴⁴Arg) and T681G (TPMT*7, His²²⁷Gln) were identified. Using a recombinant yeast expression system, kinetic parameters (K_m and V_max) of 6-thioguanine S-methylation of the four TPMT variants were determined and compared to those obtained with wild-type TPMT. This functional analysis suggests that these rare allelic variants are defective TPMT alleles. The His²²⁷Gln variant retained only 10% of the intrinsic clearance value (V_max/K_m ratio) of the wild-type enzyme. The Ser ¹²⁵Leu and Gly¹⁴⁴Arg variants were associated with a significant decrease in intrinsic clearance values, retaining about 30% of the wild-type enzyme, whereas the Glu²⁸Val variant produced a more modest decrease (57% of the wild-type enzyme). The data suggest that the sporadic contribution of the rare Glu²⁸Val, Ser¹²⁵Leu, Gly¹⁴⁴Arg, and His²²⁷Gln variants may account for the occurrence of altered metabolism of TPMT substrates. These findings improve our knowledge of the genetic basis of interindividual variability in TPMT activity and would enhance the efficiency of genotyping methods to predict patients at risk of inadequate responses to thiopurine therapy.