In vitro characterization of four novel non-functional variants of the thiopurine S-methyltransferase q Rima Hamdan-Khalil, a, * Delphine Allorge, a Jean-Marc Lo-Guidice, a Christelle Cauffiez, a Dany Chevalier, a Catherine Spire, a Nicole Houdret, a Christian Libersa, b Michel Lhermitte, a Jean-Fr  ed eric Colombel, c Jean-Luc Gala, d and Franck Broly a a Equipe d’accueil EA2679, Facult e de M edecine, P^ ole Recherche, Lille, France b Centre d’Investigation Clinique, H^ opital Cardiologique, CHRU Lille, France c Service d’H epato-Gastroent erologie, H^ opital Huriez, CHRU Lille, France d Technologies Mol eculaires Appliqu ees, D epartement des Laboratoires de la D efense, Universit e Catholique de Louvain, Brussels, Belgium Received 25 August 2003 Abstract 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, Glu 28 Val) and C374T (TPMT*12, Ser 125 Leu), and two previously described mutations, G430C (TPMT*10, Gly 144 Arg) and T681G (TPMT*7, His 227 Gln) 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 His 227 Gln variant retained only 10% of the intrinsic clearance value (V max /K m ratio) of the wild-type enzyme. The Ser 125 Leu and Gly 144 Arg variants were associated with a significant decrease in intrinsic clearance values, retaining about 30% of the wild-type enzyme, whereas the Glu 28 Val variant pro- duced a more modest decrease (57% of the wild-type enzyme). The data suggest that the sporadic contribution of the rare Glu 28 Val, Ser 125 Leu, Gly 144 Arg, and His 227 Gln 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. Ó 2003 Elsevier Inc. All rights reserved. Keywords: TPMT genetic polymorphism; Azathioprine; 6-Thioguanine; PCR-SSCP; Heterologous expression Human thiopurine S-methyltransferase (TPMT; EC2.1.1.67) exhibits a genetic polymorphism with 89% of Caucasians and African-Americans possessing a high methylator (HM) phenotype, 11% with intermediate activity (intermediate methylator, IM), and 0.33% with TPMT deficiency (deficient methylator, DM), which is inherited as an autosomal recessive trait [1]. TPMT is a cytosolic enzyme that catalyses the S-methylation of thiopurine drugs, such as azathioprine (Aza) and 6- mercaptopurine (6-MP), which are used in the treatment of leukemia and as immunosuppressants [2–4]. Several clinical studies have found that high methy- lators may be undertreated with conventional doses of thiopurine drugs, whereas intermediate and deficient methylators are recognized to be at risk for moderate to profound hematopoietic toxicity when treated with standard doses of these medications [5–10]. These ob- servations are explained by an inverse relationship be- tween TPMT activity and the production of active Biochemical and Biophysical Research Communications 309 (2003) 1005–1010 www.elsevier.com/locate/ybbrc BBRC q Rima Hamdan-Khalil is the recipient of a fellowship from the Lebanese National Center for Scientific Research. Jean-Luc Gala is supported by a grant from the Walloon region (Waleo 2002, Grant No. 215131/0938840) and Fonds National de la Recherche Scientifique (FRSM No. 3.4607.00). * Corresponding author. Fax: +33-3-20-44-47-29. E-mail address: rimahamdan@hotmail.com (R. Hamdan-Khalil). 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.08.103