Identification and functional analysis of two rare allelic variants of the thiopurine S-methyltransferase gene, TPMT*16 and TPMT*19 Rima Hamdan-Khalil a,1 , Jean-Luc Gala b,1 , Delphine Allorge a , Jean-Marc Lo-Guidice a , Yves Horsmans c , Nicole Houdret a , Franck Broly a, * a Equipe d’accueil EA2679, Faculte ´ de Me ´decine, Po ˆle Recherche, Lille, France b Technologies Mole ´culaires Applique ´es, Universite ´ Catholique de Louvain and De ´partement des Laboratoires de la De ´fense, Brussels, Belgium c Clinical Pharmacology Unit, St. Luc University Hospital, Brussels, Belgium Received 30 August 2004; accepted 27 October 2004 Abstract Human thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. TPMT is genetically polymorphic and is associated with large interindividual variations in thiopurine drug toxicity and therapeutic efficacy. During routine genotyping of patients with Crohn’s disease, one novel missense mutation, 365A > C(TPMT*19, Lys 122 Thr), and a recently described missense mutation, 488G > A(TPMT*16, Arg 163 His), were identified in a Caucasian and a Moroccan patient, respectively. Using a heterologous yeast expression system, kinetic parameters (K m and V max ) of the two variants with respect to 6-thioguanine S-methylation were determined and compared with those obtained with the wild-type enzyme. The Lys 122 Thr exchange did not significantly decrease the intrinsic clearance value (V max /K m ) of the variant enzyme. In contrast, the Arg 163 His substitution significantly decreased the intrinsic clearance value by three-fold. The Arg 163 is located in a highly conserved region of the human TPMT protein and, as such, the Arg 163 His substitution is expected to result in a marked reduction of enzyme activity, as confirmed by the in vitro data. Phenotyping by measurement of red blood cell TPMT activity indicated that the patient heterozygous for the Lys 122 Thr mutation had normal TPMT activity, whereas the patient heterozygous for the Arg 163 His mutation was an intermediate methylator, which demonstrated a positive correlation between TPMT phenotyping and the in vitro data. The identification of a novel non-functional allele of the TPMT gene improves our knowledge of the genetic basis of interindividual variability in TPMT activity. These data further enhance the efficiency of genotyping methods to predict patients at risk of an inadequate response to thiopurine therapy. # 2004 Elsevier Inc. All rights reserved. Keywords: TPMT; Genetic polymorphism; Azathioprine; 6-Thioguanine; Deficient methylator; Heterologous expression 1. Introduction The thiopurines 6-mercaptopurine, azathioprine and 6- thioguanine (6-TG) are used in the treatment of leukaemia and inflammatory bowel disease, and in the prevention of organ transplant rejection [1]. Thiopurines are metabolised extensively by both anabolic and catabolic pathways. Human thiopurine S-methyltransferase (TPMT; EC2.1.1.67) is a cytosolic enzyme that catalyses the S-methylation of hetero- cyclic and aromatic compounds, including thiopurines. TPMT exhibits a genetic polymorphism with 89% of Cau- casians and Afro-Americans exhibiting a high methylator (HM) phenotype, 11% an intermediate activity (intermediate methylator, IM) and 0.33% a TPMT deficiency (deficient methylator, DM) [2–4]. Several clinical studies have found that high methylators may be under-treated with conventional doses of thiopur- ine drugs, whereas intermediate and deficient methylators are recognized to be at risk of moderate to profound haematopoietic toxicity when treated with standard doses of these medications [5–10]. These observations are explained by an inverse relationship between TPMT activ- ity and the production of active thiopurine metabolites, designated as thioguanine nucleotides (TGN) [5–7]. www.elsevier.com/locate/biochempharm Biochemical Pharmacology 69 (2005) 525–529 Abbreviations: RBC, red blood cell; 6-TG, 6-thioguanine; TPMT, thio- purine S-methyltransferase * Corresponding author. Tel.: +33 3 20 44 49 95; fax: +33 3 20 44 47 29. E-mail address: f-broly@chru-lille.fr (F. Broly). 1 R.H.-K. and J.-L.G. contributed equally to this work. 0006-2952/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2004.10.011