ORIGINAL ARTICLESA substrate specific functional polymorphism of human γ-glutamyl hydrolase alters catalytic activity and methotrexate polyglutamate accumulation in acute lymphoblastic leukaemia cellsCheng, Qinga,b; Wu, Bainanc,b; Kager, Leoa,b; Panetta, J Carla,b; Zheng, Jiec,b; Pui, Ching-Hond,b; Relling, Mary Va,b; Evans, William Ea,bAuthor Information aDepartment of Pharmaceutical Sciences, bHematological Malignancies Program, cDepartment of Structural Biology and dDepartment of Haematology–Oncology, St Jude Children's Research Hospital, Memphis, Tennessee, USA. Sponsorship: This study was supported in part by the following NIH grants: R37 CA36401 (W.E.E., M.V.R., C.H.P.), R01 CA78224 (W.E.E., M.V.R., C.H.P.), RO1 CA51001 (M.V.R., C.H.P.), U01 GM61393 (M.V.R., W.E.E.), Cancer Center Support Grant CA21765, by a FM Kirby Clinical Research Professorship from the American Cancer Society (C.-H.P.), Erwin Schroedinger grants FWF J2110 and J2304-B13 (L.K.), and by the American Lebanese Syrian Associated Charities. Correspondence and requests for reprints to William E. Evans, Department of Pharmaceutical Sciences, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA. Tel: +1 901 495 3663; fax: +1 901 525 6869; e-mail [email protected] Received 2 March 2004 Accepted 30 April 2004 Pharmacogenetics: August 2004 - Volume 14 - Issue 8 - p 557-567 doi: 10.1097/01.fpc.0000114761.78957.7e Buy Metrics Abstract We found a significant inverse relationship between γ-glutamyl hydrolase (GGH) activity and the accumulation of long-chain methotrexate polyglutamates (MTXPG4–7) in non-hyperdiploid B-lineage acute lymphoblastic leukaemia (ALL) cells after uniform treatment with high-dose methotrexate (HDMTX) (1 g/m2 i.v.). To identify genetic polymorphisms that alter the function of human GGH, we sequenced the GGH exons of genomic DNA from children with ALL, who had a 7.8-fold range of GGH activity in their ALL cells at diagnosis. A single nucleotide polymorphism (452C>T, T127I) was found among patients with low GGH activity, but not found in patients with high GGH activity. Computational modelling indicated that the T127I substitution alters the molecular surface conformation at the catalytic cleft-tail on GGH, which is predicted to alter binding affinity with long chain but not short-chain methotrexate polyglutamates. Enzyme kinetic analysis of heterologously expressed GGH revealed a significantly higher Km (2.7-fold) and lower catalytic efficiency (Vmax/Km reduced 67%) of the T127I variant compared to wild-type GGH using long-chain MTXPG5 as substrate, but not a significant change with short-chain MTXPG2. The 452C>T single nucleotide polymorphism (SNP) was also associated with lower GGH activity in hyperdiploid B-lineage and T lineage ALL cells. Caucasians [10.0%; 95% confidence interval (CI) 6.7–13.3%; n = 155] were found to have a significantly higher frequency of the Ile127 allele than African-Americans (4.4%; 95% CI 1.2–7.5%; n = 80) (P = 0.033). These studies demonstrate a substrate specific functional SNP (452C>T) in the human GGH gene that is associated with lower catalytic activity and higher accumulation of long-chain MTX-PG in leukaemia cells of patients treated with HDMTX. © 2004 Lippincott Williams & Wilkins, Inc.