A substrate specific functional polymorphism of human gamma-glutamyl hydrolase alters catalytic activity and methotrexate polyglutamate accumulation in acute lymphoblastic leukaemia cells.

We found a significant inverse relationship between gamma-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/m 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 Ile 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.