Identification and characterization of genetic variation in the folylpolyglutamate synthase gene.
Folylpolyglutamate synthase (FPGS) catalyzes the polyglutamation of folic acid, methotrexate, and pemetrexed to produce highly active metabolites. To characterize genetic variation in the FPGS gene, FPGS, have resequenced the gene in four different ethnic populations. Thirty-four single nucleotide polymorphisms were identified including five nonsynonymous coding single nucleotide polymorphisms that altered the FPGS protein sequence: F13L and V22I polymorphisms in the mitochondrial isoform of FPGS, and R466/424C, A489/447V, and S499/457F polymorphisms, which exist in both the mitochondrial and cytosolic isoforms. When expressed in AuxB1 cells, the A447V cytosolic variant was functionally similar to the wild-type cytosolic (WT Cyt) allozyme, whereas the R424C and S457F cytosolic variants were reduced by approximately 2-fold in protein expression compared with WT Cyt (P < 0.01). The intrinsic clearance of glutamate was reduced by 12.3-fold (R424C, P < 0.01) and 6.2-fold (S457F, P < 0.01), whereas the intrinsic clearance of methotrexate was reduced by 4.2-fold (R424C, P < 0.05) and 5.4-fold (S457F, P < 0.05) in these two cytosolic variants when compared with the WT Cyt isoform. Additionally, the in vitro enzyme velocity at saturating pemetrexed concentrations was reduced by 1.6-fold (R424C, P < 0.05) and 2.6-fold (S457F, P < 0.01) compared with WT Cyt. AuxB1 cells harboring these same cytosolic variant allozymes displayed significant increases in the EC(50) for folic acid and in the IC(50) values for both methotrexate and pemetrexed relative to the WT Cyt form of FPGS. These observations suggest that genetic variations in FPGS may alter the efficacy of antifolate therapy in cancer patients.
Leil
Tarek A
TA
Department of Oncology, Mayo Clinic, Rochester, Minnesota, USA.
Endo
Chiaki
C
Adjei
Araba A
AA
Dy
Grace K
GK
Salavaggione
Oreste E
OE
Reid
Joel R
JR
Ames
Matthew M
MM
Adjei
Alex A
AA
D001483:Base Sequence; D003600:Cytosol / Q000201:enzymology; D005091:Exons; D005492:Folic Acid / Q000493:pharmacokinetics / Q000494:pharmacology; D005493:Folic Acid Antagonists / Q000493:pharmacokinetics / Q000494:pharmacology; D014644:Genetic Variation; D005971:Glutamates / Q000493:pharmacokinetics / Q000494:pharmacology; D006147:Guanine / Q000031:analogs & derivatives / Q000493:pharmacokinetics / Q000494:pharmacology; D006239:Haplotypes; D006801:Humans; D007700:Kinetics; D008727:Methotrexate / Q000493:pharmacokinetics / Q000494:pharmacology; D000068437:Pemetrexed; D010453:Peptide Synthases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D013379:Substrate Specificity
D005493:Folic Acid Antagonists; D005971:Glutamates; D000068437:Pemetrexed; D006147:Guanine; D005492:Folic Acid; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D008727:Methotrexate