Salavaggione, Oreste E.

Abstract Thiopurine methyltransferase (TPMT) is an important enzyme in the metabolism of thiopurine medications such as azathioprine. In humans, activity varies widely among individuals, primarily because of genetic polymorphisms. Low TPMT activity increases the risk of myelosuppression from azathioprine and 6-mercaptopurine, whereas high TPMT activity is associated with poor drug efficacy. The purpose of this study was to determine whether dogs also show a wide range of TPMT activity. Heparinized blood samples were obtained from 177 dogs associated with a veterinary teaching hospital. Red blood cell (RBC) TPMT activity was measured by means of a modification of a radiochemical method as established for use in people. TPMT activity varied across a 9-fold range (7.9–71.8 U of RBC per milliliter; median, 21.7). Variation in TPMT activity was not associated with age, sex, or neutering status. Giant Schnauzers had much lower TPMT activity (7.9–20 U of RBC per milliliter; median, 13.1; P < .001) than did other breeds, and Alaskan Malamutes had much higher TPMT activity (22.7–71.8 U of RBC per milliliter; median, 36.0; P < .001) than did other breeds. Such variations in TPMT activity in the canine population and within groups of related dogs could affect thiopurine drug toxicity and efficacy in canine patients.

Abstract Glutathione S-transferase P1 (GSTP1) is of importance for cancer research because of its role in detoxifying carcinogens, activating antineoplastic prodrugs, metabolizing chemotherapeutic agents, and its involvement in cell cycle and apoptosis regulation. Two common GSTP1 genetic polymorphisms have been studied extensively. However, the full range of GSTP1 genetic variation has not been systematically characterized in the absence of disease pathology. We set out to identify common GSTP1 polymorphisms in four ethnic groups, followed by functional genomic studies. All exons, splice junctions, and the 5′-flanking region of GSTP1 were resequenced using 60 DNA samples each from four ethnic groups. The 35 single-nucleotide polymorphisms (SNP) identified included six nonsynonymous SNPs and 17 previously unreported polymorphisms. GSTP1 variant allozymes were then expressed in COS-1 cells, and five displayed significantly altered levels of enzyme activity. One decreased to 22% of the wild-type (WT) activity. Four variant allozymes had Km values that differed significantly from that of the WT, and five showed altered levels of immunoreactive protein compared with WT, with a significant correlation (r = 0.79, P < 0.007) between levels of immunoreactive protein and enzyme activity in these samples. In the Mexican American population, five linked SNPs were significantly associated with GSTP1 mRNA expression, one of which was found by electrophoretic mobility shift assay to alter protein binding. These studies have identified functionally significant genetic variation, in addition to the two frequently studied GSTP1 nonsynonymous SNPs, that may influence GSTP1's contribution to carcinogen and drug metabolism, and possibly disease pathogenesis and/or drug response. [Cancer Res 2008;68(12):4791–801]

Phenylethanolamine N-methyltransferase (PNMT) catalyzes the synthesis of epinephrine from norepinephrine. We previously identified and functionally characterized common sequence variation in the PNMT gene. In the present study, we set out to determine whether common PNMT genetic polymorphisms might be associated with individual variation in circulating epinephrine levels during exercise in 74 Caucasian American subjects. Circulating epinephrine levels were measured in each subject at baseline and during two different levels of exercise, ∼40% and ∼75% of peak workload. The PNMT gene was resequenced with DNA from each study subject. Eight novel PNMT polymorphisms were identified, including a C319T (Arg107Cys) nonsynonymous single nucleotide polymorphism (SNP) and I1G(280)A, a SNP located in the first intron of the gene. The I1G(280)A SNP was significantly associated with decreased exercise-induced circulating epinephrine levels and with a decreased epinephrine-to-norepinephrine ratio. The Cys107 recombinant allozyme displayed significantly lower levels of both PNMT activity and immunoreactive protein than the wild-type allozyme after transfection into COS-1 cells, but it did not appear to be associated with level of epinephrine in these subjects. Electrophoretic mobility shift and reporter gene assays performed with the I1G(280)A SNP indicated that this polymorphism could bind nuclear proteins and might modulate gene transcription. Our studies suggest that functionally significant variant sequence in the human PNMT gene might contribute to individual variation in levels of circulating epinephrine during exercise.

Abstract Purpose: The glutathione S-transferases (GSTs) catalyze the glutathione conjugation of reactive electrophiles, including carcinogens and many antineoplastic drugs. GSTT1 and GSTM1 are polymorphically deleted, but the full range of genetic variation in these two genes has not yet been explored. We set out to systematically identify common polymorphisms in GSTT1 and GSTM1, followed by functional genomic studies. Experimental Design: First, multiplex PCR was used to determine GSTT1 and GSTM1 copy number in 400 DNA samples (100 each from 4 ethnic groups). Exons, splice junctions, and 5′-flanking regions (5′-FR) were then resequenced using DNA samples that contained at least one copy of GSTT1 or GSTM1. Results: Gene deletion frequencies among ethnic groups were from 33.5% to 73.5% for GSTT1 and from 50.5% to 78.0% for GSTM1. GSTT1 deletion data correlated with the results of mRNA microarray expression studies. The 18 single nucleotide polymorphisms (SNP) observed in GSTT1 included three nonsynonymous coding SNPs (cSNPs) and one single-nucleotide deletion, whereas the 51 GSTM1 SNPs included two nonsynonymous cSNPs. Two of the GSTT1 nonsynonymous cSNPs resulted in decreases in levels of immunoreactive protein to 56% and 12% of wild type (WT), whereas those in GSTM1 resulted in modest increases in protein levels. Reporter gene assays showed that one GSTT1 5′-FR haplotype, with a frequency of 32% in African-American subjects, resulted in an increase in transcription in JEG-3 cells to 351% of that for the WT sequence, and one GSTM1 5′-FR haplotype resulted in an increase in transcription in JEG-3 cells to 129% of WT. Conclusions: These observations suggest that functionally significant pharmacogenomic variation beyond GSTT1 and GSTM1 gene deletion may contribute to carcinogenesis or individual variation in antineoplastic drug therapy response.

Abstract 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 ∼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 EC50 for folic acid and in the IC50 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. [Cancer Res 2007;67(18):8772–82]

Abstract Purpose: Gemcitabine is a nucleoside analogue with activity against solid tumors. Gemcitabine metabolic inactivation is catalyzed by cytidine deaminase (CDA) or, after phosphorylation, by deoxycytidylate deaminase (DCTD). We set out to study the pharmacogenomics of CDA and DCTD. Experimental Design: The genes encoding CDA and DCTD were resequenced using DNA from 60 African American and 60 Caucasian American subjects. Expression constructs were created for nonsynonymous coding single nucleotide polymorphisms (cSNP) and reporter gene constructs were created for 5′-flanking region polymorphisms. Functional genomic studies were then conducted after the transfection of mammalian cells. Results: CDA resequencing revealed 17 polymorphisms, including one common nonsynonymous cSNP, 79 A>C (Lys27Gln). Recombinant Gln27 CDA had 66 ± 5.1% (mean ± SE) of the wild-type (WT) activity for gemcitabine but without a significant decrease in level of immunoreactive protein. The apparent Km (397 ± 40 μmol/L) for the Gln27 allozyme was significantly higher than that for the WT (289 ± 20 μmol/L; P < 0.025). CDA 5′-flanking region reporter gene studies showed significant differences among 5′-flanking region haplotypes in their ability to drive transcription. There were 29 SNPs in DCTD, including one nonsynonymous cSNP, 172 A>G (Asn58Asp), in Caucasian American DNA. Recombinant Asp58 DCTD had 11 ± 1.4% of WT activity for gemcitabine monophosphate with a significantly elevated level of immunoreactive protein. No DCTD polymorphisms were observed in the initial 500 bp of the 5′-flanking region. Conclusions: These results suggest that pharmacogenomic variation in the deamination of gemcitabine and its monophosphate might contribute to variation in therapeutic response to this antineoplastic agent.

Abstract Aromatase [cytochrome P450 19 (CYP19)] is a critical enzyme for estrogen biosynthesis, and aromatase inhibitors are of increasing importance in the treatment of breast cancer. We set out to identify and characterize genetic polymorphisms in the aromatase gene, CYP19, as a step toward pharmacogenomic studies of aromatase inhibitors. Specifically, we “resequenced” all coding exons, all upstream untranslated exons plus their presumed core promoter regions, all exon-intron splice junctions, and a portion of the 3′-untranslated region of CYP19 using 240 DNA samples from four ethnic groups. Eighty-eight polymorphisms were identified, resulting in 44 haplotypes. Functional genomic studies were done with the four nonsynonymous coding single nucleotide polymorphisms (cSNP) that we observed, two of which were novel. Those cSNPs altered the following amino acids: Trp39Arg, Thr201Met, Arg264Cys, and Met364Thr. The Cys264, Thr364, and double variant Arg39Cys264 allozymes showed significant decreases in levels of activity and immunoreactive protein when compared with the wild-type (WT) enzyme after transient expression in COS-1 cells. A slight decrease in protein level was also observed for the Arg39 allozyme, whereas Met201 displayed no significant changes in either activity or protein level when compared with the WT enzyme. There was also a 4-fold increase in apparent Km value for Thr364 with androstenedione as substrate. Of the recombinant allozymes, only the double mutant (Arg39Cys264) displayed a significant change from the WT enzyme in inhibitor constant for the aromatase inhibitors exemestane and letrozole. These observations indicate that genetic variation in CYP19 might contribute to variation in the pathophysiology of estrogen-dependent disease.