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Human Aromatase: Gene Resequencing and Functional Genomics

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Human Aromatase: Gene Resequencing and Functional Genomics Research Article Human Aromatase: Gene Resequencing and Functional Genomics Cynthia X. Ma,1 Araba A. Adjei,2 Oreste E. Salavaggione,2 Josefa Coronel,2 Linda Pelleymounter,2 Liewei Wang,2 Bruce W. Eckloff,3 Daniel Schaid,4 Eric D. Wieben,3 Alex A. Adjei,1 and Richard M. Weinshilboum2 Departments of 1Medical Oncology, 2Molecular Pharmacology and Experimental Therapeutics, 3Biochemistry and Molecular Biology, and 4Health Sciences Research, Mayo Clinic College of Medicine, Rochester, Minnesota Abstract selective aromatase inhibitors are being used increasingly to treat Aromatase [cytochrome P450 19 (CYP19)] is a critical enzyme postmenopausal women with estrogen-responsive breast cancer (6). for estrogen biosynthesis, and aromatase inhibitors are of CYP19 maps to chromosome 15q21.2 and has a complex increasing importance in the treatment of breast cancer. We structure (7, 8). It spans 123 kb, 30 kb of which contain the coding exons, exons 2 to 10, with a 93 kb region that contains 10 tissue- set out to identify and characterize genetic polymorphisms in the aromatase gene, CYP19, as a step toward pharmacoge- specific noncoding upstream exons with separate promoters that nomic studies of aromatase inhibitors. Specifically, we regulate transcription in different cells and tissues. Although ‘‘resequenced’’ all coding exons, all upstream untranslated CYP19 genetic polymorphisms have been reported, the possible exons plus their presumed core promoter regions, all exon- functional significance of most of those polymorphisms remains intron splice junctions, and a portion of the 3V-untranslated undefined. We set out to systematically identify and characterize region of CYP19 using 240 DNA samples from four ethnic genetic variation in CYP19 by performing complementary gene groups. Eighty-eight polymorphisms were identified, resulting resequencing and functional genomic studies. Specifically, we V in 44 haplotypes. Functional genomic studies were done with resequenced all exons, including at least 500 bp of each of the 5 - the four nonsynonymous coding single nucleotide polymor- untranslated exons, all exon-intron splice junctions, and a portion V V phisms (cSNP) that we observed, two of which were novel. of the 3 -untranslated region (3 -UTR). The samples included DNA Those cSNPs altered the following amino acids: Trp39Arg, from 60 African-American, 60 Caucasian-American, 60 Han Thr201Met, Arg264Cys, and Met364Thr. The Cys264, Thr364, and Chinese-American, and 60 Mexican-American subjects. Functional 39 264 studies were then done with allozymes encoded by the four double variant Arg Cys allozymes showed significant decreases in levels of activity and immunoreactive protein nonsynonymous coding single nucleotide polymorphisms (cSNP) when compared with the wild-type (WT) enzyme after identified during the resequencing studies. transient expression in COS-1 cells. A slight decrease in Previous population-based studies of common CYP19 poly- protein level was also observed for the Arg39 allozyme, morphisms have generated inconsistent results with regard to their whereas Met201 displayed no significant changes in either possible association with either sex hormone levels or risk for activity or protein level when compared with the WT enzyme. estrogen-dependent diseases (9–24). Selected CYP19 polymor- phisms have also been investigated for their possible association There was also a 4-fold increase in apparent Km value for Thr364 with androstenedione as substrate. Of the recombinant with the therapeutic efficacy of aromatase inhibitors (25). The fact allozymes, only the double mutant (Arg39Cys264) displayed a that past data with regard to the association of CYP19 polymor- significant change from the WT enzyme in inhibitor constant phisms with estrogen-dependent disease has been inconsistent for the aromatase inhibitors exemestane and letrozole. These (9–24), as well as the lack of functional studies of these poly- observations indicate that genetic variation in CYP19 might morphisms, underscores the importance of applying a systematic contribute to variation in the pathophysiology of estrogen- approach to identify and functionally characterize CYP19 poly- morphisms. In the present study, we have used gene resequencing dependent disease. (Cancer Res 2005; 65(23): 11071-82) to identify previously unreported genetic variation in this important gene. We then functionally characterized the four Introduction nonsynonymous cSNPs observed during the resequencing studies Aromatase [cytochrome P450 19 (CYP19)], is encoded by the and found variations in aromatase enzyme activity and quantity of CYP19 gene. Aromatase catalyzes a critical reaction for estrogen enzyme protein that were associated with those polymorphisms. biosynthesis—the formation of aromatic C18 estrogens from C19 We also studied the substrate kinetics, subcellular localization, and androgens (1). Alterations in aromatase expression have been impli- response of these variant allozymes to aromatase inhibitors. cated in the pathogenesis of estrogen-dependent disease, including breast cancer, endometrial cancer, and endometriosis (2–5). The importance of this enzyme is also highlighted by the fact that Materials and Methods DNA samples. DNA samples from 60 Caucasian-American, 60 African- American, 60 Han Chinese-American, and 60 Mexican-American subjects (sample sets HD100CAU, HD100African-American, HD100CHI, and Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). HD100MEX) were obtained from the Coriell Cell Repository (Camden, Requests for reprints: Richard M. Weinshilboum, Department of Molecular NJ). All of these DNA samples had been anonymized by the National Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, 200 Institute of General Medical Sciences before deposit and all subjects had First Street, Southwest, Rochester, MN 55905. Phone: 507-284-2246; Fax: 507-284-9111; provided written consent for the use of their DNA for experimental E-mail: [email protected]. I2005 American Association for Cancer Research. purposes. The present study was reviewed and approved by the Mayo Clinic doi:10.1158/0008-5472.CAN-05-1218 Institutional Review Board. www.aacrjournals.org 11071 Cancer Res 2005; 65: (23). December 1, 2005 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Cancer Research CYP19 gene resequencing. Each of the 240 DNA samples studied was centrifuged at 11,600 Â g for 15 minutes. The supernatant from that step used to perform PCR amplifications of the areas to be resequenced. M13 was centrifuged at 132,000 Â g for 45 minutes and the pellet was ‘‘tags’’ were added to the 5Vends of each primer to make it possible to use dye resuspended in 0.05 mol/L potassium phosphate buffer (pH 7.4) followed by primer sequencing chemistry. The sequences of all primers as well as the PCR storage at À70jC. conditions used are listed in Supplementary Data. The primer set used to To correct for variation in transfection efficiency, green fluorescence was amplify exon 10 for the Han Chinese-American samples differed from that measured in the microsomal fraction with a SPECTRAmax GEMINI XS used for the other DNA samples to avoid PCR-induced artifacts. The area dual-scanning microplate spectrofluorometer (Molecular Devices Corpora- from À643 to À137 bp upstream of exon 1.7 was amplified using a 1:10,000 tion, Sunnyvale, CA) using excitation and emission wavelengths of 395 and dilution of the reaction mixture obtained after 30 cycles of the exon 1.7 ‘‘long 507 nm, respectively. Levels of immunoreactive protein and enzyme activity PCR reaction.’’ This was done to avoid nonspecific amplification products. for these transfections were then corrected on the basis of the GFP values. Amplifications were done with AmpliTaq Gold DNA polymerase (Perkin- CYP19 Western blot analysis. A mouse anti-human aromatase Elmer, Foster City, CA), and the 25 AL reaction mixtures contained 0.75 units monoclonal antibody directed against human aromatase amino acids 376 of DNA polymerase, 0.5 AL of a 10-fold diluted DNA sample (16-19 ng DNA), 5 to 390 was purchased from Serotec (Raleigh, NC). This antibody has been to 10 pmol of each primer, 0.08 mmol/L deoxynucleotide triphosphate described in detail elsewhere (26). Aliquots of COS-1 cell microsomal (Boehringer Mannheim, Indianapolis, IN), 0% or 2% DMSO, and 2.5 ALof10Â fractions transfected with CYP19 allozyme cDNA expression constructs PCR buffer containing 15 mmol/L MgCl2 (Perkin-Elmer). Amplification were loaded onto 12.5% acrylamide SDS-PAGE gels on the basis of GFP conditions included a 10-minute ‘‘hot start’’ at 96jC, followed by 35 cycles of values to correct for transfection efficiency. After electrophoresis, proteins 96jC for 30 seconds, 30 seconds at annealing temperatures listed in Supple- were transferred to polyvinylidene difluoride membranes and were detected mentary Data, and 45 seconds at 72jC with a final 10-minute extension at using the enhanced chemiluminescence Western blotting system (ECL, 72jC. All reactions were done in Perkin-Elmer model 9700 thermal cyclers. Amersham Pharmacia, Piscataway, NJ). An AMBIS Radioanalytic Imaging Amplicons were sequenced on both strands in the Mayo Molecular Core System, Quant Probe Version 4.31 (Ambis, Inc., San Diego, CA), was used to Facility with an ABI 377 DNA sequencer using BigDye (Perkin-Elmer) dye quantitate levels of immunoreactive protein relative
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