Human Aromatase: Gene Resequencing and Functional Genomics

Research Article

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 polymorphisms 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 polymorphisms. 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

CYP19 gene resequencing. Each of the 240 DNA samples studied was centrifuged at 11,600 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 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 70jC. 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 643 to 137 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 ALof10 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 to that for the WT primer sequencing chemistry. To exclude PCR-induced artifacts, indepen- allozyme. dent amplifications were done for those samples in which a SNP was CYP19 enzyme assay, substrate kinetics, and inhibitor constants. 3 observed only once or any sample with an ambiguous chromatogram. Chro- Aromatase activity was assayed by measuring the release of H2O from matograms were analyzed using the PolyPhred 3.0 and Consed 8.0 programs [1h3H]androst-4-ene-3,17-dione (NEN Life Science Products, Boston, MA) as from the University of Washington. The University of Wisconsin GCG described elsewhere (27, 28). These reactions were carried out for 20 software package, version 10, was also used to analyze nucleotide sequence. minutes at 37jC in 0.05 mol/L Tris-HCl (pH 7.4) under air. Each reaction CYP19 GeneScan analysis. A (TTTA)n repeat at position 77 in intron 4 mixture contained either 20 or 100 nmol/L [1h3H]androst-4-ene-3,17-dione was analyzed by using GeneScan to detect polymorphism length. The (25.3 Ci/mmol), 30 to 60 ng of microsomal protein, and an NADPH primers and the PCR conditions used to perform this amplification are also regeneration system (1.5 mmol/L glucose 6-phosphate, 1 unit of glucose-6- listed in Supplementary Data. In this case, the reverse primer was labeled phosphate dehydrogenase, and 3.5 mmol/L NADPH) in a final volume of 100 with a fluorescence tag, [(3V,6V-dipivaloyfluoresceinyl)-6-carboxamidohexyl]- AL. After incubation, 6 volumes of chloroform was added to the reaction 1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (Glen Research, mixture and the mixture was vortexed for 30 seconds to terminate the Sterling, VA). An internal size standard (500-TAMPA, Perkin-Elmer) was reaction and partition the remaining substrate into the organic phase. After used to determine repeat length. These chromatogram traces were analyzed centrifugation at 14,000 g for 10 minutes, radioactivity remaining in the using GeneScan Analysis version 3 (Perkin-Elmer). aqueous phase was determined by liquid scintillation counting.

CYP19 expression constructs, COS-1 cell transfection, and micro- Apparent Km values were determined using the same radiochemical somal preparations. The aromatase wild-type (WT) cDNA sequence was assay and under the same conditions as described above. Triplicate assays cloned into the eukaryotic expression vector pCR3.1 (Invitrogen, Carlsbad, were done for each variant allozyme in the presence of eight concentrations CA). The pCR3.1 WT aromatase expression construct was then used as the of [1h3H]androst-4-ene-3,17-dione that varied from 0.3 to 40 nmol/L. For template for site-directed mutagenesis done using circular PCR to create the Thr364 allozyme, the concentration of [1h3H]androst-4-ene-3,17-dione variant constructs. The primers and amplification conditions used during ranged from 1.25 to 160 nmol/L. Ki values were also determined for each those PCR reactions are listed in Supplementary Data. Sequences of the allozyme in the presence of the aromatase inhibitors letrozole and constructs were confirmed by sequencing both strands of the insert. To help exemestane. In those experiments, triplicate assays were done using six exclude the possibility of PCR-induced alterations in the vector, in vitro concentrations of [1h3H]androst-4-ene-3,17-dione that varied from 1.25 to translation was done with each of the expression constructs using the TNT 320 nmol/L in the presence of three concentrations of letrozole (0.2, 0.4, and rabbit reticulocyte lysate system (Promega, Madison, WI) and all allozymes 0.8 nmol/L) or exemestane (1.25, 2.5, and 5 nmol/L). In the case of the showed approximately equal quantities of translated protein. Thr364 variant allozyme, the letrozole concentrations were 0.1, 0.2, and 0.4 To make it possible to correct for transfection efficiency, we also nmol/L but the exemestane concentrations were the same as those used to designed an expression construct that contained a green fluorescent protein study the other allozymes. (GFP) and human NADPH-b5 reductase (DIA1) fusion protein that would be Immunofluorescence microscopy. FITC-conjugated goat anti-mouse targeted to the endoplasmic reticulum because of the DIA1 portion of the immunoglobulin and tetramethylrhodamine isothiocyanate–conjugated construct. The DIA1 cDNA was amplified using a human liver Marathon- goat anti-rabbit immunoglobulin were purchased from Southern Biotech Ready cDNA library (BD Biosciences Clonetech, Palo Alto, CA) as template, (Birmingham, AL). COS-1 cells were subcultured to 50% to 70% confluence and was cloned into the GFP fusion TOPO TA expression vector on coverslips, were transfected with expression constructs, and were then (Invitrogen). The PCR conditions used to perform that amplification are cultured for an additional 48 hours. The cells were washed with PBS, fixed listed in Supplementary Data. with 3% paraformaldehyde for 12 minutes at room temperature, and, COS-1 cells were transfected with expression constructs for the CYP19 finally, were washed and incubated at room temperature for 5 minutes with WT and variant allozymes as well as ‘‘empty’’ pCR3.1 that lacked an insert, buffer containing 0.5% Triton X-100. The coverslips were then incubated using the TransFast reagent (Promega) at a charge ratio of 1:1. Specifically, with the primary antibodies—rabbit polyclonal antihuman antibody against 7 Ag of aromatase expression construct DNA was cotransfected with 7 Agof calnexin, an endoplasmic reticulum marker, and mouse monoclonal DIA1-GFP expression construct DNA. After 48 hours, the COS-1 cells were antihuman aromatase antibody—followed by FITC-conjugated goat harvested in 0.25 mol/L sucrose and were homogenized for 20 seconds with anti-mouse or tetramethylrhodamine isothiocyanate–conjugated goat anti- a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). The rabbit IgG antibody. The COS-1 cells were then viewed by fluorescence homogenates were centrifuged at 500 g for 5 minutes and at 6,500 g for microscopy using a Nikon 80i fluorescence microscope with 488 or 570 nm 10 minutes. The supernatant was then transferred to a new tube and was filters for excitation of the green or red fluorochrome, respectively.

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Data analysis. Values for p, u, and Tajima’s D were calculated and American, Caucasian-American, Han Chinese-American, and Mexican- corrected for length as described by Tajima (29). DVvalues for the linkage American subjects. Approximately 5,424,000 bp of DNA sequence disequilibrium analysis of polymorphism pairs were calculated as was analyzed in the course of these experiments. Eighty-eight described by Hartl and Clark (30) and Hedrick (31) and those data were polymorphisms were observed, including 85 SNPs, 2 insertion- displayed graphically. Haplotype analysis was done as described by deletion events, and 1 polymorphic TTTA repeat (Fig. 1; Table 1). Schaid et al. (32). Apparent Km values were calculated with the method of Wilkinson (33) using a computer program written by Cleland (34). There were large ethnic variations in both allele frequencies and Points that deviated from linearity on double-inverse plots (i.e., those types, with 69 polymorphisms in African-American DNA, 37 in DNA showing substrate inhibition) were not used to perform these samples from Caucasian-American subjects, 30 in Han Chinese- calculations. For the determination of Ki values, Lineweaver-Burke American subjects, and 44 in DNA from Mexican-American double-inverse plots were done at each concentration of inhibitor. Slopes subjects. Thirty-two polymorphisms were observed only in were calculated for the double-inverse plots and secondary plots of slope African-American subjects, six in Han Chinese-American subjects, against inhibitor concentration were determined. Intercepts on the six in Mexican-American subjects, and five in Caucasian-American inhibitor concentration axis were used to determine Ki values. Pearson subjects. Of the polymorphisms identified in the course of these product moment correlation coefficients were calculated using Excel and studies, 62 had not been reported previously, 31 of which were group means were compared by the use of ANOVA with the Prism ‘‘common,’’ with allele frequencies of >1% in at least one ethnic program. group. All polymorphisms were in Hardy-Weinberg equilibrium except for one polymorphism in Caucasian-American subjects that Results was located 41 bp upstream of exon 1.1. We also determined Human CYP19 gene resequencing. The CYP19 gene was ‘‘nucleotide diversity,’’ a quantitative measure of genetic variation, resequenced using 240 DNA samples, 60 each from African- adjusted for the number of alleles studied. Two standard measures

Figure 1. Human CYP19 genetic polymorphisms. Schematic representation of the CYP19 gene structure. Arrows, locations of polymorphisms. Orange rectangles, open reading frame; light blue rectangles, UTRs. Red arrows, frequencies of >10%; dark blue arrows, frequencies from 1% to 10%; black arrows, polymorphisms with frequencies of <1%. AA, African-American subjects; CA, Caucasian-American subjects; HCA, Han Chinese-American subjects; MA, Mexican-American subjects. I/D, insertion/deletion event. The GT and TCT I/D polymorphisms and the variable number of tandem repeat (TTTA)n polymorphism, as well as amino acid changes resulting from nonsynonymous cSNPs, are also indicated. www.aacrjournals.org 11073 Cancer Res 2005; 65: (23). December 1, 2005

Table 1. Human CYP19 genetic polymorphisms

db-SNP Location Nucleotide Nucleotide Amino acid Frequency of variant allele identification change change Caucasian- African- Han Chinese- Mexican- American American American American

rs7176005 5V-FR exon 1.1 588 G!A 0.142 0.408 0.150 0.175 5V-FR exon 1.1 566 C!T 0.000 0.000 0.008 0.000 5V-FR exon 1.1 554 T!C 0.000 0.008 0.000 0.008 5V-FR exon 1.1 316 T!C 0.000 0.008 0.000 0.000 5V-FR exon 1.1 278 C!T 0.000 0.000 0.283 0.416 5V-FR exon 1.1 245 G!T 0.008 0.000 0.000 0.000 rs6493497 5V-FR exon 1.1 144 C!T 0.158 0.250 0.150 0.158 Exon 1.1 35 G!A 0.000 0.008 0.000 0.008 Exon 1.1 2G!A 0.000 0.008 0.000 0.000 5V-FR exon 2a 639 G!A 0.008 0.000 0.000 0.000 5V-FR exon 2a 632 C!T 0.000 0.042 0.000 0.000 rs4774585 5V-FR exon 2a 468 C!T 0.175 0.183 0.000 0.300 5V-FR exon 2a 429 T!C 0.042 0.000 0.000 0.000 5V-FR exon 2a 149 C!G 0.000 0.008 0.000 0.000 5V-FR exon 2a 125 C!T 0.000 0.150 0.000 0.025 5V-FR exon 2a 124 G!A 0.000 0.008 0.000 0.000 Exon 2a 38 A!G 0.000 0.000 0.000 0.008 Exon 2a 21 C!A 0.008 0.092 0.000 0.000 5V-FR exon 1.4 563 G!A 0.000 0.042 0.000 0.000 5V-FR exon 1.4 562 C!A 0.000 0.042 0.000 0.000 Exon 1.4 241 G!T 0.000 0.000 0.008 0.000 5V-FR exon 1.5 638 C!T 0.000 0.000 0.000 0.008 rs936308 5V-FR exon 1.5 628 C!G 0.867 0.433 0.675 0.875 rs1902582 5V-FR exon 1.5 334 T!C 0.050 0.350 0.167 0.100 rs2470175 5V-FR exon 1.5 317 G!C 0.092 0.075 0.000 0.025 rs1902581 5V-FR exon 1.5 128 C!T 0.000 0.016 0.000 0.000 Exon 1.5 80 A!T 0.000 0.025 0.000 0.000 5V-FR exon 1.7 651 C!T 0.000 0.025 0.000 0.000 5V-FR exon 1.7 550 G!A 0.000 0.008 0.000 0.000 5V-FR exon 1.7 543 G!A 0.000 0.042 0.000 0.000 5V-FR exon 1.7 495 G!A 0.000 0.033 0.000 0.000 5V-FR exon 1.7 439 A!C 0.008 0.000 0.000 0.008 5V-FR exon 1.7 428 G!A 0.008 0.000 0.000 0.000 5V-FR exon 1.7 408 G!A 0.000 0.017 0.000 0.000 5V-FR exon 1.7 194 G!T 0.000 0.008 0.000 0.000 Exon 1.7 26 C!T 0.000 0.017 0.000 0.000 3V-FR exon 1.7 25 G!A 0.000 0.033 0.000 0.000 rs7181886 3V-FR exon 1.7 54 G!C 0.058 0.367 0.325 0.092 5V-FR exon 1.f 739 C!A 0.000 0.042 0.000 0.000 5V-FR exon 1.f 725 G!A 0.092 0.150 0.000 0.025 5V-FR exon 1.f 690 A!C 0.092 0.158 0.000 0.025 rs1902586 5V-FR exon 1.f 649 C!T 0.058 0.442 0.317 0.117 5V-FR exon 1.f 425 C!T 0.000 0.000 0.000 0.008 5V-FR exon 1.f 391 T!G 0.000 0.008 0.000 0.000 Exon 1.f 108 C!T or A 0.000 0.008 (T) 0.000 0.008 (A) Exon 1.f 66 C!T 0.000 0.033 0.000 0.000 Exon 1.f 35 A!G 0.000 0.008 0.000 0.000 5V-FR exon 1.2 827 A!G 0.000 0.008 0.000 0.008 5V-FR exon 1.2 757 G!A 0.000 0.017 0.000 0.000

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Table 1. Human CYP19 genetic polymorphisms (Cont’d)

db-SNP Location Nucleotide Nucleotide Amino acid Frequency of variant allele identification change change Caucasian- African- Han Chinese- Mexican- American American American American

rs2008691 5V-FR exon 1.2 596 T!C 0.125 0.392 0.225 0.108 5V-FR exon 1.2 555 T!A 0.000 0.017 0.000 0.000 rs1062033 Exon 1.2 224 G!C 0.450 0.125 0.441 0.267 Exon 1.2 217 G!A 0.000 0.000 0.000 0.008 Exon 1.2 125 C!T 0.000 0.016 0.000 0.000 5V-FR exon 1.6 362 C!T 0.000 0.000 0.000 0.008 5V-FR exon 1.6 301 T!G 0.000 0.008 0.000 0.000 5V-FR exon 1.6 273 T!A 0.008 0.000 0.000 0.000 rs10459592 5V-FR exon 1.6 196 A!C 0.6 0.308 0.442 0.517 rs4775936 Exon 1.6 77 G!A 0.492 0.117 0.425 0.267 Intron 1.6 53 C!A 0.000 0.000 0.017 0.000 Intron 1.6 61 C!T 0.025 0.033 0.000 0.033 Intron 1.6 353 GT(I!D) 0.000 0.000 0.017 0.000 Exon PII 83 C!A 0.008 0.067 0.008 0.025

Exon 2 42 C!G 0.000 0.033 0.000 0.000 Exon 2 109 T!C 0.000 0.033 0.000 0.000 Published (9) Exon 2 115 T!C Trp39Arg 0.000 0.000 0.067 0.000 rs3759811 Intron 2 59 A!G 0.542 0.175 0.450 0.283 Intron 2 27 T!C 0.000 0.100 0.000 0.000 Exon 3 186 C!T 0.008 0.067 0.008 0.033 rs700518 Exon 3 240 A!G 0.542 0.167 0.450 0.283 Intron 3 48 G!A 0.000 0.008 0.000 0.000 Intron 4 8 G!A 0.000 0.008 0.000 0.000 rs11575899 Intron 4 27 TCT(I!D) 0.333 0.308 0.333 0.417 Published (9) Intron 4 77 (TTTA)n n = 7 0.475 0.833 0.533 0.658 n = 8 0.125 0.025 0.008 0.092 n = 10 0.008 0.008 0.017 0.017 n = 11 0.342 0.125 0.350 0.200 n = 12 0.033 0.000 0.092 0.033 n = 13 0.008 0.008 0.000 0.000 Exon 5 602 C!T Thr201Met 0.050 0.050 0.000 0.008 rs4324076 Intron 5 16 T!G 0.530 0.200 0.508 0.317 Exon 6 633 T!C 0.000 0.000 0.000 0.008 Published (19) Intron 6 36 A!T 0.542 0.200 0.508 0.317 Intron 6 44 G!C 0.000 0.008 0.000 0.008 rs2304463 Intron 6 106 T!G 0.542 0.192 0.542 0.308 rs700519 Exon 7 790 C!T Arg264Cys 0.025 0.225 0.117 0.050 rs2289105 Intron 7 26 C!T 0.100 0.033 0.000 0.225 Published (19) Intron 7 79 A!G 0.542 0.183 0.508 0.317 Exon 8 963 C!G 0.000 0.017 0.000 0.000 Intron 8 29 C!T 0.000 0.008 0.000 0.000 Exon 9 1,091 T!CMet364Thr 0.000 0.000 0.008 0.000 rs10046 3V-UTR 1,531 C!T 0.558 0.192 0.542 0.317 rs4646 3V-UTR 1,673 G!T 0.292 0.308 0.333 0.533

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Table 1. Human CYP19 genetic polymorphisms (Cont’d)

Caucasian- African- Han Chinese- Mexican- American American American American

p, 104 9.95 F 5.04 8.16 F 4.20 8.75 F 4.47 7.87 F 4.05 u, 104 11.5 F 3.00 6.22 F 1.77 5.01 F 1.47 7.33 F 2.01 Tajima’s D 0.43 0.95 2.22 0.23

NOTE: Polymorphism locations, alterations in nucleotide and amino acid sequences, and frequencies of polymorphisms observed are listed for each of the four ethnic groups studied. Polymorphisms in exons are ‘‘boxed.’’ (I!D) represents an insertion/deletion event. The numbering scheme for nucleotides located within introns 5Vand 3Vof the exons is based on their distance from splice junctions, with the use of negative and positive numbers, respectively. Nucleotides within the untranslated upstream exons are numbered relative to the 3Vsplice junction for that exon. Nucleotides in the region upstream of exon 2, which includes the two most proximal initial untranslated exons (E1.3 and PII), all of the translated exons (2-10) and the 3V-UTR are numbered relative to the ‘‘A’’ in the translation initiation codon. The table also includes estimates of two measures of nucleotide diversity, p and u,as well as Tajima’s D, a test of the ‘‘neutral’’ mutation hypothesis.

of nucleotide diversity are p, average heterozygosity per site, and immunoreactive protein levels were then determined using and u, a population mutation measure that is theoretically equal microsomes isolated from these cells. Because one of the DNA to the neutral mutation variable (35). These values are listed in samples that we had resequenced contained two nonsynonymous Table 1. In addition, values for Tajima’s D, a test of the ‘‘neutral’’ cSNPs, resulting in both Trp39Arg and Arg264Cys alterations in mutation hypothesis (29), were estimated for each population encoded amino acids, an expression construct was created that we (Table 1). Only the value for Tajima’s D in Han Chinese-American designated as a double-mutant construct. This construct contained subjects differed significantly from values for the other ethnic both cSNPs although it was not possible to determine unequivo- groups. cally that a single allele that included both polymorphisms was Four nonsynonymous cSNPs, polymorphisms that altered the present in this subject. Finally, to make it possible to correct for encoded amino acids, were observed: Trp39Arg, Thr201Met, transfection efficiency, an expression vector for a GFP and DIA1 Arg264Cys, and Met364Thr (Fig. 1; Table 1). The SNPs resulting in fusion protein that would be targeted to the endoplasmic reticulum Trp39Arg and Arg264Cys alterations in amino acid sequence had was also created and cotransfected with the aromatase allozyme been described previously. Two of these polymorphisms, Trp39Arg constructs. and Met364Thr, were observed only in the Han Chinese-American Six independent transfections were done for each allozyme. As samples, with allele frequencies of 6.7% and 0.8%, respectively. shown graphically in Fig. 3A, the Cys264, Thr364, and double-mutant The Arg264Cys polymorphism was common, with a frequency >2.5% allozymes had 72%, 15%, and 21% of the WT enzyme activity, in all four populations. Thr201Met polymorphism was observed respectively, all of which differed significantly from the WT value. in three of the four ethnic groups, with an allele frequency of 5% in Values for neither the Arg39 nor Met201 allozymes differed both African-American and Caucasian-American and 0.8% in significantly from that for WT. Very similar results were obtained Mexican-American subjects. Homozygous samples were observed when a 5-fold higher substrate concentration, 100 nmol/L andros- only for Arg264Cys, in both African-American and Han Chinese- tenedione rather than 20 nmol/L, was used to perform the assays American subjects. (data not shown). Haplotype and linkage disequilibrium analysis. There is Substrate and inhibitor kinetic studies. One possible increasing appreciation for the importance of linkage disequilib- explanation for the decreased levels of enzyme activity observed rium and haplotype data for application to association studies with several of the variant allozymes would involve an alteration in (36). Therefore, we did population-specific linkage disequilibrium substrate kinetics. Therefore, apparent Km values were determined and haplotype analysis for the CYP19 polymorphisms. Figure 2 for the WT and variant allozymes with androstenedione as the shows a graphical representation of pairwise DV values, a measure substrate. An elevated Km value when compared with that for the of linkage disequilibrium, in all four ethnic groups that we WT allozyme was observed only with the Thr364 variant (Table 3). studied. DV values are 1.0 when polymorphisms are maximally However, although the increase in apparent Km for this allozyme associated and zero when they are randomly associated might have contributed to the observed decrease in activity (30, 31). Haplotypes can be determined unequivocally only observed, the major mechanism involved a decrease in protein if not more than one polymorphism in an allele is heterozygous level as described subsequently and as shown by the lack of a but it is possible to ‘‘infer’’ haplotypes computationally (32). significant increase in activity when the substrate concentration Ethnic-group-specific haplotype analysis for CYP19 showed 12 was increased 5-fold from 20 to 100 nmol/L. unequivocal haplotypes and 32 inferred haplotypes—with striking We also determined whether alterations in the amino acid variations among the four ethnic groups in haplotype frequencies sequences of the variant allozymes might influence response to two (Table 2). aromatase inhibitors, letrozole and exemestane. We selected these Recombinant allozyme enzyme activity. The possible func- two drugs as representatives of nonsteroidal and steroidal tional significance of the four CYP19 nonsynonymous cSNPs aromatase inhibitors, respectively. IC50 values for the WT allozyme observed during the gene resequencing experiments was studied by were found to be 0.6 and 4.5 nmol/L for these two inhibitors, expressing each variant allozyme in COS-1 cells. Enzyme activity respectively. Ki values for letrozole and exemestane were then

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determined with the recombinant variant allozymes (Table 3). Ki translation studies were done with all expression constructs using a values were similar for all of the allozymes studied, with only the rabbit reticulocyte lysate system. Similar quantities of recombinant value for letrozole for the double-mutant allozyme being protein were produced for all of the allozymes studied (data not significantly different from that for the WT enzyme. An example shown). of the data used to calculate the Ki value for letrozole with WT Subcellular localization. Aromatase, like other eukaryotic aromatase is shown in Fig. 3D. cytochrome P450 enzymes, is localized to the endoplasmic Western blot analysis. We have previously reported that a reticulum (38). Therefore, another mechanism that might explain common mechanism for the functional effects of nonsynonymous decreased levels of the variant allozymes in microsomes would cSNPs is an alteration in protein quantity (37). Therefore, involve changes in subcellular localization. Amino acids 20 to 39 in quantitative Western blot analysis was done using monoclonal CYP19 are hydrophobic and represent a putative transmembrane antibody against a polypeptide corresponding to CYP19 amino domain that is located in the endoplasmic reticulum (1, 39). acids 376 to 390, an area that did not include any of the amino Because of the possibility that the change from Trp to the more acids altered by the four nonsynonymous cSNPs. As shown in hydrophilic Arg at amino acid 39 might alter the subcellular Fig. 3B, levels of recombinant protein corresponded to levels of localization of the Trp39Arg allozyme, we also studied subcellular enzyme activity for the variant allozymes. When level of enzyme localization using fluorescence microscopy. Two other allozymes— activity was plotted against level of immunoreactive protein for the those with the lowest levels of microsomal activity and protein, WT enzyme and all five of the variant allozymes, including the Thr364 and the double-mutant allozyme—were also studied. With double-mutant construct, a significant correlation was observed calnexin as an endoplasmic reticulum marker, immunofluorescent (Rp = 0.937, P = 0.006; Fig. 3C). This observation suggests that a studies were done using COS-1 cells transiently transfected with major mechanism by which these genetic polymorphisms influence constructs encoding the WT or the three variant allozymes. All of aromatase activity, at least after the transient transfection of the allozymes colocalized with calnexin (Fig. 4), indicating that mammalian cells, is through a reduction in the quantity of enzyme they were localized to the endoplasmic reticulum. Therefore, the protein. To exclude the possibility that a defect in the expression decreased levels of immunoreactive protein that we observed for vector introduced during site-directed mutagenesis might have these allozymes could not be explained by alterations in their caused the decreased levels of immunoreactive protein, in vitro subcellular localization.

Figure 2. Human CYP19 linkage disequilibrium (LD). The extent of population-based linkage disequilibrium within the area of CYP19 resequenced, shown as pairwise |DV| values, is depicted graphically. www.aacrjournals.org 11077 Cancer Res 2005; 65: (23). December 1, 2005

Discussion junctions. To do that, we resequenced all exons and their adjacent splice The cytochrome P450 enzyme aromatase, CYP19, is required for junctions, as well as at least 500 bp flanking the 5V-terminus of each of estrogen biosynthesis in both premenopausal and postmenopausal the untranslated upstream exons and portions of the gene encoding the women (1). Molecular epidemiology studies done with a relatively small 3V-UTR using 240 DNA samples from four different ethnic groups. This number of common CYP19 polymorphisms have produced inconsistent in-depth resequencing strategy allowed us to discover previously results with regard to the possible role of genetic variation in CYP19 in unreported polymorphisms of possible functional significance and influencing levels of sex hormones and/or risk for estrogen-dependent also to infer haplotype patterns in different ethnic groups. Two of the disease. In the present study, we set out to systematically identify four nonsynonymous cSNPs identified, those that resulted in Thr201Met genetic polymorphisms in this gene, especially within exons and splice and Met364Thr alterations in amino acid sequence, were novel.

Table 2. CYP19 haplotypes with frequencies of 1% or greater

NOTE: Nucleotide positions are numbered as described in Table 1. Variant nucleotides compared with the ‘‘reference sequence’’ (i.e., the most common sequence in African-American subjects) are highlighted as white on black. Initial haplotype designations (*1, *2, *3, *4, and *5) are made on the basisof amino acids that vary, with the WT sequence designated *1. Subsequent assignments/letter designations were made within ethnic groups based on decreasing frequencies. , unequivocal haplotypes.

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Figure 3. CYP19 recombinant allozyme enzyme activity, immunoreactive protein levels, and inhibitor kinetics. A, average levels of enzyme activity for each of the recombinant allozymes assayed with 20 nmol/L androstenedione as substrate. DM, a double mutant that included the Arg39 and Cys264 polymorphisms. All values have been corrected for transfection efficiency. Bars, mean of six independent transfections; bars, SE. *, P < 0.05; **, P < 0.001, compared with the WT allozyme. The Arg39, Met201, and Cys264 variants also differed significantly from the Thr362 and double-mutant allozymes (P < 0.05). B, average levels of immunoreactive protein on the basis of Western blot analysis. Bars, mean of six independent transfections; bars, SE. *, P < 0.05; **, P < 0.001, compared with the WT allozyme. In addition, the Met201 variant differed significantly (P < 0.05) from the Cys264, Thr364, and double-mutant allozymes, whereas the Arg39 allozyme differed significantly (P < 0.05) only from the Thr362 and the double-mutant variants. C, correlation of levels of CYP19 enzyme activity and immunoreactive protein for recombinant allozymes. The correlation was still significant (Rp = 0.92, P < 0.03) even if the double mutant data were not included in the analysis. D, Letrozole inhibitor kinetics done with WT CYP19. The double-inverse plots show the effect of various concentrations of letrozole on CYP19 enzyme activity. These data were used to calculate the Ki value listed in Table 3.

We also did functional genomic studies, including determin- mechanism by which nonsynonymous cSNPs affect the activity ations of activity and immunoreactive protein levels, substrate and of this enzyme is to alter the level of enzyme protein. The 4-fold 364 inhibitor kinetics, and subcellular localization for all four of the elevation in apparent Km for the Thr allozyme represents an nonsynonymous cSNPs. Levels of aromatase enzyme activity were additional possible explanation for the low level of activity observed dramatically decreased for the Thr364 and the double mutant that with this variant. Finally, with the exception of the double-mutant included both the Arg39 and Cys264 polymorphisms (Fig. 3A). There allozyme, there were no significant differences among the variant was also a slight decrease in activity for the Cys264 allozyme. It allozymes in their response to the aromatase inhibitors letrozole should be emphasized once again that it is unclear whether the two and exemestane (Table 3). In addition to polymorphisms within the polymorphisms present in the double-mutant construct occur coding region, multiple polymorphisms were also identified outside naturally within a single allele; however, because of this uncertainty, of exons (Fig. 1; Table 1), all of which could potentially contribute to we created the construct and studied the double-mutant allozyme. variation in gene expression. Obviously, any of these polymorphisms These relative levels of enzyme activity correlated well with levels of could also be linked to functionally important variation in DNA immunoreactive protein (Fig. 3C), suggesting that one important sequence located within areas of the gene that we did not sequence. www.aacrjournals.org 11079 Cancer Res 2005; 65: (23). December 1, 2005

Our functional genomic studies included two novel allozymes, those Table 3. Human aromatase allozyme substrate and 201 364 inhibitor kinetic data containing Met and Thr alterations in amino acid sequence, as well as two that had been reported previously, Arg39 and Cys264.The 264 Aromatase Substrate Inhibitor kinetics, Cys variant was similar to the WTallozyme with regard to substrate

allozyme kinetics, apparent Ki (nmol/L) and inhibitor kinetics, as reported in one previous study (9). However, Km, (nmol/L) we observed a slight decrease in the enzyme activity of this variant Letrozole Exemestane (75% of WT), whereas the previous study, which also used microsome preparations from transiently transfected COS-1 cells, did not (9). WT 6.7 F 2.0 0.21 F 0.05 1.05 F 0.41 One possible explanation for this slight difference might be our use of Arg39 6.0 F 2.0 0.18 F 0.06 0.94 F 0.59 GFP-DIA1, a fusion protein targeted to the endoplasmic reticulum, 201 F F F Met 7.1 1.9 0.22 0.06 1.16 0.09 to correct for transfection efficiency. The previous study used 264 F F F Cys 5.9 2.6 0.21 0.08 1.04 0.33 the cytosolic marker enzyme, h-galactosidase, for this purpose (9). Thr364 26.0 F 10* 0.29 F 0.09 2.86 F 1.29 c A more striking difference occurred with the Arg39 variant, which Double mutant 7.3 F1.9 0.46 F 0.12 1.09 F 0.10 had previously been reported to be inactive after transient expression in human embryonic kidney cells (19). We observed NOTE: The substrate used in these experiments was androstenedione. only slight decreases in both activity and quantity of allozyme Each value is the mean F SE for three determinations. protein (Fig. 3). We have no explanation for this striking difference. *P < 0.001, compared with values for all other allozymes. Obviously, no previous studies had been done with the Met201 and cP < 0.05, compared with WT, Arg39, Cys264,orMet201. the Thr364 variant allozymes because those variant alleles were discovered during the present experiments. The low levels of both

Figure 4. Subcellular localization of recombinant CYP19 allozymes. Red, location of the endoplasmic reticular marker calnexin; green, location of CYP19; Merge, colocalization of calnexin and CYP19.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Human Aromatase Gene Resequencing enzyme activity and immunoreactive protein levels for the Thr364 alleles with nonsynonymous cSNPs showed a significant correlation variant indicate that it would also be of interest to identify subjects between level of activity and immunoreactive protein (Fig. 3C), an who carry this variant and to assess their risk for estrogen- observation compatible with a growing body of data that indicate dependent disease. that alteration in protein quantity is a common mechanism One of the more striking observations made in the course of our responsible for the functional effects of this type of polymorphism, studies was the significant correlation between levels of activity and most often as a result of accelerated protein degradation (37) but also, protein for CYP19 variant allozymes—a phenomenon that confirms at times, involving intracellular protein aggregation (42). Obviously, that a common, although certainly not the only, mechanism by our results must be confirmed in the future by in vivo genotype- which nonsynonymous cSNPs influence function is by altering phenotype correlation studies. Finally, the CYP19 genomic and func- levels of protein—most often as a result of accelerated degradation, tional genomic data included in the present study are of particular at times with protein aggregation and aggresome formation (37, 40– importance in light of the rapidly expanding use of aromatase 42). Obviously, it would be ideal to know the structure of CYP19 to inhibitors during the adjuvant therapy of breast cancer (50). pursue our functional observations. Unfortunately, no mammalian aromatase structure is available. Homology models have been published that are based on soluble bacterial cytochrome P450s, Acknowledgments which have only 13% to 18% amino acid identity to human CYPs Received 4/11/2005; revised 6/23/2005; accepted 7/25/2005. (43–46). Even with the recent publication of the human CYP2C8 and Grant support: NIH grants R01 GM28157 (L. Wang and R.M. Weinshilboum), R01 GM35720 (O.E. Salavaggione and R.M. Weinshilboum), and U01 GM61388; Pharma- CYP2C9 crystal structures (47–49), homology modeling might cogenetics Research Network (O.E. Salavaggione, L. Pelleymounter, L. Wang, B.W. remain problematic because those two enzymes are only 25% to Eckloff, D. Schaid, E.D. Wieben, and R.M. Weinshilboum); CA82267 (Araba A. Adjei and R.M. Weinshilboum); and Pfizer (Alex A. Adjei and Araba A. Adjei). 27% identical to aromatase in amino acid sequence. The costs of publication of this article were defrayed in part by the payment of page In summary, we have resequenced the human CYP19 gene using charges. This article must therefore be hereby marked advertisement in accordance DNA samples from four ethnic groups. In the course of these studies, with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Luanne Wussow for her assistance with the preparation of the we observed 88 polymorphisms and 44 common CYP19 haplotypes. manuscript and Alexander Vandell for his assistance with the creation of the Functional characterization of the four variant allozymes encoded by aromatase allozyme expression constructs.

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Cynthia X. Ma, Araba A. Adjei, Oreste E. Salavaggione, et al.

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