Human Aromatase: Gene Resequencing and Functional Genomics · significant change from the WT enzyme in inhibitor constant for the aromatase inhibitors exemestane and letrozole. These

Human Aromatase: Gene Resequencing and Functional Genomics

Cynthia X. Ma,1Araba A. Adjei,

2Oreste E. Salavaggione,

2Josefa Coronel,

2

Linda Pelleymounter,2Liewei Wang,

2Bruce W. Eckloff,

3Daniel Schaid,

4

Eric D. Wieben,3Alex A. Adjei,

1and Richard M. Weinshilboum

2

Departments of 1Medical Oncology, 2Molecular Pharmacology and Experimental Therapeutics, 3Biochemistry andMolecular Biology, and 4Health Sciences Research, Mayo Clinic College of Medicine, Rochester, Minnesota

Abstract

Aromatase [cytochrome P450 19 (CYP19)] is a critical enzymefor estrogen biosynthesis, and aromatase inhibitors are ofincreasing importance in the treatment of breast cancer. Weset out to identify and characterize genetic polymorphisms inthe aromatase gene, CYP19 , as a step toward pharmacoge-nomic studies of aromatase inhibitors. Specifically, we‘‘resequenced’’ all coding exons, all upstream untranslatedexons plus their presumed core promoter regions, all exon-intron splice junctions, and a portion of the 3V-untranslatedregion of CYP19 using 240 DNA samples from four ethnicgroups. Eighty-eight polymorphisms were identified, resultingin 44 haplotypes. Functional genomic studies were done withthe four nonsynonymous coding single nucleotide polymor-phisms (cSNP) that we observed, two of which were novel.Those cSNPs altered the following amino acids: Trp39Arg,Thr201Met, Arg264Cys, and Met364Thr. The Cys264, Thr364, anddouble variant Arg39Cys264 allozymes showed significantdecreases in levels of activity and immunoreactive proteinwhen compared with the wild-type (WT) enzyme aftertransient expression in COS-1 cells. A slight decrease inprotein level was also observed for the Arg39 allozyme,whereas Met201 displayed no significant changes in eitheractivity or protein level when compared with the WT enzyme.There was also a 4-fold increase in apparent Km value forThr364 with androstenedione as substrate. Of the recombinantallozymes, only the double mutant (Arg39Cys264) displayed asignificant change from the WT enzyme in inhibitor constantfor the aromatase inhibitors exemestane and letrozole. Theseobservations indicate that genetic variation in CYP19 mightcontribute to variation in the pathophysiology of estrogen-dependent disease. (Cancer Res 2005; 65(23): 11071-82)

Introduction

Aromatase [cytochrome P450 19 (CYP19)], is encoded by theCYP19 gene. Aromatase catalyzes a critical reaction for estrogenbiosynthesis—the formation of aromatic C18 estrogens from C19androgens (1). Alterations in aromatase expression have been impli-cated in the pathogenesis of estrogen-dependent disease, includingbreast cancer, endometrial cancer, and endometriosis (2–5). Theimportance of this enzyme is also highlighted by the fact that

selective aromatase inhibitors are being used increasingly to treatpostmenopausal women with estrogen-responsive breast cancer (6).CYP19 maps to chromosome 15q21.2 and has a complex

structure (7, 8). It spans 123 kb, 30 kb of which contain the codingexons, exons 2 to 10, with a 93 kb region that contains 10 tissue-specific noncoding upstream exons with separate promoters thatregulate transcription in different cells and tissues. AlthoughCYP19 genetic polymorphisms have been reported, the possiblefunctional significance of most of those polymorphisms remainsundefined. We set out to systematically identify and characterizegenetic variation in CYP19 by performing complementary generesequencing and functional genomic studies. Specifically, weresequenced all exons, including at least 500 bp of each of the 5V-untranslated exons, all exon-intron splice junctions, and a portionof the 3V-untranslated region (3V-UTR). The samples included DNAfrom 60 African-American, 60 Caucasian-American, 60 HanChinese-American, and 60 Mexican-American subjects. Functionalstudies were then done with allozymes encoded by the fournonsynonymous coding single nucleotide polymorphisms (cSNP)identified during the resequencing studies.Previous population-based studies of common CYP19 poly-

morphisms have generated inconsistent results with regard to theirpossible association with either sex hormone levels or risk forestrogen-dependent diseases (9–24). Selected CYP19 polymor-phisms have also been investigated for their possible associationwith the therapeutic efficacy of aromatase inhibitors (25). The factthat past data with regard to the association of CYP19 polymor-phisms with estrogen-dependent disease has been inconsistent(9–24), as well as the lack of functional studies of these poly-morphisms, underscores the importance of applying a systematicapproach to identify and functionally characterize CYP19 poly-morphisms. In the present study, we have used gene resequencingto identify previously unreported genetic variation in thisimportant gene. We then functionally characterized the fournonsynonymous cSNPs observed during the resequencing studiesand found variations in aromatase enzyme activity and quantity ofenzyme protein that were associated with those polymorphisms.We also studied the substrate kinetics, subcellular localization, andresponse of these variant allozymes to aromatase inhibitors.

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, andHD100MEX) were obtained from the Coriell Cell Repository (Camden,

NJ). All of these DNA samples had been anonymized by the National

Institute of General Medical Sciences before deposit and all subjects hadprovided written consent for the use of their DNA for experimental

purposes. The present study was reviewed and approved by the Mayo Clinic

Institutional Review Board.

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

Requests for reprints: Richard M. Weinshilboum, Department of MolecularPharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, 200First Street, Southwest, Rochester, MN 55905. Phone: 507-284-2246; Fax: 507-284-9111;E-mail: [email protected].

I2005 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-1218

www.aacrjournals.org 11071 Cancer Res 2005; 65: (23). December 1, 2005

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CYP19 gene resequencing. Each of the 240 DNA samples studied wasused to perform PCR amplifications of the areas to be resequenced. M13

‘‘tags’’ were added to the 5Vends of each primer to make it possible to use dye

primer sequencing chemistry. The sequences of all primers as well as the PCR

conditions used are listed in Supplementary Data. The primer set used toamplify exon 10 for the Han Chinese-American samples differed from that

used for the other DNA samples to avoid PCR-induced artifacts. The area

from �643 to �137 bp upstream of exon 1.7 was amplified using a 1:10,000

dilution of the reaction mixture obtained after 30 cycles of the exon 1.7 ‘‘longPCR reaction.’’ This was done to avoid nonspecific amplification products.

Amplifications were done with AmpliTaq Gold DNA polymerase (Perkin-

Elmer, Foster City, CA), and the 25 AL reaction mixtures contained 0.75 units

of DNA polymerase, 0.5 AL of a 10-fold diluted DNA sample (16-19 ng DNA), 5

to 10 pmol of each primer, 0.08 mmol/L deoxynucleotide triphosphate

(Boehringer Mannheim, Indianapolis, IN), 0% or 2% DMSO, and 2.5 AL of 10�PCR buffer containing 15 mmol/L MgCl2 (Perkin-Elmer). Amplification

conditions included a 10-minute ‘‘hot start’’ at 96jC, followed by 35 cycles of

96jC for 30 seconds, 30 seconds at annealing temperatures listed in Supple-

mentary Data, and 45 seconds at 72jC with a final 10-minute extension at

72jC. All reactions were done in Perkin-Elmer model 9700 thermal cyclers.

Amplicons were sequenced on both strands in the Mayo Molecular Core

Facility with an ABI 377 DNA sequencer using BigDye (Perkin-Elmer) dye

primer sequencing chemistry. To exclude PCR-induced artifacts, indepen-

dent amplifications were done for those samples in which a SNP was

observed only once or any sample with an ambiguous chromatogram. Chro-

matograms were analyzed using the PolyPhred 3.0 and Consed 8.0 programs

from the University of Washington. The University of Wisconsin GCG

software package, version 10, was also used to analyze nucleotide sequence.

CYP19 GeneScan analysis. A (TTTA)n repeat at position 77 in intron 4

was analyzed by using GeneScan to detect polymorphism length. Theprimers and the PCR conditions used to perform this amplification are also

listed in Supplementary Data. In this case, the reverse primer was labeled

with a fluorescence tag, [(3V,6V-dipivaloyfluoresceinyl)-6-carboxamidohexyl]-

1-O -(2-cyanoethyl)-(N,N -diisopropyl)-phosphoramidite (Glen Research,Sterling, VA). An internal size standard (500-TAMPA, Perkin-Elmer) was

used to determine repeat length. These chromatogram traces were analyzed

using GeneScan Analysis version 3 (Perkin-Elmer).CYP19 expression constructs, COS-1 cell transfection, and micro-

somal preparations. The aromatase wild-type (WT) cDNA sequence was

cloned into the eukaryotic expression vector pCR3.1 (Invitrogen, Carlsbad,

CA). The pCR3.1 WT aromatase expression construct was then used as thetemplate for site-directed mutagenesis done using circular PCR to create

variant constructs. The primers and amplification conditions used during

those PCR reactions are listed in Supplementary Data. Sequences of the

constructs were confirmed by sequencing both strands of the insert. To helpexclude the possibility of PCR-induced alterations in the vector, in vitro

translation was done with each of the expression constructs using the TNT

rabbit reticulocyte lysate system (Promega, Madison, WI) and all allozymes

showed approximately equal quantities of translated protein.To make it possible to correct for transfection efficiency, we also

designed an expression construct that contained a green fluorescent protein

(GFP) and human NADPH-b5 reductase (DIA1) fusion protein that would betargeted to the endoplasmic reticulum because of the DIA1 portion of the

construct. The DIA1 cDNA was amplified using a human liver Marathon-

Ready cDNA library (BD Biosciences Clonetech, Palo Alto, CA) as template,

and was cloned into the GFP fusion TOPO TA expression vector(Invitrogen). The PCR conditions used to perform that amplification are

listed in Supplementary Data.

COS-1 cells were transfected with expression constructs for the CYP19

WT and variant allozymes as well as ‘‘empty’’ pCR3.1 that lacked an insert,using the TransFast reagent (Promega) at a charge ratio of 1:1. Specifically,

7 Ag of aromatase expression construct DNA was cotransfected with 7 Ag ofDIA1-GFP expression construct DNA. After 48 hours, the COS-1 cells wereharvested in 0.25 mol/L sucrose and were homogenized for 20 seconds with

a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). The

homogenates were centrifuged at 500 � g for 5 minutes and at 6,500� g for

10 minutes. The supernatant was then transferred to a new tube and was

centrifuged at 11,600 � g for 15 minutes. The supernatant from that stepwas centrifuged at 132,000 � g for 45 minutes and the pellet was

resuspended in 0.05 mol/L potassium phosphate buffer (pH 7.4) followed by

storage at �70jC.To correct for variation in transfection efficiency, green fluorescence was

measured in the microsomal fraction with a SPECTRAmax GEMINI XS

dual-scanning microplate spectrofluorometer (Molecular Devices Corpora-

tion, Sunnyvale, CA) using excitation and emission wavelengths of 395 and

507 nm, respectively. Levels of immunoreactive protein and enzyme activityfor these transfections were then corrected on the basis of the GFP values.

CYP19 Western blot analysis. A mouse anti-human aromatase

monoclonal antibody directed against human aromatase amino acids 376

to 390 was purchased from Serotec (Raleigh, NC). This antibody has beendescribed in detail elsewhere (26). Aliquots of COS-1 cell microsomal

fractions transfected with CYP19 allozyme cDNA expression constructs

were loaded onto 12.5% acrylamide SDS-PAGE gels on the basis of GFPvalues to correct for transfection efficiency. After electrophoresis, proteins

were transferred to polyvinylidene difluoride membranes and were detected

using the enhanced chemiluminescence Western blotting system (ECL,

Amersham Pharmacia, Piscataway, NJ). An AMBIS Radioanalytic ImagingSystem, Quant Probe Version 4.31 (Ambis, Inc., San Diego, CA), was used to

quantitate levels of immunoreactive protein relative to that for the WT

allozyme.

CYP19 enzyme assay, substrate kinetics, and inhibitor constants.

Aromatase activity was assayed by measuring the release of 3H2O from

[1h3H]androst-4-ene-3,17-dione (NEN Life Science Products, Boston, MA) as

described elsewhere (27, 28). These reactions were carried out for 20

minutes at 37jC in 0.05 mol/L Tris-HCl (pH 7.4) under air. Each reaction

mixture contained either 20 or 100 nmol/L [1h3H]androst-4-ene-3,17-dione

(25.3 Ci/mmol), 30 to 60 ng of microsomal protein, and an NADPH

regeneration system (1.5 mmol/L glucose 6-phosphate, 1 unit of glucose-6-

phosphate dehydrogenase, and 3.5 mmol/L NADPH) in a final volume of 100

AL. After incubation, 6 volumes of chloroform was added to the reaction

mixture and the mixture was vortexed for 30 seconds to terminate the

reaction and partition the remaining substrate into the organic phase. After

centrifugation at 14,000 � g for 10 minutes, radioactivity remaining in the

aqueous phase was determined by liquid scintillation counting.

Apparent Km values were determined using the same radiochemical

assay and under the same conditions as described above. Triplicate assayswere done for each variant allozyme in the presence of eight concentrations

of [1h3H]androst-4-ene-3,17-dione that varied from 0.3 to 40 nmol/L. For

the Thr364 allozyme, the concentration of [1h3H]androst-4-ene-3,17-dione

ranged from 1.25 to 160 nmol/L. K i values were also determined for eachallozyme in the presence of the aromatase inhibitors letrozole and

exemestane. In those experiments, triplicate assays were done using six

concentrations of [1h3H]androst-4-ene-3,17-dione that varied from 1.25 to320 nmol/L in the presence of three concentrations of letrozole (0.2, 0.4, and

0.8 nmol/L) or exemestane (1.25, 2.5, and 5 nmol/L). In the case of the

Thr364 variant allozyme, the letrozole concentrations were 0.1, 0.2, and 0.4

nmol/L but the exemestane concentrations were the same as those used tostudy the other allozymes.

Immunofluorescence microscopy. FITC-conjugated goat anti-mouse

immunoglobulin and tetramethylrhodamine isothiocyanate–conjugated

goat anti-rabbit immunoglobulin were purchased from Southern Biotech(Birmingham, AL). COS-1 cells were subcultured to 50% to 70% confluence

on coverslips, were transfected with expression constructs, and were then

cultured for an additional 48 hours. The cells were washed with PBS, fixed

with 3% paraformaldehyde for 12 minutes at room temperature, and,finally, were washed and incubated at room temperature for 5 minutes with

buffer containing 0.5% Triton X-100. The coverslips were then incubated

with the primary antibodies—rabbit polyclonal antihuman antibody againstcalnexin, an endoplasmic reticulum marker, and mouse monoclonal

antihuman aromatase antibody—followed by FITC-conjugated goat

anti-mouse or tetramethylrhodamine isothiocyanate–conjugated goat anti-

rabbit IgG antibody. The COS-1 cells were then viewed by fluorescencemicroscopy using a Nikon 80i fluorescence microscope with 488 or 570 nm

filters for excitation of the green or red fluorochrome, respectively.

Cancer Research

Cancer Res 2005; 65: (23). December 1, 2005 11072 www.aacrjournals.org



Data analysis. Values for p, u , and Tajima’s D were calculated andcorrected for length as described by Tajima (29). DVvalues for the linkage

disequilibrium analysis of polymorphism pairs were calculated as

described by Hartl and Clark (30) and Hedrick (31) and those data were

displayed graphically. Haplotype analysis was done as described bySchaid et al. (32). Apparent Km values were calculated with the method

of Wilkinson (33) using a computer program written by Cleland (34).

Points that deviated from linearity on double-inverse plots (i.e., those

showing substrate inhibition) were not used to perform thesecalculations. For the determination of K i values, Lineweaver-Burke

double-inverse plots were done at each concentration of inhibitor. Slopes

were calculated for the double-inverse plots and secondary plots of slope

against inhibitor concentration were determined. Intercepts on theinhibitor concentration axis were used to determine K i values. Pearson

product moment correlation coefficients were calculated using Excel and

group means were compared by the use of ANOVA with the Prismprogram.

Results

Human CYP19 gene resequencing. The CYP19 gene wasresequenced using 240 DNA samples, 60 each from African-

American, Caucasian-American, Han Chinese-American, and Mexican-American subjects. Approximately 5,424,000 bp of DNA sequencewas analyzed in the course of these experiments. Eighty-eightpolymorphisms were observed, including 85 SNPs, 2 insertion-deletion events, and 1 polymorphic TTTA repeat (Fig. 1; Table 1).There were large ethnic variations in both allele frequencies andtypes, with 69 polymorphisms in African-American DNA, 37 in DNAsamples from Caucasian-American subjects, 30 in Han Chinese-American subjects, and 44 in DNA from Mexican-Americansubjects. Thirty-two polymorphisms were observed only inAfrican-American subjects, six in Han Chinese-American subjects,six in Mexican-American subjects, and five in Caucasian-Americansubjects. Of the polymorphisms identified in the course of thesestudies, 62 had not been reported previously, 31 of which were‘‘common,’’ with allele frequencies of >1% in at least one ethnicgroup. All polymorphisms were in Hardy-Weinberg equilibriumexcept for one polymorphism in Caucasian-American subjects thatwas located �41 bp upstream of exon 1.1. We also determined‘‘nucleotide diversity,’’ a quantitative measure of genetic variation,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, polymorphismswith frequencies of <1%. AA, African-American subjects; CA, Caucasian-American subjects; HCA, Han Chinese-American subjects; MA, Mexican-Americansubjects. 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 acidchanges resulting from nonsynonymous cSNPs, are also indicated.

Human Aromatase Gene Resequencing




Table 1. Human CYP19 genetic polymorphisms

db-SNP

identification

Location Nucleotide Nucleotide

change

Amino acid

change

Frequency of variant allele

Caucasian-

American

African-

American

Han Chinese-

American

Mexican-

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 0.0085V-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 0.000rs6493497 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 �2 G!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 0.000rs4774585 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 0.0005V-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 0.875rs1902582 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 0.0005V-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 0.0005V-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 0.0255V-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

(Continued on the following page)

Cancer Research




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

db-SNP

identification

Location Nucleotide Nucleotide

change

Amino acid

change

Frequency of variant allele

Caucasian-

American

African-

American

Han Chinese-

American

Mexican-

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 0.033Intron 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 0.000Exon 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 0.283Intron 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 0.417Published (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 0.017n = 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 0.008rs2304463 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!C Met364Thr 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

(Continued on the following page)





of nucleotide diversity are p , average heterozygosity per site,and u, a population mutation measure that is theoretically equalto the neutral mutation variable (35). These values are listed inTable 1. In addition, values for Tajima’s D , a test of the ‘‘neutral’’mutation hypothesis (29), were estimated for each population(Table 1). Only the value for Tajima’s D in Han Chinese-Americansubjects differed significantly from values for the other ethnicgroups.Four nonsynonymous cSNPs, polymorphisms that altered the

encoded amino acids, were observed: Trp39Arg, Thr201Met,Arg264Cys, and Met364Thr (Fig. 1; Table 1). The SNPs resulting inTrp39Arg and Arg264Cys alterations in amino acid sequence hadbeen described previously. Two of these polymorphisms, Trp39Argand Met364Thr, were observed only in the Han Chinese-Americansamples, with allele frequencies of 6.7% and 0.8%, respectively.The Arg264Cys polymorphism was common, with a frequency >2.5%in all four populations. Thr201Met polymorphism was observedin three of the four ethnic groups, with an allele frequency of 5% inboth African-American and Caucasian-American and 0.8% inMexican-American subjects. Homozygous samples were observedonly for Arg264Cys, in both African-American and Han Chinese-American subjects.Haplotype and linkage disequilibrium analysis. There is

increasing appreciation for the importance of linkage disequilib-rium and haplotype data for application to association studies(36). Therefore, we did population-specific linkage disequilibriumand haplotype analysis for the CYP19 polymorphisms. Figure 2shows a graphical representation of pairwise DV values, a measureof linkage disequilibrium, in all four ethnic groups that westudied. DV values are 1.0 when polymorphisms are maximallyassociated and zero when they are randomly associated(30, 31). Haplotypes can be determined unequivocally onlyif not more than one polymorphism in an allele is heterozygousbut it is possible to ‘‘infer’’ haplotypes computationally (32).Ethnic-group-specific haplotype analysis for CYP19 showed 12unequivocal haplotypes and 32 inferred haplotypes—with strikingvariations among the four ethnic groups in haplotype frequencies(Table 2).Recombinant allozyme enzyme activity. The possible func-

tional significance of the four CYP19 nonsynonymous cSNPsobserved during the gene resequencing experiments was studied byexpressing each variant allozyme in COS-1 cells. Enzyme activity

and immunoreactive protein levels were then determined usingmicrosomes isolated from these cells. Because one of the DNAsamples that we had resequenced contained two nonsynonymouscSNPs, resulting in both Trp39Arg and Arg264Cys alterations inencoded amino acids, an expression construct was created that wedesignated as a double-mutant construct. This construct containedboth cSNPs although it was not possible to determine unequivo-cally that a single allele that included both polymorphisms waspresent in this subject. Finally, to make it possible to correct fortransfection efficiency, an expression vector for a GFP and DIA1fusion protein that would be targeted to the endoplasmic reticulumwas also created and cotransfected with the aromatase allozymeconstructs.Six independent transfections were done for each allozyme. As

shown graphically in Fig. 3A , the Cys264, Thr364, and double-mutantallozymes had 72%, 15%, and 21% of the WT enzyme activity,respectively, all of which differed significantly from the WT value.Values for neither the Arg39 nor Met201 allozymes differedsignificantly from that for WT. Very similar results were obtainedwhen a 5-fold higher substrate concentration, 100 nmol/L andros-tenedione rather than 20 nmol/L, was used to perform the assays(data not shown).Substrate and inhibitor kinetic studies. One possible

explanation for the decreased levels of enzyme activity observedwith several of the variant allozymes would involve an alteration insubstrate kinetics. Therefore, apparent Km values were determinedfor the WT and variant allozymes with androstenedione as thesubstrate. An elevated Km value when compared with that for theWT allozyme was observed only with the Thr364 variant (Table 3).However, although the increase in apparent Km for this allozymemight have contributed to the observed decrease in activityobserved, the major mechanism involved a decrease in proteinlevel as described subsequently and as shown by the lack of asignificant increase in activity when the substrate concentrationwas increased 5-fold from 20 to 100 nmol/L.We also determined whether alterations in the amino acid

sequences of the variant allozymes might influence response to twoaromatase inhibitors, letrozole and exemestane. We selected thesetwo drugs as representatives of nonsteroidal and steroidalaromatase inhibitors, respectively. IC50 values for the WT allozymewere found to be 0.6 and 4.5 nmol/L for these two inhibitors,respectively. K i values for letrozole and exemestane were then

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

Caucasian-

American

African-

American

Han Chinese-

American

Mexican-

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 fornucleotides 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 , aswell as Tajima’s D , a test of the ‘‘neutral’’ mutation hypothesis.

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determined with the recombinant variant allozymes (Table 3). K i

values were similar for all of the allozymes studied, with only thevalue for letrozole for the double-mutant allozyme beingsignificantly different from that for the WT enzyme. An exampleof the data used to calculate the K i value for letrozole with WTaromatase is shown in Fig. 3D .Western blot analysis. We have previously reported that a

common mechanism for the functional effects of nonsynonymouscSNPs is an alteration in protein quantity (37). Therefore,quantitative Western blot analysis was done using monoclonalantibody against a polypeptide corresponding to CYP19 aminoacids 376 to 390, an area that did not include any of the aminoacids altered by the four nonsynonymous cSNPs. As shown inFig. 3B , levels of recombinant protein corresponded to levels ofenzyme activity for the variant allozymes. When level of enzymeactivity was plotted against level of immunoreactive protein for theWT enzyme and all five of the variant allozymes, including thedouble-mutant construct, a significant correlation was observed(Rp = 0.937, P = 0.006; Fig. 3C). This observation suggests that amajor mechanism by which these genetic polymorphisms influencearomatase activity, at least after the transient transfection ofmammalian cells, is through a reduction in the quantity of enzymeprotein. To exclude the possibility that a defect in the expressionvector introduced during site-directed mutagenesis might havecaused the decreased levels of immunoreactive protein, in vitro

translation studies were done with all expression constructs using arabbit reticulocyte lysate system. Similar quantities of recombinantprotein were produced for all of the allozymes studied (data notshown).Subcellular localization. Aromatase, like other eukaryotic

cytochrome P450 enzymes, is localized to the endoplasmicreticulum (38). Therefore, another mechanism that might explaindecreased levels of the variant allozymes in microsomes wouldinvolve changes in subcellular localization. Amino acids 20 to 39 inCYP19 are hydrophobic and represent a putative transmembranedomain that is located in the endoplasmic reticulum (1, 39).Because of the possibility that the change from Trp to the morehydrophilic Arg at amino acid 39 might alter the subcellularlocalization of the Trp39Arg allozyme, we also studied subcellularlocalization using fluorescence microscopy. Two other allozymes—those with the lowest levels of microsomal activity and protein,Thr364 and the double-mutant allozyme—were also studied. Withcalnexin as an endoplasmic reticulum marker, immunofluorescentstudies were done using COS-1 cells transiently transfected withconstructs encoding the WT or the three variant allozymes. All ofthe allozymes colocalized with calnexin (Fig. 4), indicating thatthey were localized to the endoplasmic reticulum. Therefore, thedecreased levels of immunoreactive protein that we observed forthese allozymes could not be explained by alterations in theirsubcellular 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.





Discussion

The cytochrome P450 enzyme aromatase, CYP19, is required forestrogen biosynthesis in both premenopausal and postmenopausalwomen (1). Molecular epidemiology studies done with a relatively smallnumber of commonCYP19 polymorphisms have produced inconsistentresults with regard to the possible role of genetic variation in CYP19 ininfluencing levels of sex hormones and/or risk for estrogen-dependentdisease. In the present study, we set out to systematically identifygenetic polymorphisms in this gene, especially within exons and splice

junctions. To do that, we resequenced all exons and their adjacent splice

junctions, as well as at least 500 bp flanking the 5V-terminus of each of

the untranslated upstream exons and portions of the gene encoding the

3V-UTR using 240 DNA samples from four different ethnic groups. Thisin-depth resequencing strategy allowed us to discover previously

unreported polymorphisms of possible functional significance and

also to infer haplotype patterns in different ethnic groups. Two of the

four nonsynonymous cSNPs identified, those that resulted in Thr201Met

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 commonsequence in African-American subjects) are highlighted as white on black. Initial haplotype designations (*1, *2, *3, *4, and *5) are made on the basis of

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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We also did functional genomic studies, including determin-ations of activity and immunoreactive protein levels, substrate andinhibitor kinetics, and subcellular localization for all four of thenonsynonymous cSNPs. Levels of aromatase enzyme activity weredramatically decreased for the Thr364 and the double mutant thatincluded both the Arg39 and Cys264 polymorphisms (Fig. 3A). Therewas also a slight decrease in activity for the Cys264 allozyme. Itshould be emphasized once again that it is unclear whether the twopolymorphisms present in the double-mutant construct occurnaturally within a single allele; however, because of this uncertainty,we created the construct and studied the double-mutant allozyme.These relative levels of enzyme activity correlated well with levels ofimmunoreactive protein (Fig. 3C), suggesting that one important

mechanism by which nonsynonymous cSNPs affect the activityof this enzyme is to alter the level of enzyme protein. The 4-foldelevation in apparent Km for the Thr364 allozyme represents anadditional possible explanation for the low level of activity observedwith this variant. Finally, with the exception of the double-mutantallozyme, there were no significant differences among the variantallozymes in their response to the aromatase inhibitors letrozoleand exemestane (Table 3). In addition to polymorphisms within thecoding region, multiple polymorphisms were also identified outsideof exons (Fig. 1; Table 1), all of which could potentially contribute tovariation in gene expression. Obviously, any of these polymorphismscould also be linked to functionally important variation in DNAsequence located within areas of the gene that we did not sequence.

Figure 3. CYP19 recombinant allozyme enzyme activity, immunoreactive protein levels, and inhibitor kinetics. A, average levels of enzyme activity for each of therecombinant allozymes assayed with 20 nmol/L androstenedione as substrate. DM, a double mutant that included the Arg39 and Cys264 polymorphisms. Allvalues have been corrected for transfection efficiency. Bars, mean of six independent transfections; bars, SE. *, P < 0.05; **, P < 0.001, compared with the WTallozyme. The Arg39, Met201, and Cys264 variants also differed significantly from the Thr362 and double-mutant allozymes (P < 0.05). B, average levels ofimmunoreactive 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 WTallozyme. In addition, the Met201 variant differed significantly (P < 0.05) from the Cys264, Thr364, and double-mutant allozymes, whereas the Arg39 allozyme differedsignificantly (P < 0.05) only from the Thr362 and the double-mutant variants. C, correlation of levels of CYP19 enzyme activity and immunoreactive protein forrecombinant 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 inhibitorkinetics done with WT CYP19. The double-inverse plots show the effect of various concentrations of letrozole on CYP19 enzyme activity. These data were usedto calculate the Ki value listed in Table 3.





Our functional genomic studies included two novel allozymes, thosecontaining Met201 and Thr364 alterations in amino acid sequence, aswell as two that had been reported previously, Arg39 and Cys264. TheCys264 variantwas similar to theWTallozyme with regard to substrateand inhibitor kinetics, as reported in one previous study (9). However,we observed a slight decrease in the enzyme activity of this variant(75% of WT), whereas the previous study, which also used microsomepreparations from transiently transfected COS-1 cells, did not (9).One possible explanation for this slight difference might be our use ofGFP-DIA1, a fusion protein targeted to the endoplasmic reticulum,to correct for transfection efficiency. The previous study usedthe cytosolic marker enzyme, h-galactosidase, for this purpose (9).A more striking difference occurred with the Arg39 variant, which

had previously been reported to be inactive after transientexpression in human embryonic kidney cells (19). We observedonly slight decreases in both activity and quantity of allozymeprotein (Fig. 3). We have no explanation for this striking difference.Obviously, no previous studies had been done with the Met201 andthe Thr364 variant allozymes because those variant alleles werediscovered during the present experiments. The low levels of both

Table 3. Human aromatase allozyme substrate andinhibitor kinetic data

Aromataseallozyme

Substratekinetics, apparent

Km, (nmol/L)

Inhibitor kinetics,K i (nmol/L)

Letrozole Exemestane

WT 6.7 F 2.0 0.21 F 0.05 1.05 F 0.41

Arg39 6.0 F 2.0 0.18 F 0.06 0.94 F 0.59Met201 7.1 F 1.9 0.22 F 0.06 1.16 F 0.09

Cys264 5.9 F 2.6 0.21 F 0.08 1.04 F 0.33

Thr364 26.0 F 10* 0.29 F 0.09 2.86 F 1.29Double mutant 7.3 F1.9 0.46 F 0.12

c1.09 F 0.10

NOTE: The substrate used in these experiments was androstenedione.

Each value is the mean F SE for three determinations.

*P < 0.001, compared with values for all other allozymes.cP < 0.05, compared with WT, Arg39, Cys264, or Met201.

Figure 4. Subcellular localization of recombinantCYP19 allozymes. Red, location of theendoplasmic reticular markercalnexin; green, location of CYP19; Merge,colocalization of calnexin and CYP19.

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enzyme activity and immunoreactive protein levels for the Thr364

variant indicate that it would also be of interest to identify subjectswho carry this variant and to assess their risk for estrogen-dependent disease.One of the more striking observations made in the course of our

studies was the significant correlation between levels of activity andprotein for CYP19 variant allozymes—a phenomenon that confirmsthat a common, although certainly not the only, mechanism bywhich nonsynonymous cSNPs influence function is by alteringlevels of protein—most often as a result of accelerated degradation,at times with protein aggregation and aggresome formation (37, 40–42). Obviously, it would be ideal to know the structure of CYP19 topursue our functional observations. Unfortunately, no mammalianaromatase structure is available. Homology models have beenpublished that are based on soluble bacterial cytochrome P450s,which have only 13% to 18% amino acid identity to human CYPs(43–46). Even with the recent publication of the human CYP2C8 andCYP2C9 crystal structures (47–49), homology modeling mightremain problematic because those two enzymes are only 25% to27% identical to aromatase in amino acid sequence.In summary, we have resequenced the human CYP19 gene using

DNA samples from four ethnic groups. In the course of these studies,we observed 88 polymorphisms and 44 common CYP19 haplotypes.Functional characterization of the four variant allozymes encoded by

alleles with nonsynonymous cSNPs showed a significant correlationbetween level of activity and immunoreactive protein (Fig. 3C), anobservation compatible with a growing body of data that indicatethat alteration in protein quantity is a common mechanismresponsible for the functional effects of this type of polymorphism,most often as a result of accelerated protein degradation (37) but also,at times, involving intracellular protein aggregation (42). Obviously,our results must be confirmed in the future by in vivo genotype-phenotype correlation studies. Finally, the CYP19 genomic and func-tional genomic data included in the present study are of particularimportance in light of the rapidly expanding use of aromataseinhibitors during the adjuvant therapy of breast cancer (50).

Acknowledgments

Received 4/11/2005; revised 6/23/2005; accepted 7/25/2005.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-cogenetics Research Network (O.E. Salavaggione, L. Pelleymounter, L. Wang, B.W.Eckloff, D. Schaid, E.D. Wieben, and R.M. Weinshilboum); CA82267 (Araba A. Adjei andR.M. Weinshilboum); and Pfizer (Alex A. Adjei and Araba A. Adjei).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Luanne Wussow for her assistance with the preparation of themanuscript and Alexander Vandell for his assistance with the creation of thearomatase allozyme expression constructs.

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Human Aromatase: Gene Resequencing and Functional Genomics · significant change from the WT enzyme in inhibitor constant for the aromatase inhibitors exemestane and letrozole. These