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[Cancer Research 65, 11071-11082, December 1, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Human Aromatase: Gene Resequencing and Functional Genomics

Cynthia X. Ma1, Araba A. Adjei2, Oreste E. Salavaggione2, Josefa Coronel2, Linda Pelleymounter2, Liewei Wang2, Bruce W. Eckloff3, Daniel Schaid4, Eric D. Wieben3, Alex A. Adjei1 and Richard M. Weinshilboum2

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

Requests for reprints: Richard M. Weinshilboum, Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, 200 First Street, Southwest, Rochester, MN 55905. Phone: 507-284-2246; Fax: 507-284-9111; E-mail: weinshilboum.richard{at}mayo.edu.


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


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Aromatase [cytochrome P450 19 (CYP19)], is encoded by the CYP19 gene. Aromatase catalyzes a critical reaction for estrogen biosynthesis—the formation of aromatic C18 estrogens from C19 androgens (1). Alterations in aromatase expression have been implicated 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 selective aromatase inhibitors are being used increasingly to treat postmenopausal 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 coding exons, exons 2 to 10, with a 93 kb region that contains 10 tissue-specific noncoding upstream exons with separate promoters that regulate transcription in different cells and tissues. Although CYP19 genetic polymorphisms have been reported, the possible functional significance of most of those polymorphisms remains undefined. We set out to systematically identify and characterize genetic variation in CYP19 by performing complementary gene resequencing and functional genomic studies. Specifically, we resequenced all exons, including at least 500 bp of each of the 5'-untranslated exons, all exon-intron splice junctions, and a portion of the 3'-untranslated region (3'-UTR). The samples included DNA from 60 African-American, 60 Caucasian-American, 60 Han Chinese-American, and 60 Mexican-American subjects. Functional studies were then done with allozymes encoded by the four nonsynonymous coding single nucleotide polymorphisms (cSNP) identified during the resequencing studies.

Previous population-based studies of common CYP19 polymorphisms have generated inconsistent results with regard to their possible association with either sex hormone levels or risk for estrogen-dependent diseases (9–24). Selected CYP19 polymorphisms have also been investigated for their possible association with the therapeutic efficacy of aromatase inhibitors (25). The fact that past data with regard to the association of CYP19 polymorphisms with estrogen-dependent disease has been inconsistent (9–24), as well as the lack of functional studies of these polymorphisms, underscores the importance of applying a systematic approach to identify and functionally characterize CYP19 polymorphisms. In the present study, we have used gene resequencing to identify previously unreported genetic variation in this important gene. We then functionally characterized the four nonsynonymous cSNPs observed during the resequencing studies and found variations in aromatase enzyme activity and quantity of enzyme protein that were associated with those polymorphisms. We also studied the substrate kinetics, subcellular localization, and response of these variant allozymes to aromatase inhibitors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 HD100MEX) 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 had provided 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.

CYP19 gene resequencing. Each of the 240 DNA samples studied was used to perform PCR amplifications of the areas to be resequenced. M13 "tags" were added to the 5' ends 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 to amplify 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 "long PCR 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 µL reaction mixtures contained 0.75 units of DNA polymerase, 0.5 µL 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 µL of 10x PCR buffer containing 15 mmol/L MgCl2 (Perkin-Elmer). Amplification conditions included a 10-minute "hot start" at 96°C, followed by 35 cycles of 96°C for 30 seconds, 30 seconds at annealing temperatures listed in Supplementary Data, and 45 seconds at 72°C with a final 10-minute extension at 72°C. 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, independent amplifications were done for those samples in which a SNP was observed only once or any sample with an ambiguous chromatogram. Chromatograms 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. The primers 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, [(3',6'-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 microsomal 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 the template 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 help exclude 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 be targeted 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 µg of aromatase expression construct DNA was cotransfected with 7 µg of DIA1-GFP expression construct DNA. After 48 hours, the COS-1 cells were harvested 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 x g for 5 minutes and at 6,500 x g for 10 minutes. The supernatant was then transferred to a new tube and was centrifuged at 11,600 x g for 15 minutes. The supernatant from that step was centrifuged at 132,000 x g for 45 minutes and the pellet was resuspended in 0.05 mol/L potassium phosphate buffer (pH 7.4) followed by storage at –70°C.

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 Corporation, Sunnyvale, CA) using excitation and emission wavelengths of 395 and 507 nm, respectively. Levels of immunoreactive protein and enzyme activity for 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 been described 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 GFP values 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 Imaging System, 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 [1ß3H]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 37°C in 0.05 mol/L Tris-HCl (pH 7.4) under air. Each reaction mixture contained either 20 or 100 nmol/L [1ß3H]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 µL. 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 x 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 assays were done for each variant allozyme in the presence of eight concentrations of [1ß3H]androst-4-ene-3,17-dione that varied from 0.3 to 40 nmol/L. For the Thr364 allozyme, the concentration of [1ß3H]androst-4-ene-3,17-dione ranged from 1.25 to 160 nmol/L. Ki values were also determined for each allozyme in the presence of the aromatase inhibitors letrozole and exemestane. In those experiments, triplicate assays were done using six concentrations of [1ß3H]androst-4-ene-3,17-dione that varied from 1.25 to 320 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 to study 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 against calnexin, 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 fluorescence microscopy using a Nikon 80i fluorescence microscope with 488 or 570 nm filters for excitation of the green or red fluorochrome, respectively.

Data analysis. Values for {pi}, {theta}, and Tajima's D were calculated and corrected for length as described by Tajima (29). D' values 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 by Schaid 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 these calculations. For the determination of Ki 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 the inhibitor concentration axis were used to determine Ki values. Pearson product moment correlation coefficients were calculated using Excel and group means were compared by the use of ANOVA with the Prism program.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human CYP19 gene resequencing. The CYP19 gene was resequenced 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 sequence was analyzed in the course of these experiments. Eighty-eight polymorphisms 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 and types, with 69 polymorphisms in African-American DNA, 37 in DNA samples from Caucasian-American subjects, 30 in Han Chinese-American subjects, and 44 in DNA from Mexican-American subjects. Thirty-two polymorphisms were observed only in African-American subjects, six in Han Chinese-American subjects, six in Mexican-American subjects, and five in Caucasian-American subjects. Of the polymorphisms identified in the course of these studies, 62 had not been reported previously, 31 of which were "common," with allele frequencies of >1% in at least one ethnic group. All polymorphisms were in Hardy-Weinberg equilibrium except for one polymorphism in Caucasian-American subjects that was 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 of nucleotide diversity are {pi}, average heterozygosity per site, and {theta}, a population mutation measure that is theoretically equal to the neutral mutation variable (35). These values are listed in Table 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-American subjects differed significantly from values for the other ethnic groups.



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  		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.

 

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  		Table 1. Human CYP19 genetic polymorphisms

 
Four nonsynonymous cSNPs, polymorphisms that altered the encoded amino acids, were observed: Trp39Arg, Thr201Met, Arg264Cys, and Met364Thr (Fig. 1; Table 1). The SNPs resulting in Trp39Arg and Arg264Cys alterations in amino acid sequence had been described previously. Two of these polymorphisms, Trp39Arg and Met364Thr, were observed only in the Han Chinese-American samples, 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 observed in three of the four ethnic groups, with an allele frequency of 5% in both African-American and Caucasian-American and 0.8% in Mexican-American subjects. Homozygous samples were observed only 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 disequilibrium and haplotype data for application to association studies (36). Therefore, we did population-specific linkage disequilibrium and haplotype analysis for the CYP19 polymorphisms. Figure 2 shows a graphical representation of pairwise D' values, a measure of linkage disequilibrium, in all four ethnic groups that we studied. D' values are 1.0 when polymorphisms are maximally associated and zero when they are randomly associated (30, 31). Haplotypes can be determined unequivocally only if not more than one polymorphism in an allele is heterozygous but it is possible to "infer" haplotypes computationally (32). Ethnic-group-specific haplotype analysis for CYP19 showed 12 unequivocal haplotypes and 32 inferred haplotypes—with striking variations among the four ethnic groups in haplotype frequencies (Table 2).



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  		Figure 2. Human CYP19 linkage disequilibrium (LD). The extent of population-based linkage disequilibrium within the area of CYP19 resequenced, shown as pairwise |D'| values, is depicted graphically.

 

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

 
Recombinant allozyme enzyme activity. The possible functional significance of the four CYP19 nonsynonymous cSNPs observed during the gene resequencing experiments was studied by expressing each variant allozyme in COS-1 cells. Enzyme activity and immunoreactive protein levels were then determined using microsomes isolated from these cells. Because one of the DNA samples that we had resequenced contained two nonsynonymous cSNPs, resulting in both Trp39Arg and Arg264Cys alterations in encoded amino acids, an expression construct was created that we designated as a double-mutant construct. This construct contained both cSNPs although it was not possible to determine unequivocally that a single allele that included both polymorphisms was present in this subject. Finally, to make it possible to correct for transfection efficiency, an expression vector for a GFP and DIA1 fusion protein that would be targeted to the endoplasmic reticulum was also created and cotransfected with the aromatase allozyme constructs.

Six independent transfections were done for each allozyme. As shown graphically in Fig. 3A, the Cys264, Thr364, and double-mutant allozymes 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 differed significantly from that for WT. Very similar results were obtained when a 5-fold higher substrate concentration, 100 nmol/L androstenedione rather than 20 nmol/L, was used to perform the assays (data not shown).



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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.

 

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  		Table 3. Human aromatase allozyme substrate and inhibitor kinetic data

 
Substrate and inhibitor kinetic studies. One possible explanation for the decreased levels of enzyme activity observed with several of the variant allozymes would involve an alteration in substrate kinetics. Therefore, apparent Km values were determined for the WT and variant allozymes with androstenedione as the substrate. An elevated Km value when compared with that for the WT allozyme was observed only with the Thr364 variant (Table 3). However, although the increase in apparent Km for this allozyme might have contributed to the observed decrease in activity observed, the major mechanism involved a decrease in protein level as described subsequently and as shown by the lack of a significant increase in activity when the substrate concentration was 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 two aromatase inhibitors, letrozole and exemestane. We selected these two drugs as representatives of nonsteroidal and steroidal aromatase inhibitors, respectively. IC50 values for the WT allozyme were found to be 0.6 and 4.5 nmol/L for these two inhibitors, respectively. Ki values for letrozole and exemestane were then determined with the recombinant variant allozymes (Table 3). Ki values were similar for all of the allozymes studied, with only the value for letrozole for the double-mutant allozyme being significantly different from that for the WT enzyme. An example of the data used to calculate the Ki value for letrozole with WT aromatase is shown in Fig. 3D.

Western blot analysis. We have previously reported that a common mechanism for the functional effects of nonsynonymous cSNPs is an alteration in protein quantity (37). Therefore, quantitative Western blot analysis was done using monoclonal antibody against a polypeptide corresponding to CYP19 amino acids 376 to 390, an area that did not include any of the amino acids altered by the four nonsynonymous cSNPs. As shown in Fig. 3B, levels of recombinant protein corresponded to levels of enzyme activity for the variant allozymes. When level of enzyme activity was plotted against level of immunoreactive protein for the WT enzyme and all five of the variant allozymes, including the double-mutant construct, a significant correlation was observed (Rp = 0.937, P = 0.006; Fig. 3C). This observation suggests that a major mechanism by which these genetic polymorphisms influence aromatase activity, at least after the transient transfection of mammalian cells, is through a reduction in the quantity of enzyme protein. To exclude the possibility that a defect in the expression vector introduced during site-directed mutagenesis might have caused the decreased levels of immunoreactive protein, in vitro translation studies were done with all expression constructs using a rabbit reticulocyte lysate system. Similar quantities of recombinant protein were produced for all of the allozymes studied (data not shown).

Subcellular localization. Aromatase, like other eukaryotic cytochrome P450 enzymes, is localized to the endoplasmic reticulum (38). Therefore, another mechanism that might explain decreased levels of the variant allozymes in microsomes would involve changes in subcellular localization. Amino acids 20 to 39 in CYP19 are hydrophobic and represent a putative transmembrane domain that is located in the endoplasmic reticulum (1, 39). Because of the possibility that the change from Trp to the more hydrophilic Arg at amino acid 39 might alter the subcellular localization of the Trp39Arg allozyme, we also studied subcellular localization 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. With calnexin as an endoplasmic reticulum marker, immunofluorescent studies were done using COS-1 cells transiently transfected with constructs encoding the WT or the three variant allozymes. All of the allozymes colocalized with calnexin (Fig. 4), indicating that they were localized to the endoplasmic reticulum. Therefore, the decreased levels of immunoreactive protein that we observed for these allozymes could not be explained by alterations in their subcellular localization.



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  		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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The cytochrome P450 enzyme aromatase, CYP19, is required for estrogen biosynthesis in both premenopausal and postmenopausal women (1). Molecular epidemiology studies done with a relatively small number of common CYP19 polymorphisms have produced inconsistent results with regard to the possible role of genetic variation in CYP19 in influencing levels of sex hormones and/or risk for estrogen-dependent disease. In the present study, we set out to systematically identify genetic 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 5'-terminus of each of the untranslated upstream exons and portions of the gene encoding the 3'-UTR using 240 DNA samples from four different ethnic groups. This in-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.

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

Our functional genomic studies included two novel allozymes, those containing Met201 and Thr364 alterations in amino acid sequence, as well as two that had been reported previously, Arg39 and Cys264. The Cys264 variant was similar to the WT allozyme with regard to substrate and 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 microsome preparations from transiently transfected COS-1 cells, did not (9). One possible explanation for this slight difference might be our use of GFP-DIA1, a fusion protein targeted to the endoplasmic reticulum, to correct for transfection efficiency. The previous study used the cytosolic marker enzyme, ß-galactosidase, for this purpose (9).

A more striking difference occurred with the Arg39 variant, which had previously been reported to be inactive after transient expression in human embryonic kidney cells (19). We observed only slight decreases in both activity and quantity of allozyme protein (Fig. 3). We have no explanation for this striking difference. Obviously, no previous studies had been done with the Met201 and the Thr364 variant allozymes because those variant alleles were discovered during the present experiments. The low levels of both enzyme activity and immunoreactive protein levels for the Thr364 variant indicate that it would also be of interest to identify subjects who 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 and protein for CYP19 variant allozymes—a phenomenon that confirms that a common, although certainly not the only, mechanism by which nonsynonymous cSNPs influence function is by altering levels 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 to pursue our functional observations. Unfortunately, no mammalian aromatase structure is available. Homology models have been published 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 and CYP2C9 crystal structures (47–49), homology modeling might remain problematic because those two enzymes are only 25% to 27% 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 correlation between level of activity and immunoreactive protein (Fig. 3C), an observation compatible with a growing body of data that indicate that alteration in protein quantity is a common mechanism responsible 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 functional genomic data included in the present study are of particular importance in light of the rapidly expanding use of aromatase inhibitors during the adjuvant therapy of breast cancer (50).


   Acknowledgments
 
Grant support: NIH grants R01 GM28157 (L. Wang and R.M. Weinshilboum), R01 GM35720 (O.E. Salavaggione and R.M. Weinshilboum), and U01 GM61388; Pharmacogenetics 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 and R.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 page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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


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

Received 4/11/05. Revised 6/23/05. Accepted 7/25/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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