JMD GMP oligos for in vitro Diagnostics
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Journal of Molecular Diagnostics 2008, Vol. 10, No. 3
Copyright © 2008 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2008.070133

A Simple and Rapid Genotyping Assay for Simultaneous Detection of Two ADRB2 Allelic Variants Using Fluorescence Resonance Energy Transfer Probes and Melting Curve Analysis

M. Fernanda Sábato*, Anne-Marie Irani{dagger}, Bonny L. Bukaveckas*{ddagger}§, Lawrence B. Schwartz, David S. Wilkinson* and Andrea Ferreira-Gonzalez*

From the Department of Pathology, * Division of Molecular Diagnostics, the Department of Pediatrics, {dagger} Division of Allergy, Immunology and Rheumatology, the Department of Pharmacy, {ddagger} and Internal Medicine, § Division of Rheumatology, Allergy and Immunology, Virginia Commonwealth University Medical Center, Richmond, Virginia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Allelic variants at codons 16 and 27 of the β2-adrenergic receptor gene (ADRB2) have shown clinical and pharmacological implications in asthma, hypertension, ischemic heart failure, diabetes, obesity, and cystic fibrosis. We have developed a simultaneous genotyping assay for the c.46A>G and c.79C>G allelic variants using hybridization probes and melting curve analysis. The assay was optimized on a panel of 30 DNA samples of known ADRB2 genotype as determined by sequencing with 100% concordance between the two techniques. Melting temperature (Tm) ranges for the different genotypes were obtained using data from three independent experiments. Single peaks for p.Arg16Arg (Tm = 57.76°C ± 0.10°C) and p.Gly16Gly (Tm = 66.73°C ± 0.18°C) and two melting peaks for p.Arg16Gly were obtained. Similarly, single peaks for p.Gln27Gln (Tm = 53.98°C ± 0.19°C) and p.Glu27Glu (Tm = 64.93°C ± 0.16°C) and two peaks for p.Gln27Glu were detected. Independent operators easily assigned genotypes in a sample set of 385 asthmatic patients. Haplotype and allele frequencies were in concordance with previously published data: Arg allele frequencies in children/adults were 0.34/0.30 in Caucasians and 0.45/0.52 in African Americans, and Gln allele frequencies were 0.58/0.52 in Caucasians and 0.82/0.84 in African Americans. Thus, the ADRB2 genotyping assay represents a highly reliable and rapid technique for routine clinical use in the simultaneous detection of ADRB2 variants.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The human β2-adrenergic receptor is a G-protein-coupled receptor found in a wide variety of tissue types and is a target for many β2-adrenoreceptor agonists and antagonists currently used in the treatment of many disorders. Individual variations in physiological responses, expression and function of the receptor, as well as individual differences in response to drugs that act on these receptors may, at least in part, relate to polymorphic variants of the receptor.

The β2-adrenergic receptor is encoded by an intronless gene (ADRB2) located on chromosome 5 (5q31-q32). At least nine different allelic variants have been identified,1 of which two are more frequent and give rise to amino acid exchanges in the putative extracellular amino-terminus region of the gene. Both variants ADRB2 NM000024.4: c46A>G (p.Arg16Gly) and NM000024.4: c.79C>G (p.Gln27Glu), display differences in allele frequencies among different biogeographical ancestry groups (http://www.pharmgkb.org/search/annotatedGene/adrb2/variant.jsp; accessed December 2007). The effects of these allelic variants on the function of the β2-adrenergic receptor have been studied in vitro and in vivo.2 They have been shown to alter the receptor function in the cardiovascular and respiratory systems. In blood vessels, where β2-adrenergic receptors mediate vasodilatation, the c.46A>G is associated with enhanced agonist mediated desensitization in the vasculature, whereas the c.79C>G is associated with increased agonist mediated responsiveness.3 Most clinical association studies examining effects of the c.46A>G and the c.79C>G sequence variants have been in relation to asthma and are aimed at identifying their role as either risk factors, modifiers of disease or as determinants of the response to β-adrenergic agonists.4, 5, 6, 7, 8, 9, 10, 11, 12 Genotype-phenotype correlations have indicated that the presence of homozygous p.Arg16Arg genotype (about 15% of patients with asthma in the United States6 and in the United Kingdom13 ) confers relative protection in vitro against down-regulation of β-2-adrenoreceptor by β-agonists14 and reverses the benefits from the regular use of short- and long-acting β-agonists in children, young adults, and adult asthmatic patients.4, 5, 6, 8, 10, 11, 13, 15, 16, 17 However, a recent retrospective study suggests that p.Arg16Arg patients taking combination therapy with inhaled corticosteroids and a long-acting β-agonist may respond equally well to those with the p.Gly16Gly genotype, suggesting that inhaled steroids may protect from the potential pharmacogenetic effect driven by this locus.18 Several factors that might account for the difference of this analysis have been identified, such as, limiting the duration of the study to 12 weeks, simultaneous initiation of treatment with both an inhaled corticosteroid and long-acting β-agonist in patients not being treated with inhaled corticosteroids; no long-acting β-agonist placebo arm; and bias toward β-2-adrenoreceptor responders.11, 19, 20 The Long-Acting β-Agonists Response by Genotype (LARGE) study, run by the Asthma Clinical Research Network (http://www.acrn.org/), is underway in the United States to evaluate prospectively the effects of inhaled corticosteroids plus long-acting β-agonists or placebo in p.Arg16Arg versus p.Gly16Gly homozygous genotypes, looking at the effects on peak flow over 18 weeks. The c.46A>G and the c.79C>G variants have also been associated with hypertension,21 heart failure22, 23 obesity and metabolic alterations,24, 25 dislipoproteinemia, and type II diabetes,26 in predicting survival associated with β-blocker therapy after acute coronary syndrome27 and cystic fibrosis.28

These findings have generated considerable interest in the development of molecular assays to detect ADRB2 allelic variants. These assays have relied on preliminary amplification of ADRB2 sequences by polymerase chain reaction (PCR) and subsequent identification by a variety of methods such as direct sequencing,29 allele-specific oligonucleotide hybridization,10, 30 and allele-specific multiplex PCR with agarose gel detection.31, 32 All of these methods require multiple manual steps with post-PCR manipulation and are difficult to optimize, time-consuming, and relatively expensive to use in routine clinical laboratory testing. Real-time PCR-based ADRB2 genotyping assays have also been recently developed. They use either fluorescently labeled oligonucleotide probes33 or the double-stranded DNA selective fluorescent dye, SYBR Green I,34 in combination with allele-specific amplification. These are multiple tube genotyping methods. Real-time PCR amplification that uses hybridization probes and the principle of fluorescence resonance energy transfer (FRET) with melting curve analysis by the LightCycler (Roche Applied Science, Indianapolis, IN) provides an elegant system for homogenous amplification and multiple genotyping in a single tube within 1 hour. Assays using the LightCycler have been developed for multiple sequence variants influencing drug sensitivity or for multiple haplotyping.35, 36 However, we are not aware of any studies that describe rapid and accurate assays for routine clinical use that are specific for the ADRB2 allelic variants that have clinical and pharmacological implications.

We therefore developed a robust and accurate assay that uses FRET probes and melting curve analysis to identify the c.46A>G (p.Arg16Gly) and the c.79C>G (p.Gln27Glu) variants in less than 1 hour. The assay has the advantage of using a single primer set, a single double-labeled anchor probe and two sensor probes. Furthermore, it is being performed in a closed system and avoids post-PCR processing common in other assay strategies. This assay was initially developed for the ADRB2 genotyping in asthma patients, but it should be applicable to other diseases associated with these allelic variants.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Samples
To develop and validate the new ADRB2 genotyping assay, a test panel of 30 DNA samples was generated. These samples were selected from our DNA bank and anonymized for the present study. The ADRB2 genotype of each sample was determined by sequencing the forward and the reverse strands. This sample set was categorized into distinct biogeographical ancestries: 12 Caucasians, 17 African Americans, and 1 Asian. Of these 30 samples, three samples including a homozygous p.Arg16Arg–p.Gln27Gln, heterozygous p.Arg16Gly–p.Gln27Glu, and homozygous p.Gly16Gly–p.Glu27Glu were used as control samples to develop and optimize the new assay. The remaining 27 samples were used for the validation of the ADRB2 genotyping assay. Haplotype frequencies were determined in a second sample set consisted of asthmatic patients with unknown ADRB2 genotype (n = 385) attending outpatient allergy/immunology and pulmonary clinics or the adult and pediatric emergency rooms at Virginia Commonwealth University Medical Center. Whole-blood specimens in EDTA were prospectively collected from these patients. The study was reviewed and approved by the Virginia Commonwealth University Medical Center Institutional Review Board.

DNA Extraction
DNA was extracted from 50 µl of peripheral blood using the MagNA Pure LC DNA isolation kit I (Roche Applied Science, Indianapolis, IN) and the MagNA Pure LC instrument (Roche Applied Science) according to the manufacturer’s instructions and by following the DNA I blood cells fast protocol.

Direct DNA Sequence Analysis
The target DNA sequence of the ADRB2 NM_000024.4, was amplified using a set of primers that were previously described3 (forward, nucleotides 188-212: 5'-AGCCAGTGCGCTCACCTGCCAGACT-3'; reverse, nucleotides 406-383: 5'-GCTCGAACTTGGCAATGGCTGTGA-3') to generate an amplicon of 219 bp in length. PCR was performed on a 96-well GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA) using 10 µl (ranging from 1 to 25 ng/µl) of genomic DNA in 40 µl of a PCR buffer containing 1 µmol/L each of the forward and reverse primers, 0.8 mmol/L deoxynucleoside-5'-triphosphates, 1.5 mmol/L Mg2Cl, and 1.25 U of AmpliTaq gold DNA polymerase (Applied Biosystems). PCR conditions were as follow: initial denaturation at 95°C for 10 minutes followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and elongation at 72°C for 1 minute, with a final extension at 72°C for 7 minutes. Electrophoresis in a 2% agarose gel and staining with ethidium bromide were performed using 10% of the PCR reaction product to confirm the expected amplification product. The remaining PCR product was purified using ExoSAP-IT (USB Corporation, Cleveland, OH) and then analyzed by DNA sequencing of the forward and the reverse strand using the ABI PRISM BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems). Cycle sequencing was performed in the ABI 9700 thermocycler using the following parameters: initial denaturation at 98°C for 1 minute followed by 25 cycles at 97°C for 15 seconds, 50°C for 5 seconds, and 60°C for 4 minutes. DNA sequencing products were purified using spin columns (Edge Biosystems, Gaithersburg, MD) and both DNA strands were resolved on the ABI PRISM 3100 genetic analyzer (Applied Biosystems).

Genotyping by Melting Curve Analysis Using the LightCycler
The ADRB2 genotyping assay using fluorescence hybridization probes and melting curve analysis was performed in the LightCycler 1.2 instrument (Roche Applied Science). A schematic representation of the assay design is given in Figure 1Go . The ADRB2 target was amplified from genomic DNA with the primer set that was used for sequencing. The hybridization probes were designed and synthesized by TIB MolBiol (Adelphia, NJ) following specific guidelines.38 Briefly, the LC-Red 705 and the LC-Red 640 probes were designed to be complementary to the guanine (corresponding to the polymorphic variant) at nucleotide positions 46 and 79 in the target DNA, respectively. The anchor probe was made longer than the sensor probes resulting in a higher Tm for the anchor probe. A gap of one base was left between the hybridization probes to leave space for the fluorophores and to allow the energy to be transferred between them. To prevent the formation of dimers, the sequence of the hybridization probes were not complementary to the primers. The LC-Red 705 probe was phosphorylated at the 3' end to prevent probe elongation by the Taq polymerase during PCR. The sequences for the probes used are shown in Table 1Go .


Figure 1
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  		Figure 1. Diagram showing positions of primers and hybridization probes for the ADRB2 genotyping assay. An amplicon that spans the area of the allelic variants is created with one set of primers. A single anchor probe and two sensor probes anneal to the forward strand internal to the primer set. During FRET, the long anchor probe labeled at the 5' and 3' ends with fluorescein is excited by the light source of the LightCycler instrument and then passes on part of its excitation energy via dipole-dipole interactions to the adjacent acceptors, the LC-Red 705 and LC-Red 640 in the so-called sensor probes that cover the allelic variants at codons 16 and 27 of the β-2-adrenergic receptor, respectively. The excited fluorophores emit measurable light in channels 3 and 2 of the LightCycler instrument, respectively. The DNA sequence changes (c.46A>G and c.79C>G) are shown in capital letters.

 

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  		Table 1. Hybridization Probes Sequence for the ADRB2 Genotyping Assay

 
PCR was performed in glass capillaries with a final volume of 20 µl containing 1X LightCycler FastStart DNA MasterPLUS HybProbe (Roche Applied Science), 1 µmol/L forward and reverse primers, 0.2 µmol/L each of the anchor and the two sensor probes, and 5 µl of genomic DNA (ranging from 5 to 125 ng). The following LightCycler experimental protocol was used: initial preincubation step for 1 cycle at 95°C for 10 minutes to activate the FastStart TaqDNA polymerase, programmed temperature transition rate of 20°C/second; amplification of the target DNA for 45 cycles of 95°C for 10 seconds, 60°C for 10 seconds, and 72°C for 12 seconds, each with a temperature transition rate of 20°C/seconds; followed by melting curve analysis with initial denaturation at 95°C for 90 seconds, cooling to 40°C for 60 seconds to achieve maximum hybridization of the probes, and then an increase in temperature at a rate of 0.1°C/second, with continual fluorescence detection up to 80°C. Amplification and detection occurred in the same closed tube in approximately 50 minutes. Data analysis was performed using LightCycler Software 3.5 and choosing the F3/back-F1 and F2/back-F1 settings for genotyping analysis of the c.46A>G and c.79C>G variants, respectively. The melting curves were converted by the software to melting peaks by plotting the negative derivatives of fluorescence against temperature ([-d(F3/Back-F1)/dT] and [-d(F2/Back-F1)/dT], respectively). All analyses were performed with background correction and polynomial calculation. A color compensation file was created as described by the manufacturer (LightCycler color compensation set, Roche Applied Science) and was included in each run to eliminate problems associated with spectral overlap of the different fluorochromes.

Statistical Analysis
Statistical analysis was performed using Microsoft Excel (Microsoft Office 2003; Microsoft Corp., Redmond, WA). Student’s t-test was used to assess whether the different allelic variants were statistically different from each other. Allele frequencies were calculated from genotype frequencies using the formula: frequency of A = p = f(AA) + 1/2f(Aa).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ADRB2 Genotyping Assay Development
Three residual DNA samples of known genotypes consisting of a homozygous p.Arg16Arg–p.Gln27Gln, heterozygous p.Arg16Gly–p.Gln27Glu, and homozygous p.Gly16Gly–p.Glu27Glu, as determined earlier by sequencing were used as reference samples to develop the new assay. Melting curve analysis of the c.46A>G (p.Arg16Gly) variant for the LC Red-705 labeled probe and the c.79C>G (p.Gln27Glu) variant for the LC Red-640 labeled probe is shown in Figure 2 A and BGo ; respectively. Melting temperatures for the homozygous p.Arg16Arg and the homozygous p.Gly16Gly were 57.8°C and 66.8°C, respectively. For the homozygous p.Gln27Gln and the homozygous p.Glu27Glu, melting temperatures were 54.5°C and 65.0°C, respectively.


Figure 2
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  		Figure 2. Melting curves and melting peaks for each sensor probe. One DNA representing each genotype was used to optimize PCR conditions and LightCycler protocol as described in Materials and Methods. Amplicons were denatured and the probes melted at the rate of 0.1°C/second. A: Melting curves (top) and derivative melting peaks (bottom) for genotypes detected by the LC Red-705 sensor probe. The asterisk represents an unexplained elevation (shoulder) during the melting analysis of the p.Gly16Gly genotype. B: Melting curves (top) and derivative melting peaks (bottom) for genotypes detected by the LC Red-640 sensor probe.

 
Validation
The remaining 27 of the 30 DNA samples (10 Caucasians, 16 African Americans, and one Asian) that were previously genotyped by sequencing were blinded and subjected to amplification and melting curve analysis along with the three reference samples and a non-template control sample. Genotype results for the reference samples and the non-template control were as expected and the genotypes assigned by the melting curve analysis for the 27 DNA samples showed 100% concordance with the sequencing results, confirming the accuracy of the ADRB2 genotyping method. Melting curves and the derivate melting peaks for each genotype were consistently comparable with the pattern established earlier for the reference samples (Figure 2 A and B)Go . Interestingly, the resolution of the melting curves was not affected by various DNA template concentrations in the PCR (ranging from 5 to 125 ng of DNA per reaction). Therefore, we do not routinely quantify the template DNA used for this assay.

We encountered an unexplained elevation (shoulder) (Figure 2AGo , asterisk) close to the expected original curve during the melting curve analysis for the c.46A>G variant, when the sensor probe and the template were in perfect match. This unexplained melting curve behavior created a change in the plotting of the negative derivate of fluorescence against temperature that appeared as a small elevation in the melting peak analysis. Although the shoulder was always out of range (Tm ~53°C) and would not lead to a misinterpretation, we attempted to correct the problem by examining different variables. We were unsuccessful in eliminating the unexplained shoulder despite using combinations of primers titration and ratios (forward/reverse 1:1 to 5:1), different annealing temperatures and faster temperature transition rates during melting (from 0.1°C/second to 1°C/second).

Reproducibility
A subset of 15 DNA samples previously genotyped by sequencing were used as controls to evaluate the reproducibility of the ADRB2 genotyping assay and Tm acceptance ranges for the different genotypes. The non-template control was included in each run to control contamination. The homozygous samples always displayed one melting peak and the heterozygous samples always displayed two peaks when analyzed for the p.Arg16Gly and the p.Gln27Glu genotypes in F3 channel and F2 channel, respectively. The average Tms and SDs of the control samples were determined in both the F3 and F2 channels (Table 2)Go using data from three independent experiments. The separation of the allelic variants was significantly defined (P < 0.001). Tm acceptance ranges for genotyping of patient samples were defined at ±2.5 SD of the average Tm value for each ADRB2 variant, which is well within the temperature ranges recommended by the College of American Pathologists (http://www.cap.org/apps/docs/laboratory_accreditation/checklists/molecular_pathology_december2006.pdf; accessed December 2007).


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  		Table 2. Average Tm and SD of Control Samples

 
Haplotype and Allele Frequency Results in Asthmatic Patients
Asthmatic patients (n = 385) of different ages and biogeographical ancestries presenting at the Virginia Commonwealth University Medical Center were genotyped for the ADRB2 allelic variants by melting curve analysis. Melting curves and derivative melting peaks for each genotype were consistently comparable with the pattern obtained with the control samples. Genotypes were easily assigned by independent operators on consecutive days. The estimated haplotypes frequencies by biogeographical ancestry group for the 385 asthmatic patients samples analyzed in this study are shown in Table 3Go . No individuals with the Arg/Arg16-Glu/Glu27 haplotype were observed, thus confirming tight linkage disequilibrium at these two loci. We also estimated the allele frequencies for both variants (c.46A>G and c.79G>C) in our asthmatic population (Table 4)Go .


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  		Table 3. Estimated Haplotype Frequencies of p.Arg16Gly and p.Gln27Glu in Asthmatic Patients by Biogeographical Ancestry

 

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  		Table 4. Estimated Allelic Frequencies of the p.Arg16Gly and p.Gln27Glu by Age and Biogeographical Ancestry

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We used FRET hybridization probes to identify clinically significant sequence variants at codon 16 and codon 27 of the β2-adrenergic receptor with excellent performance compared with sequencing as the gold standard method. The fact that the allelic variants are 33 nucleotides apart in the ADRB2 gene sequence, allowed us to apply a probe system previously published by Herrmann et al39 in which two sensor probes at each polymorphic position were labeled with acceptor fluorophores and the third probe was a dual-labeled fluorescein donor probe that spanned the distance between the sensor probes. Therefore, with the primer-probe combination used in this study, we were able to perform a simultaneous genotyping of the two allelic variants by using two discriminating parameters, hybridization probe color and melting temperature.

Reference materials for ADRB2 genotyping were not commercially available; therefore, we generated our own reference samples by sequencing to develop and validate the new assay. Our probes’ design combined with the set of primers used for the PCR reaction for sequencing were successful in our initial studies, allowing accurate and easy detection and high discrimination of the two allelic variants as shown by agreement with sequencing results. The sensor probes were designed to be complementary to the polymorphic variant (G) at nucleotide positions 46 and 79 in the target DNA. This resulted in a higher melting temperature (Tm ~66°C and ~64°C for the c.46A>G and the c.79C>G, respectively) for a perfect match corresponding to the variant, which provided greater stability than a mismatch corresponding to the wild type (Tm ~57°C and ~54°C for the c.46A>G and the c.79C>G, respectively). The ADRB2 genotyping assay was highly reproducible with Tm, for each allele tested, within a very narrow range.

The assay presented herein has several advantages over previously described methods that are based on sequencing29 or allelic discrimination.31, 32, 33, 34 A major advantage of the melting curve analysis is its simplicity as compared to the gold standard method of sequencing, which is time consuming because it involves PCR and post-PCR steps. Allelic discrimination based assays, although relatively simple, cannot distinguish between allelic variants in a single-tube reaction and might require separate PCR reactions using allelic specific primers to make that distinction33, 34 or using multiplex allele specific PCR with post-PCR handling.31, 32 The assay is rapid and reliable and can be performed in a single capillary tube without intervening steps and post-PCR handling, which limits associated problems, including contamination.

We performed ADRB2 genotyping in 385 DNA samples from asthma patients attending outpatient allergy immunology and pulmonary clinics or the adult and pediatric emergency rooms at the Virginia Commonwealth University Medical Center. Genotypes were easily assigned by independent operators and the relative haplotypes distribution for the two ADRB2 allelic variants reported for our asthmatic population. No individuals were observed with the compound diplotype of p.Arg16Arg–p.Glu27Glu, thus confirming the tight linkage disequilibrium at these two loci. Results of allelic frequencies in our asthmatic population, were in concordance with previously reported frequencies in asthmatic African-American adults,39 who have an increased frequency of the Arg allele, and in Caucasian adult and pediatric populations.40, 41 We are not aware of any previous data on allele frequencies for pediatric African-American asthmatics population but the frequency that we found was similar to adult African Americans.

We have developed a single-step assay based on FRET probes and melting curve analysis in which two sensor probes at each polymorphic position are applied in combination with one anchor probe. This assay that is performed on the LightCycler thermocycler, enables the rapid and reliable genotyping of the c.46A>G and the c.79C>G variants of ADRB2. Genotype calls generated by FRET probes-melting analysis for the control samples were in 100% concordance with those identified by the sequencing method, thus, confirming the validity of this assay to simultaneously genotype the ADRB2 allelic variants. As the clinical relevance of these variants become better appreciated in asthma and other disorders, reliable and efficient genotype determinations will be of great value.


   Footnotes
 
Address reprint requests to Andrea Ferreira-Gonzalez, Ph.D., Virginia Commonwealth University Medical Center, Professor of Pathology, Director, Molecular Diagnostics Laboratory, CSC Building, Room 246, 409 North 13th Street, Richmond VA 23298-0248. E-mail: aferreira-gonzalez{at}mcvh-vcu.edu

Supported by National Institutes of Health grant R01 AI27517 (to L.B.S.) and by the Philip Morris Foundation (to L.B.S.).

L.B.S. is supported by Glaxo Smith Kline (grants MHE100901, MHE100185, MHE104317) and is participating in a randomized, placebo-controlled, double-blind Phase II study of the safety and efficacy of rhuC1inh for the treatment of acute attacks in patients with hereditary angioedema. L.B.S. has received speaker’s honoraria from Genentech/Novartis, is on the Advisory Board of Genentech/Novartis, and is a Scientific Advisor for Mast Cell Pharmaceutical Company. A.M.I. receives grant and other funding support from Novartis, Genentech, Astra-Zeneca, Aventis, and Schering-Plough. A.M.I. is on the Speaker’s Bureau for Astra-Zeneca, Merck, Novartis, Genentech, and Aventis and is on the Advisory Boards of Astra-Zeneca and Merck.

Accepted for publication January 15, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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