Combination allele-specific real-time PCR for differentiation of [[beta].sub.2]-adrenergic receptor coding single-nucleotide polymorphisms.
ADRB2 is transcribed from an intronless gene on chromosome 5q32 that encodes a 413-residue protein with 7 transmembrane domains (2). Within the ADRB2 gene, 13 single-nucleotide polymorphisms (SNPs) have been identified (3), including 3 coding ADRB2 missense polymorphisms that alter protein function (4). A threonine-toisoleucine (T164I) substitution in the fourth transmembrane domain alters ligand binding and decreases adenylate cyclase activation but is rare in the population (5). The extracellular domain N-terminal arginine-to-glycine (R16G) and glutamine-to-glutamic acid (Q27E) polymorphisms are common and widely distributed within the population. These polymorphisms strongly influence the degree of agonist-mediated receptor down-regulation. Whereas Q27E induces complete resistance to downregulation in combination with R16, R16G increases agonist-mediated receptor down-regulation in both haplotypes (5).
The importance of developing rapid ADRB2 screening protocols that differentiate codon 16/27 diplotypes is emphasized by the number of studies linking these genotypes to clinical and pharmacologic responses. SNPs representing the partial haplotype at codons 16 and 27 are variously associated with altered vasodilation (6) and high blood pressure (7), play roles in the development of type II diabetes (8), and have been linked to preterm delivery (9), agonist desensitization (10), albuterol responses (11), increased IgE in asthmatic families (12), obesity (13), hypertriglyceridemia, and development of fatty liver (14).
Methods to routinely detect the common codon 16 and 27 polymorphisms have relied on preliminary amplification of ADRB2 sequences by PCR and subsequent identification by a variety of methods (15). Codon 16/27 genotyping is accomplished with dot-blot oligonucleotide hybridization (16), single-strand conformation polymorphism analysis (9), direct sequencing (17), multiplex PCR (18), denaturation selective amplification and subtractive genotyping (19), allele-specific codon 16 PCR combined with restriction fragment length polymorphism analysis (20), or strand displacement amplification (21).
The increasing clinical relevance of ADRB2 SNP phenotypes makes automated screening methods important as tools for routine assessment of ADRB2 polymorphisms. A new assay to simultaneously detect coding polymorphisms in ADRB2 must accurately distinguish between A(R) and G(G) residues in codon 16 (nucleotide 265) and identify the G(E) and C(Q) polymorphisms in codon 27 (nucleotide 298). We describe here single-step ADRB2 codon 16/27 genotyping on chromosomal DNA based on a combination of allele-specific codon 16 amplification primers in a real-time PCR combined with allele-differentiating fluorescent probes.
After receiving approval from the ethics commission of the University of Vienna and informed consent from the donors, we collected peripheral blood from 249 healthy male Caucasian donors between 19 and 35 years of age in citrate-coated tubes (Becton Dickinson) and isolated chromosomal DNA with the Perfect gDNA blood mini reagent set (Eppendorf). DNA concentrations were measured with the RediPlate 96 PicoGreen dsDNA Assay Kit (Molecular Probes) in a Victor2 multilabel plate reader (EG&G Wallac).
To generate ADRB2 PCR products for subsequent sequencing, we amplified a 219-bp ADRB2 fragment from 300 ng of DNA with 10 pmol of forward (5'-AGCCAGTGCGCTTACCTGCCAGACT-3'; nucleotides 188-212) and reverse (5'-GCTCGAACTTGGCAATGGCTGTGA-3'; nucleotides 406-383) primers (10) by initial denaturation at 95[degrees]C for 2 min, followed by 35 cycles of amplification at 95[degrees]C, denaturation for 30 s, annealing at 60[degrees]C for 30 s, and extension at 72[degrees]C for 1 min, with a final extension at 72[degrees]C for 7 min. The 50-[micro]L reaction contained 200 [micro]M deoxynucleotide triphosphates, 1.5 mM Mg[Cl.sub.2], 20 mM Tris-HCl (pH 8), 50 mM KCl, and 1 U of Platinum Taq polymerase (Invitrogen) and was performed in a GenAmp Thermocycler 2400 (Applied Biosystems, Perkin-Elmer). Nucleotide positions were based on the human ADRB2 GenBank sequence accession number (NM_000024.3). Fragments were gel-purified with the QIAquick PCR Purification Kit (Qiagen), and the genotypes of the polymorphic region were determined by sequencing of both strands with the amplification primers on an ABIPrism 3100 Genetic Analyzer (Applied Biosystems).
Chromosomal DNA was amplified in two independent PCR reactions, each containing an allele-specific codon 16 amplification primer. Presence of the correct nucleotide enables initiation of amplification in a real-time PCR reaction containing both 6-carboxyfluorescein (FAM)-labeled E-specific and VIC[R]-labeled Q-specific fluorescent probes. Detection of FAM or VIC fluorescence in either the codon 16A(R) or codon 16G(G) PCR reactions then identifies the codon 27 allele as G(E) or C(Q), respectively, as shown in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol50/issue4/.
Individual real-time PCR amplifications were performed on 50-100 ng of chromosomal DNA in a PRISM 7700 sequence detector (Applied Biosystems). Each reaction contained 5 pmol each of the fluorescent codon-27-differentiating FAM-labeled (5'-TCACGCAGGAAAG-3'; nucleotides 290-302) and VIC-labeled (5'-TCACGCAGCAAAG-3'; nucleotides 290-302) probes (the bolded nucleotides represent the site of the SNP), 23 pmol of a reverse primer (5'-GGCAATGGCTGTGATGACC-3'; nucleotides 396-378), and 23 pmol of either the allelespecific codon 16A (5'-TCTTGCTGGCACCCTATA-3'; nucleotides 248-265) or codon 16G (5'-TCTTGCTGGCACCCTATG-3'; nucleotides 248-265) forward primers in a 25-[micro]L reaction containing TaqMan Universal PCR master mixture (Applied Biosystems). Similar to previous studies incorporating PCR with restriction fragment length polymorphism analysis (11, 20), the codon 16 allele-differentiating PCR primers that had been optimized for specificity during real-time PCR cycling conditions were designed to include an A3T substitution at nucleotide 262, which considerably improved specificity for the terminal primer nucleotide (data not shown). Real-time PCR was initiated at 50[degrees]C for 2 min and 95[degrees]C for 10 min, followed by 40 cycles of 95[degrees]C for 15 s and 60[degrees]C for 1 min. Threshold cycle values were generated with Sequence Detector (Ver. 1.7) software (Applied Biosystems), and positive loci amplifications were defined as signals exceeding a normalized reporter signal of [10.sup.-1] within 40 cycles.
Typical amplification plots obtained are shown in Fig. 1 and the online Data Supplement. Compared with the directly sequenced ADRB2 locus, which was routinely determined in 20% of the DNA samples, this assay correctly identified all base combinations at these positions and distinguished G16E27, G16Q27, and R16Q27 homozygotes from the simple G16Q27/R16Q27 and compound G16Q27/G16E27 heterozygotes. All allelic variations were detected within 34 cycles of real-time PCR, and background amplification was minimal and never exceeded threshold cycle values within 40 cycles. This method also accurately differentiated allelic variants from chromosomal DNA templates, thus eliminating the need for pre-assay PCR amplification of the locus. In contrast to gel-based assays, combination of 96-well format DNA isolation, quantification, and real-time amplification procedures shortens the time needed to conduct large-scale ADRB2 phenotyping with an increase in accuracy because compound heterozygous phenotypes are additionally precisely defined.
[FIGURE 1 OMITTED]
ADRB2 polymorphism frequencies are known to differ among Caucasian, African-American, and Chinese populations (15, 22). To determine the allelic frequencies within the male Caucasian Austrian population, we performed real-time PCR on 249 chromosomal DNA samples from Caucasian males (Table 1). In all cases (n = 249), genotypes were assigned correctly by independent operators on consecutive days, and there was no ambiguity for any sample. The total number of polymorphic alleles detected (Table 1) was similar to those detected in other previously described Caucasian populations. Of the 498 alleles analyzed in this study, 65.1% were G16 compared with 61-61.6% in US (11, 15), 63.8% in Swedish (23), and 67% in United Kingdom (12) Caucasian populations. Similarly, 42% of alleles analyzed were polymorphic E27 compared with 35.9-42% (11, 15) in American, 45.5% in Swedish (23), and 47.5% in United Kingdom (12) Caucasian populations. In the Austrian population, a slightly greater (35.5%) linkage between G16 and E27 alleles was seen than in Swedish [28% (23)] and in American [26.3% (12)] Caucasians, and 18.9% homozygosity for G16E27 was observed compared with 13% in the American population (11). Similar to the Swedish population (23), we detected no examples of R16E27 in the present cohort as R16E27/G16E27, R16Q27/R16E27, or R16E27/G16E27, although the assay correctly identified SNPs at both positions.
In summary, we describe here the development of a novel assay that differentiates codon 16/27 polymorphisms in chromosomal DNA in parallel PCR reactions within 2 h. This method may also find universal application for the simultaneous detection of adjacent SNPs. Use of this assay could allow the rapid, cost-effective, and unequivocal identification of coding genotypes and dramatically decrease the time needed to conduct ADRB2 phenotyping. In conjunction with other recently reported assays (19, 21), this method could also form an integral part of strategies to routinely generate full ADRB2 haplotypes and may find use in future clinical diagnostics.
We thank Dr. Masahiro Hiratsuka (Tohoku Pharmaceutical University, Tohoku, Japan) and Dr. Julie Johnson (University of Florida, Gainesville, FL) for generously providing DNA for initial experiments. This work was supported by the "Kamillo Eisner Stiftung" and the "Hygiene Funds".
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Trevor Lucas, [1,2] ([dagger])* Doris Losert,  ([dagger]) Matthew Allen, [1,2] Julius Halaschek-Wiener,  Barbara Pratscher, [1,2] Cornelia Friedrich, [1,2] Markus Wolschek,  Gabriele Fuchsjager-Mayrl, [1,3] Leopold Schmetterer, [1,4] Hubert Pehamberger,  Markus Muller,  and Volker Wacheck  ( Department of Clinical Pharmacology, Section of Experimental Oncology/ Molecular Pharmacology,  Department of Dermatology, Division of General Dermatology, and  Department of Ophthalmology, University of Vienna, Vienna, Austria;  Institute of Medical Physics, Vienna, Austria; ([dagger]) these authors contributed equally to this work; * address correspondence to this author at: Department of Clinical Pharmacology, University of Vienna, Vienna General Hospital, Waehringer Guertel 18-20, Vienna, Austria A-1090; fax 43-1-40400-2998, e-mail firstname.lastname@example.org
Table 1. Allelic frequencies of coding ADRB2 polymorphisms in a healthy Austrian population (n = 249) detected by allele-specific real-time PCR. (a) Allelic composition n Frequency R16Q27/R16Q27 24 0.096 G16Q27/G16Q27 13 0.052 G16E27/G16E27 47 0.189 G16Q27/G16E27 39 0.157 R16Q27/G16Q27 50 0.201 R16Q27/G16E27 76 0.305 (a) The rare R16E27 allele as R16E27/G16E27 was not detected.
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|Title Annotation:||Technical Briefs|
|Author:||Lucas, Trevor; Losert, Doris; Allen, Matthew; Halaschek-Wiener, Julius; Pratscher, Barbara; Friedric|
|Date:||Apr 1, 2004|
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