title: A simple and rapid genotyping assay for simultaneous detection of two ADRB2 allelic variants using fluorescence resonance energy transfer probes and melting curve analysis.
abstract: Allelic variants at codons 16 and 27 of the beta(2)-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 degrees C +/- 0.10 degrees C) and p.Gly16Gly (Tm = 66.73 degrees C +/- 0.18 degrees C) and two melting peaks for p.Arg16Gly were obtained. Similarly, single peaks for p.Gln27Gln (Tm = 53.98 degrees C +/- 0.19 degrees C) and p.Glu27Glu (Tm = 64.93 degrees C +/- 0.16 degrees 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.
authors.affiliation: Department of Pathology, Virginia Commonwealth University Medical Center, Richmond VA, USA.
authors.forename: M Fernanda
authors.initials: MF
authors.lastname: Sábato
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Irani
authors.forename: Bonny L
authors.initials: BL
authors.lastname: Bukaveckas
authors.forename: Lawrence B
authors.initials: LB
authors.lastname: Schwartz
authors.forename: David S
authors.initials: DS
authors.lastname: Wilkinson
authors.forename: Andrea
authors.initials: A
authors.lastname: Ferreira-Gonzalez
mesh_terms: D000328:Adult; D001741:Black or African American; D000483:Alleles; D001249:Asthma / Q000235:genetics; D002648:Child; D015342:DNA Probes / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D009691:Nucleic Acid Denaturation; D011110:Polymorphism, Genetic; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D015342:DNA Probes; D018343:Receptors, Adrenergic, beta-2