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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

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.






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Copyright © 2008 by the American Society for Investigative Pathology and the Association for Molecular Pathology.