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1 The University of Virginia, Department of Pathology, PO Box 800214, Charlottesville, VA 22908.
2 Serologicals Corporation, 202 Perry Parkway, Gaithersburg, MD 20877.
3 University "Vita e Salute San Raffaele", Division of Nephrology, Dialysis, and Hypertension, San Raffaele Hospital, 20132 Milan, Italy.
4 Meharry Medical College, Department of Microbiology, Nashville, TN 37208-3599.
5 Georgetown University Medical Center, Department of Pediatrics and Physiology and Biophysics, 3800 Reservoir Rd., Washington, DC 20007.
aAddress correspondence to this author at: University of Virginia, Department of Pathology/MARC, PO Box 800214, Charlottesville, VA 22908. Fax 434-924-5718; e-mail raf7k{at}virginia.edu.
Background: Human hypertension is a complex, multifactorial disease with a heritability of more than 30–50%. A genetic screening test based on analysis of multiple single-nucleotide polymorphisms (SNPs) to assess the likelihood of developing hypertension would be helpful for disease management.
Methods: Tailed allele-specific primers were designed to amplify by PCR six biallelic SNP loci [three in G protein-coupled receptor kinase type 4 (GRK4): R65L, A142V, and A486V; two in angiotensinogen: -6G
A and M235T; and one in aldosterone synthase: -344CT] associated with essential hypertension. PCRs of SNP loci were coupled (via a common sequence of 21 nucleotide tails) to incorporate Universal AmplifluorTM primers labeled with fluorescein or sulforhodamine in a homogeneous format. Use of Amplifluors in SNP PCRs produced labeled amplicons, the fluorescence of which was quantified by a microplate reader and then analyzed via an Excel macro to provide genotypes for all six SNP loci. Unique restriction endonucleases were identified for five SNP loci that could independently confirm homogeneous PCR results when needed.
Results: We developed six homogeneous PCR assays that were set up, performed, and fluorometrically analyzed in 96-well microplates. Allele frequencies were determined for six SNPs in 60 Italian hypertensive patients and a control group of 60 normotensive persons. A significant correlation (P = 0.034) between one SNP [GRK4 (A486V)] and the hypertensive patients was observed. Genotyping results for five of six SNPs were confirmed by digesting corresponding amplicons with locus-specific restriction endonucleases.
Conclusions: We developed a simple and homogeneous fluorescent protocol that has been used to determine the SNP genotype for six loci in a population of hypertensive and normotensive persons. We also observed a significant association (P = 0.034) between one SNP (A486V) and an Italian population of mildly hypertensive patients.
The following articles in journals at HighWire Press have cited this article:
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  C. Zeng, V. A. M. Villar, G. M. Eisner, S. M. Williams, R. A. Felder, and P. A. Jose G Protein-Coupled Receptor Kinase 4: Role in Blood Pressure Regulation Hypertension, June 1, 2008; 51(6): 1449 - 1455. [Full Text] [PDF]
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T. V. Pereira, A. C.F. Nunes, M. Rudnicki, Y. Yamada, A. C. Pereira, and J. E. Krieger Meta-Analysis of the Association of 4 Angiotensinogen Polymorphisms With Essential Hypertension: A Role Beyond M235T? Hypertension, March 1, 2008; 51(3): 778 - 783. [Abstract] [Full Text] [PDF]
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 Z. Wang, I. Armando, L. D. Asico, C. Escano, X. Wang, Q. Lu, R. A. Felder, C. G. Schnackenberg, D. R. Sibley, G. M. Eisner, et al. The elevated blood pressure of human GRK4{gamma} A142V transgenic mice is not associated with increased ROS production Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2083 - H2092. [Abstract] [Full Text] [PDF]
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 M. Desai, E. Zeggini, V. A. Horton, K. R. Owen, A. T. Hattersley, J. C. Levy, G. A. Hitman, M. Walker, R. R. Holman, M. I. McCarthy, et al. The Variable Number of Tandem Repeats Upstream of the Insulin Gene Is a Susceptibility Locus for Latent Autoimmune Diabetes in Adults Diabetes, June 1, 2006; 55(6): 1890 - 1894. [Abstract] [Full Text] [PDF]
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H. Sanada, J. Yatabe, S. Midorikawa, T. Katoh, S. Hashimoto, T. Watanabe, J. Xu, Y. Luo, X. Wang, C. Zeng, et al. Amelioration of Genetic Hypertension by Suppression of Renal G Protein-Coupled Receptor Kinase Type 4 Expression Hypertension, June 1, 2006; 47(6): 1131 - 1139. [Abstract] [Full Text] [PDF]
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 H. Sanada, J. Yatabe, S. Midorikawa, S. Hashimoto, T. Watanabe, J. H. Moore, M. D. Ritchie, S. M. Williams, J. C. Pezzullo, M. Sasaki, et al. Single-Nucleotide Polymorphisms for Diagnosis of Salt-Sensitive Hypertension Clin. Chem., March 1, 2006; 52(3): 352 - 360. [Abstract] [Full Text] [PDF]
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D. R. Warden and H. Refsum Detection of Single-Nucleotide Polymorphisms by PCR with Universal Energy Transfer-Labeled Primers: Application to Folate- and Cobalamin-Related Genes Clin. Chem., September 1, 2005; 51(9): 1713 - 1716. [Full Text] [PDF]
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L. M. Silverman and T. M. Mifflin Genotype-Phenotype Correlations (II): Assessing the Influence of Sequence Variants on the Clinical Phenotype in Multifactorial Disorders Clin. Chem., June 1, 2005; 51(6): 929 - 930. [Full Text] [PDF]
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 C. Zeng, H. Sanada, H. Watanabe, G. M. Eisner, R. A. Felder, and P. A. Jose Functional genomics of the dopaminergic system in hypertension Physiol Genomics, November 17, 2004; 19(3): 233 - 246. [Abstract] [Full Text] [PDF]
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S.-S. Wang, K. Thornton, A. M. Kuhn, J. G. Nadeau, and T. J. Hellyer Homogeneous Real-Time Detection of Single-Nucleotide Polymorphisms by Strand Displacement Amplification on the BD ProbeTec ET System Clin. Chem., October 1, 2003; 49(10): 1599 - 1607. [Abstract] [Full Text] [PDF]
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