Genotyping of essential hypertension single-nucleotide polymorphisms by a homogeneous PCR method with universal energy transfer primers.
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: -344C-->T] associated with essential hypertension. PCRs of SNP loci were coupled (via a common sequence of 21 nucleotide tails) to incorporate Universal Amplifluor(TM) 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.
Bengra
Chikh
C
Department of Pathology, The University of Virginia, PO Box 800214, Charlottesville, VA 22908, USA.
Mifflin
Theodore E
TE
Khripin
Yuri
Y
Manunta
Paolo
P
Williams
Scott M
SM
Jose
Pedro A
PA
Felder
Robin A
RA
D000808:Angiotensinogen / Q000235:genetics; D019405:Cytochrome P-450 CYP11B2 / Q000235:genetics; D017931:DNA Primers; D031541:Fluorescence Resonance Energy Transfer; D005470:Fluorometry; D054771:G-Protein-Coupled Receptor Kinase 4; D005838:Genotype; D006801:Humans; D006973:Hypertension / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D017346:Protein Serine-Threonine Kinases / Q000235:genetics
D017931:DNA Primers; D000808:Angiotensinogen; D019405:Cytochrome P-450 CYP11B2; D017346:Protein Serine-Threonine Kinases; D054771:G-Protein-Coupled Receptor Kinase 4; C517855:GRK4 protein, human