Genotyping of essential hypertension single-nucleotide polymorphisms by a homogeneous PCR method with universal energy transfer primers

Chikh Bengra, Theodore E. Mifflin, Yuri Khripin, Paolo Manunta, Scott M. Williams, Pedro A. Jose, Robin A. Felder

Research output: Contribution to journalArticlepeer-review

Abstract

Background: Human hypertension is a complex, multi-factorial 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™ 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 geno-types 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.

Original languageEnglish
Pages (from-to)2131-2140
Number of pages10
JournalClinical Chemistry
Volume48
Issue number12
Publication statusPublished - Dec 1 2002

ASJC Scopus subject areas

  • Clinical Biochemistry

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