Genotyping of Essential Hypertension Single-Nucleotide Polymorphisms by a Homogeneous PCR Method with Universal Energy Transfer Primers

Abstract

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™ 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.

Association Study of G Protein‐Coupled Receptor Kinase 4 Gene Variants with Essential Hypertension in Northern Han Chinese

“…Although we have found that the A486V polymorphism is associated with HT in the northern Chinese Han population, this result contradicts those found in studies of Caucasian Italians (Bengra et al 2002) and Caucasian Australians (Speirs et al 2004). In their studies the V486 allele was found to be the risk factor for HT, while in ours A486 was shown to be associated with HT.…”

“…Three variants of the γ isoform of GRK4 , R65L , A142V and A486V have been reported to affect GRK activity, resulting in increased serine phosphorylation of D 1 receptors and uncoupling of the receptor from its G‐protein complex. Bengra et al (2002) deserved a significant association ( P = 0.034) between V486 and an Italian population of mildly hypertensive patients. In another study the V486 allele was also found to be associated with HT ( P = 0.02), and additionally the L65 and the V142 alleles tracked with elevation in diastolic blood pressure (DBP), even though this was seen only in male HTs ( P = 0.009; P = 0.002, respectively) (Speirs et al 2004).…”

Variants in Genes Involved in Functional Pathways Associated with Hypertension in African Americans

“…More recently GRK4 variants have been associated with a decreased response to beta-blocker treatment for HBP in African American males. 28 32 A metaanalysis of the major studies involving GRK4 variants and HBP showed that the allele 486V increases the risk for HBP with an OR of 1.5 (95% CI: 1.2-1.9). 33 Other studies have found associations between the A142V variant 34 and HBP in a Japanese population.…”

G Protein–Coupled Receptor Kinases: Crucial Regulators of Blood Pressure

“…The GRK4 locus on human chromosome 4p16.3 is linked to essential hypertension and salt sensitivity. [79][80][81][82][83][84][85] Three missense SNPs ( ie, 65R>L, 142A>V, and 486A>V) in the coding region of GRK4c are associated with increased blood pressure. Depending on the genetic background of the mouse, mice overexpressing the GRK4c wild-type transgene are normotensive and salt-resistant, whereas GRK4c142V transgenic mice have high blood pressure even with normal NaCl intake.…”

“…The association between GRK4486V and essential hypertension was found in Italian and Euro-Australian populations. 79,80 In a study of northern Han Chinese participants, the GRK465L, GRK4142V, and GRK4A486 haplotypes are associated with a 6-fold higher risk of systolic and diastolic hypertension. 81 In these same Han Chinese participants, GRK4486V alone is associated with hypertension.…”