Bengra Chikh, Mifflin Theodore E, Khripin Yuri, Manunta Paolo, Williams Scott M, Jose Pedro A, Felder Robin A
Department of Pathology, The University of Virginia, PO Box 800214, Charlottesville, VA 22908, USA.
Clin Chem. 2002 Dec;48(12):2131-40.
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.
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.
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.
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.
人类高血压是一种复杂的多因素疾病，遗传度超过30%-50%。基于多个单核苷酸多态性（SNP）分析的基因筛查试验，用于评估患高血压的可能性，将有助于疾病管理。
设计带尾的等位基因特异性引物，通过聚合酶链反应（PCR）扩增6个双等位基因SNP位点[3个位于4型G蛋白偶联受体激酶（GRK4）：R65L、A142V和A486V；2个位于血管紧张素原：-6G→A和M235T；1个位于醛固酮合酶：-344C→T]，这些位点与原发性高血压相关。SNP位点的PCR与（通过21个核苷酸尾的共同序列）掺入用荧光素或磺基罗丹明标记的通用扩增荧光引物以均相形式偶联。在SNP PCR中使用扩增荧光素产生标记的扩增子，其荧光通过微孔板读数器定量，然后通过Excel宏分析以提供所有6个SNP位点的基因型。为5个SNP位点鉴定了独特的限制性内切酶，在需要时可独立确认均相PCR结果。
我们开发了6种均相PCR检测方法，在96孔微孔板中进行设置、操作和荧光分析。测定了60例意大利高血压患者和60例血压正常对照组人群中6个SNP的等位基因频率。观察到1个SNP[GRK4（A486V）]与高血压患者之间存在显著相关性（P = 0.034）。通过用位点特异性限制性内切酶消化相应的扩增子，证实了6个SNP中5个的基因分型结果。
我们开发了一种简单的均相荧光方法，用于确定高血压和血压正常人群中6个位点的SNP基因型。我们还观察到1个SNP（A486V）与意大利轻度高血压患者人群之间存在显著关联（P = 0.034）。
