The only compelling evidence supporting the linkage of a human gene with essential hypertension is for angiotensinogen (AGT). In initial reports, the allele frequency of the variant (M235T) was significantly higher in hypertensive versus control populations and was associated with elevated levels of plasma AGT. However, numerous studies have reported disparately conflicting results over its importance in hypertensive populations. Indeed, the notion that the 235 variant is causative is founded entirely on a statistical correlation of a higher frequency of the 235T allele vs. 235M allele in hypertensives. The variant at position 235 was reported to be in linkage disequilibrium with another variant at position -6, resulting in the definition of two common haplotypes of the AGT gene (-6A:235T and -6G:235M) in humans. These data have led many to propose the following hypothesis: In humans, the AGT gene is linked to hypertension, and abnormalities in the AGT gene or its expression may cause a genetic predisposition to hypertension or an altered responsiveness to vasoactive factors. Specifically, the haplotype generated by the presence of the -6A and 235T variants may predispose to hypertension. The purpose of this proposal is to directly challenge this hypothesis by using gene targeting strategies that will provide an opportunity to directly compare cardiovascular responses in mice containing either the - 6A:235T or -6G:235M haplotypes of AGT. To accomplish this, we propose to: 1) Use a novel gene targeting strategy to generate transgenic mice containing human AGT (HAGT) constructs that are identical except for sequence variants identified in humans. Each construct will be targeted in a single copy to an identical location in the genome. 2) Examine the abundance of haplotype- specific HAGT mRNA and HAGT protein in these mice. 3) Compare resting blood pressure and cardiovascular and renal responses in the models. 4) Directly test the hypothesis that -6A augments AGT promoter activity in vivo by generating single-copy unique insertion site transgenic mice containing allelic variants (-6A vs. -6G) of the HAGT promoter fused to the LacZ (B-Gal) reporter gene. These studies may provide a paradigm for the functional testing of gene variants identified in other genes and for other polygenic diseases.
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