This is a revised proposal to study angiotensinogen and how mutations of either its gene or protein might predispose to hypertension. The investigators have tested the involvement of genes of the renin-angiotensin system in human hypertension by linkage analysis in pairs of affected siblings. In two large (379 pairs) series of patients form Utah and France, they have obtained evidence of genetic linkage between the angiotensinogen gene (AGT) and hypertension, demonstrated association of a common molecular variant of the gene (T235) with the disease, and found significant differences in plasma concentrations of angiotensinogen among hypertensive subjects as a function of AGT genotype at residue 235. Subsequently, they have reported a similar association with preeclampsia (or 'toxemia'), a common hypertensive disorder of pregnancy. These and other data support the hypothesis that molecular variants of AGT constitute inherited predispositions to essential hypertension. This hypothesis is the focus of the renewal application with the long- term objective of obtaining some clues about pathophysiological mechanisms. Initially, they define four specific aims: (1) to test the functional significance of naturally-occurring missense variants of human angiotensinogen through in vitro mutagenesis and expression followed by physical, biochemical, and metabolic analyses of recombinant products; (2) to produce and purify human angiotensinogen on a scale amenable to physical studies; (3) to investigate systematically determinants of the substrate which affect the reaction rate of mouse renin with mouse angiotensinogen, using synthetic peptides and in vitro recombinant products as substrates and recombinant mouse renin as enzyme; (4) to exploit homologous recombination in mice in order to generate transgenic animals with increased or decreased reactivity or expression of angiotensinogen for future physiological and biochemical studies on the role of angiotensinogen in the regulation of blood pressure and the pathogenesis of hypertension. Since their initial submission, they have found that another molecular variant occurring at the -6 position of the transcription start site was present in most genes coding for T235. This region is known to harbor elements that critically affects the basal transcription rate and initiating using segments of the angiotensinogen promoter with each of the two alleles observed at residue -6.
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