| Association studies on Single Nucleotide Polymorphisms (SNPs) within the Angiotensin II type 1 Receptor gene (AGTR1) attempt to identify variations as key predictors of an individual's susceptibility to or presence of hypertension. We developed a library of 14 Variable Combinations of mRNA (VCmRNA) sequences from 62 hypertensive and normotensive patients. Competition binding assays, used to estimate AT1expression, showed no significant differences between the means +/- S.E. values. The change in the means after standardization with either CAT activity or EGFP expression, depending on the vector, did not alter significance between VCmRNA.; Secondary structure predictions were determined using MFold. This analysis defined a limited number of unique secondary structures. The variations at +1150T/G, +1166A/C, and +1517G/T were the most effective at altering predicted structures. Analyzing binding results, segregated by these groups, showed no significant variance in Bmax.; Maximum expression of AT1 was achieved by transfection of the entire Ex 1/5 sequence. Minimal expression was observed by transfection of the Coding sequence (Cds). Removal of the 5'-LS and 3'-UTR, from the Ex 1/5 construct, resulted in significant reductions in AT1 expression, (p < 0.0001) and (p < 0.0035), respectively. Addition of the 5'-LS to the Cds showed significantly increases in B max (p < 0.0005); however, addition of the 3'-UTR had no effect.; This work suggests the influence of SNPs do not alter AT1 expression significantly. Variations in the 5'-LS would likely have a greater influence on AT1 expression than those in the 3'-UTR; although, the 3'-UTR is functionally significant. Although none of the VCmRNAs showed significant changes in AT1 expression, the limited population analyzed does not rule out the possibility that VCmRNAs may exist within certain individuals which effect AT1 expression. This work does not consider changes occurring outside of steady-state. Potential long term effects of minor variations in AT1 expression on the functioning of the Renin-Angiotensin System (RAS) are beyond the scope of this study. |
