Hypertension affects more than 50 million Americans. Despite many treatments, blood pressure remains largely uncontrolled in large number of patients in North America leading to increased incidence of stroke, heart and renal disease and escalating health care costs. Thus, there is considerable interest in better defining the genetic basis of hypertension in humans and experimental animal models. In preliminary experiments, we found that introgression of chromosome 13 (Chr 13) from the BN into the SS genetic background (SS-13BN) attenuates the development of hypertension. We subsequently developed 26 and phenotyped 23 overlapping congenic strains with various regions of Chr 13 from a BN rat introgressed into the SS genetic background and found that 4 of these congenic strains (Strains 1, 5, 9 and 26) had lower blood pressure and proteinuria than SS rats. We selected the Chr 13 congenic strain 5 for further study and confirmed by telemetry that this strain was protected from the development of hypertension and proteinuria when fed a high salt diet for 4 weeks. In contrast, an overlapping congenic strain 6, with a shorter introgressed region, exhibited no protection. These results narrow the region of interest to a 10 Mbp segment on Chr 13 (32.4-42.5Mbp) that contains between 110 and 159 named and predicted genes. This region is homologous to a region on human chromosome 2 linked to blood pressure in the QuAbec Family Study and the Family Blood Pressure Program. The goal of Project 2 is to identify and prioritize the candidate genes in the region responsible for the protection from the development of salt-sensitive hypertension in the Chr 13 congenic strain 5 and then to test whether they can alter blood pressure in the SS genetic background using transgenic techniques.
The Specific Aims are: 1) To create and phenotype subcongenic strains to narrow the region responsible for the protection from the development of salt-sensitive hypertension to 1-2 Mbp. We have already created 12 overlapping subcongenic lines from the Chr 13 strain 5 congenic strain. These strains will be challenged with a high salt diet and sequentially phenotyped for blood pressure and proteinuria to narrow the region responsible for the lowering of blood pressure from 10 Mbp containing from 110 to 159 genes to a region less than 2 Mbp containing 10-20 genes. 2) To prioritize, using sequencing and gene expression analysis, which of the positional candidate genes (1-5 expected) to evaluate further in functional studies. We will compare the cDNA sequence of all of the genes in the region of interest that are present in several tissues of the subcongenic rats protected from hypertension versus the susceptible SS strain to identify sequence variants that could alter the function of the protein. In parallel, we will perform real time PCR expression studies to look for differentially expressed genes in the interval in subcongenic rats versus the susceptible SS strain. Finally, we will sequence through the entire region of interest in SS rats and the congenic strain to identify potential causal sequence variants in the 5'or 3'regions of the differential expressed genes or in highly conserved flanking regions. 3) To test which of the prioritized candidate genes alter blood pressure in the SS genetic background using transgenic techniques. If the expression of the gene is downregulated or there is an inactivating mutation in SS rats, we will test if global upregulation of the expression of the gene using lentiviral transgenesis can reduce blood pressure in SS rats. Alternatively, we will overexpress the gene in the subcongenic strain if the expression of the gene or the activity of the protein is found to be upregulated in SS rats. Final functional validation of candidate genes with a potential causal mutation that alters blood pressure in the screen will rely on the creation of a BAG transgenic line that expresses the correct allele under the control of the native promoter in a permissive genetic background (SS or the subcongenic strain) using pronuclear injection. Characterization of candidate gene(s) in this region that influence blood pressure may identify novel pathways contributing to the control of arterial pressure and the development of new therapeutic approaches for the treatment of hypertension.
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