Genomic Region Influencing Blood Pressure in SS-13bn Rat

Medical College Of Wisconsin, Milwaukee WI

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Abstract

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