Coronary Artery Disease (CAD) is the most common form of heart disease, a condition that effects around 18.2 million people[i] and kills hundreds of thousands of Americans every year[ii]. Through Genome Wide Association studies, a gene called ARNT found in humans has been identified as a new risk locus for CAD.[iii] ARNT is known to play an essential role in transcriptional regulation in vertebrates.[iv] It has also been found that, on a biological level, ARNT plays a rolle in positive regulation of endothelial cell proliferation[v]. What is left unknown is how these two properties of ARNT are related and how they contribute to the development of CAD. If we were better able to understand this relationship, it would create potential for the development of new medicines, gene therapies, and other treatments for this genetic mutation and in turn a reduction in its role in the development of CAD.
We will test the hypothesis that a mutation in the ARNT gene in humans increases specificity of its regulatory properties, leading to an increase in endothelial cell proliferation. We are led to this hypothesis by the identification of ARNT as a risk locus for CAD and by its known function as a transcriptional regulator.
The model organism we will be using is Danio rerio due the visibility of this organism’s heart, the organ effected by CAD.
The primary goal of this research is to identify genes that play a causative rolel in the development of coronary artery disease. The long-term goal of this project is to develop new gene therapies that can prevent the onset of CAD in those with ARNT mutations. The objective of this proposal is to determine the relationship between mutations at the ARNT locus and CAD through three specific aims:
AIM 1: Identify the different mutations in ARNT that lead to the CAD phenotype
Approach: We would first conduct sequencing on Danio rRerio which exhibit the CAD phenotype using Sanger Sequencing for the ARNT locus. After identifying all mutations in ARNT in these individuals I will use CRISPR/Cas9 to develop a strain of Danio rRerio for each of these mutations and determine if the specific mutation is causative or silent. By making these observations I can then create a comprehensive list of SNPs and other mutations in ARNT that lead to CAD. Rationale: By identifying all mutations in ARNT that lead to the CAD phenotype, it allows for a comprehensive gene therapy to be developed. I hypothesize: there will be a variety of mutations in ARNT that lead to the CAD phenotype with differing levels of severity.
AIM 2: Determine which genes, are differentially expressed in the Coronary Artery that lead to CAD.
Approach: Using RNA-seq on coronary artery tissue from both wild type Danio rRerio and those with CAD, I will be able to determine which genes are upregulated and down regulated in the coronary artery that are involved in endothelial cell proliferation including ARNT. Once I discover these genes, I will use CRISPR/Cas9 to develop Danio rRerio strains that are mutated for one of these genes to see if the gene is directly involved in CAD. Rationale: From this information, I will discover what genes are involved in the development of CAD which will verify ARNT’s role and will lead to new genes of interest. I hypothesize: that there will be multiple genes that contribute to the CAD phenotype, including ARNT and including genes that code for associated proteins found in Aim 3.
AIM 3: Identify proteins that interact with ARNT that assist with endothelial proliferation and CAD.
Approach: In order to identify proteins that interact with ARNT during endothelial cell proliferation, I will used TAP tagging method. I will TAP-tag both wild type and ARNT mutant Danio rRerio in order to identify proteins associated with the ARNT protein. I will then use CRISPR/Cas9 to create strains of Danio rRerio that are mutant for each of the proteins and ARNT and monitor the effect on development of CAD. Rationale: By Identifying interacting proteins we can identify other potential targets for gene therapy for preventing onset of CAD. I hypothesize: there will be multiple proteins that ARNT interacts with which will give us better insight to its function and potential ways to treat CAD.
[i] Fryar, C. D., Chen, T.-C., & Li, X. (2012, August). Prevalence of uncontrolled risk factors for cardiovascular disease: United States, 1999-2010. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/23101933
[ii] Benjamin, E. J., Muntner, P., Alonso, A., Bittencourt, M. S., Callaway, C. W., Carson, A. P., … American Heart Association Council on Epidemiology and Prevention Statistics Committee and Stroke Statistics Subcommittee. (2019, March 5). Heart Disease and Stroke Statistics-2019 Update: A Report From the American Heart Association. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/30700139
[iii] Deloukas, P., Kanoni, S., Willenborg, C. et al. Large-scale association analysis identifies new risk loci for coronary artery disease. Nat Genet 45, 25–33 (2013). https://doi.org/10.1038/ng.2480
[iv] Scheel, J., Hussong, R., Schrenk, D., & Schmitz, H.-J. (n.d.). Variability of the human aryl hydrocarbon receptor nuclear translocator (ARNT) gene. Retrieved from https://www.nature.com/articles/jhg200235
[v] Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D., & Semenza, G. L. (1996, September). Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC231459/
We will test the hypothesis that a mutation in the ARNT gene in humans increases specificity of its regulatory properties, leading to an increase in endothelial cell proliferation. We are led to this hypothesis by the identification of ARNT as a risk locus for CAD and by its known function as a transcriptional regulator.
The model organism we will be using is Danio rerio due the visibility of this organism’s heart, the organ effected by CAD.
The primary goal of this research is to identify genes that play a causative rolel in the development of coronary artery disease. The long-term goal of this project is to develop new gene therapies that can prevent the onset of CAD in those with ARNT mutations. The objective of this proposal is to determine the relationship between mutations at the ARNT locus and CAD through three specific aims:
AIM 1: Identify the different mutations in ARNT that lead to the CAD phenotype
Approach: We would first conduct sequencing on Danio rRerio which exhibit the CAD phenotype using Sanger Sequencing for the ARNT locus. After identifying all mutations in ARNT in these individuals I will use CRISPR/Cas9 to develop a strain of Danio rRerio for each of these mutations and determine if the specific mutation is causative or silent. By making these observations I can then create a comprehensive list of SNPs and other mutations in ARNT that lead to CAD. Rationale: By identifying all mutations in ARNT that lead to the CAD phenotype, it allows for a comprehensive gene therapy to be developed. I hypothesize: there will be a variety of mutations in ARNT that lead to the CAD phenotype with differing levels of severity.
AIM 2: Determine which genes, are differentially expressed in the Coronary Artery that lead to CAD.
Approach: Using RNA-seq on coronary artery tissue from both wild type Danio rRerio and those with CAD, I will be able to determine which genes are upregulated and down regulated in the coronary artery that are involved in endothelial cell proliferation including ARNT. Once I discover these genes, I will use CRISPR/Cas9 to develop Danio rRerio strains that are mutated for one of these genes to see if the gene is directly involved in CAD. Rationale: From this information, I will discover what genes are involved in the development of CAD which will verify ARNT’s role and will lead to new genes of interest. I hypothesize: that there will be multiple genes that contribute to the CAD phenotype, including ARNT and including genes that code for associated proteins found in Aim 3.
AIM 3: Identify proteins that interact with ARNT that assist with endothelial proliferation and CAD.
Approach: In order to identify proteins that interact with ARNT during endothelial cell proliferation, I will used TAP tagging method. I will TAP-tag both wild type and ARNT mutant Danio rRerio in order to identify proteins associated with the ARNT protein. I will then use CRISPR/Cas9 to create strains of Danio rRerio that are mutant for each of the proteins and ARNT and monitor the effect on development of CAD. Rationale: By Identifying interacting proteins we can identify other potential targets for gene therapy for preventing onset of CAD. I hypothesize: there will be multiple proteins that ARNT interacts with which will give us better insight to its function and potential ways to treat CAD.
[i] Fryar, C. D., Chen, T.-C., & Li, X. (2012, August). Prevalence of uncontrolled risk factors for cardiovascular disease: United States, 1999-2010. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/23101933
[ii] Benjamin, E. J., Muntner, P., Alonso, A., Bittencourt, M. S., Callaway, C. W., Carson, A. P., … American Heart Association Council on Epidemiology and Prevention Statistics Committee and Stroke Statistics Subcommittee. (2019, March 5). Heart Disease and Stroke Statistics-2019 Update: A Report From the American Heart Association. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/30700139
[iii] Deloukas, P., Kanoni, S., Willenborg, C. et al. Large-scale association analysis identifies new risk loci for coronary artery disease. Nat Genet 45, 25–33 (2013). https://doi.org/10.1038/ng.2480
[iv] Scheel, J., Hussong, R., Schrenk, D., & Schmitz, H.-J. (n.d.). Variability of the human aryl hydrocarbon receptor nuclear translocator (ARNT) gene. Retrieved from https://www.nature.com/articles/jhg200235
[v] Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D., & Semenza, G. L. (1996, September). Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC231459/