Identifying Genes Related to Anxiety Spurthi Jayadeva, International Academy East, Troy, MI, 48083 & BioScience Project, Wakefield, MA 01880 Introduction • Occasional anxiety is normal and people experience it before they are in stressful situations such as interviews, tests, public speaking, etc. • Anxiety disorders, however, extend further than occasional anxiety. The stress does not go away and it can get worse over time. There are types of anxiety disorders such as generalized anxiety disorder, panic disorder, and social anxiety disorder. • People with generalized anxiety disorder worry for months and face many symptoms that make it harder to continue everyday activities. • People with panic disorder experience several panic attacks where they go through sudden periods of intense fear. • Social anxiety disorder causes people to have a fear of social or performance situations where they have to be around other people. (Source: https://www.nimh.nih.gov/health/topics/anxiety-disorders/index.shtml ) • Studies have shown that there is heightened amygdala activation in response to stress (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3055419/) .. In this study gene expression patterns in the amaygdala were evaluated to identify genes related to anxiety. Methods • The Allen Brain Atlas (http://www.brain-map.org/ ) was used to find the gene expression data. The Differential Search was used in the Allen Human Brain Atlas to find the expression of particular genes in a particular area of the brain, or target structure (Amygdala), versus the other areas known as contrast structures (gray matter). The search showed a heat map and data was collected from four of the donors (H0351.2001, H0351.2002, H0351.1009, and H0351.1012). • Interactive Histogram (http://www.shodor.org/interactivate/activities/Histogram/ ) was used to find the statistics for the fold-change values found in the Allen Brain Atlas for the four donors. • Venny 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/ ) was used to find common genes that are shared by all four of the donors. • DAVID (https://david.ncifcrf.gov/ ) is a functional annotation tool that helped give a biological meaning behind a large list of genes. This was used for cluster analysis. Genes related to anxiety were found through keywords such as stress and heart. • The STRING database (https://string-db.org/ ) was used to find genes that are associated with the genes that were found in the DAVID database. Results 1. Gene Expression Profiling Donor A (H0351.2001) Donor B (H0351.2002) Donor C (H0351.1009) Donor D (H0351.1012) The heat maps show the gene expression data for the four chosen donors. The genes are sorted from the highest to the lowest fold-change. The bar at the top represents the donor and the bar under it represents different parts of the amygdala. Each row of the heat map represents a probe. Each column represents a tissue or anatomical brain structure. Red represents relatively high expression and green represents relatively low expression in the heat map. The top two hundred genes were then downloaded and analyzed. The gene symbol, p-value, and fold-change were used for the following steps in the project. 2. Common Genes After creating a Venn Diagram using the top twenty highest expressing genes for each donor, the results showed that there are thirty three common genes that are shared by all of the donors. This means that 16.8% of the data was common among all of the donors. Common Genes DCSTAMP MYB PDYN KCTD4 UTS2D NPY PTGER3 MOXD1 GDA ZNF831 FOXG1 LOC392145 GABRQ TRHR HTR2C CARTPT CD36 ZCCHC12 CXXC11 DIRAS3 MYH6 ARHGAP6 BCL11B C6orf118 TFAP2D ANO3 NR2F2 GLRA3 PRSS35 PCDH11Y GABRA2 TNFAIP8L3 KCNG3 3. Top 20 Highest Expressing Genes Donor A (H0351.2001) Donor B (H0351.2002) Donor C (H0351.1009) The charts show graphs for the top twenty highest expressing genes for each donor. The blue graphs show a relationship between the fold-change and the gene symbol while the red graphs show a relationship between the p-value and the gene symbol. 4. Data Distribution In the histograms, the x-axis shows the fold-change values and the y-axis shows the frequency of the fold-change values. The histograms for all four donors are skewed right because most of the genes have lower fold-change values. The average fold- change ranges between seven and twelve. 5. Genes of Interest The common genes between all of the donors were put into the DAVID database. No genes of interest for anxiety in humans were found. Genes from the common gene list related to stress were found in a study with mice (www.geneweaver.org). Key word search for terms related to anxiety such as “stress” and “addiction” led to the genes of interest. HTR2C MYH6 CARTPT CD36 PDYN The genes of interest were put into the STRING database to identify interacting partners . Networks were created based on experimentally validated interactions. The related genes in the network from the STRING database were plugged back into the DAVID database to see if any of the genes were directly elated to anxiety. Each gene of interest’s list was plugged in separately. Although non of the lists directly related to anxiety, MYH6’s list showed many genes of interest for the keyword “stress.” This was then narrowed down to one gene that related the most to stress. This was protein phosphatase 3 catalytic subunit alpha or PPP3CA. It has “cardiac muscle hypertrophy in response to stress”, “multicellular organismal response to stress”, and “positive regulation of cardiac muscle hypertrophy in response to stress.” It also has “cellular response to drug” which is helpful because drugs increase the risk of anxiety. This gene of interest related the most to anxiety and stress. Conclusion • Anxiety is a disorder that is becoming more and more common every single day. There is still a lot of research that needs to be done because there are many factors that contribute to it. • The amygdala is a region of the brain that is affected by anxiety and stress. There are other regions that may be researched to find more information about it. • Six genes of interest were identified and then expanded using the DAVID and STRING databases. Since there are many other factors that related and contribute to stress and anxiety, there are most likely many more genes of interest. • PPP3CA was found to be related to stress and it should be further researched to see if it has a role in everyday stress or if it has a larger role in contributing to anxiety. This is a good candidate for another study. • Other genes that were identified throughout the project could also be researched further in order to determine if they contribute to stress or anxiety.