Introduction

Integrative Genomics Analysis of PTSD In the AmygdalaShyam Soundararajan, Dubai Gem Private School, Dubai, United Arab Emirates & BioScience Project, Wakefield, MA, US. 01880

● Posttraumatic stress disorder (PTSD) is a psychiatric disorder that can occur in people who have experienced or witnessed a traumatic event.

● Symptoms of PTSD include: 1. Intrusive thoughts; distressing dreams; or flashbacks 2. Avoiding people, places, activities, objects and situations that bring on distressing memories 3. Negative thoughts and distorted beliefs about others 4. Reactive symptoms such as being irritable and having angry outbursts 5. Having problems concentrating or sleeping.

● PTSD often occurs with other related conditions, such as depression, substance use, memory problems and other physical and mental health problems.

● Of an estimated 1.45 billion people having experienced war between 1989 and 2015, an average of 354 million have suffered PTSD during that lifespan.

● Many hormones are involved in PTSD. Hormones having a direct impact on PTSD include epinephrine (adrenaline), norepinephrine, serotonin and corticotropin.

● Certain structures of the brain are closely related to some of the symptoms of PTSD. These structures include the amygdala, hippocampus and several parts of the prefrontal cortex such as the mid-anterior cingulate cortex and the right inferior frontal gyrus.

● Both the amygdala and the mid-anterior cingulate cortex become over-stimulated when a person suffers from PTSD. However, the hippocampus and right inferior frontal gyrus becomes hypoactive when a person suffers from PTSD.

● For this project, we observed and examined gene expression patterns in the amygdala in order to identify candidate genes for PTSD. The amygdala is part of the limbic system which is responsible for emotions, memory and the processing of fear. Recent studies have found that PTSD leads to increased brain matter volume of the amygdala

Methods● The Human Brain Atlas from the Allen Brain Atlas (www.brain-map.org) is a database which was used

as the primary source of gene expression data for the amygdala. The Human Brain Atlas was used to find the heatmaps of the brains that were donated to the Allen Institute for Brain Science which shows the gene expression data required. A differential search was conducted which compared the amygdala (Amg) compared to grey matter (GM) for the donors. The data was collected from four individual donors using the differential search option: H0351.2001, H0351.1016, H0351.1009, H0351.1015.

● Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/) was used to find the common genes from the 4 gene lists of the individual donors. A Venn diagram was created for visual depiction of the data.

● PythonAnywhere (https://www.pythonanywhere.com/) is an online integrated development environment (IDE) based on Python. It was used as a tool for statistical analysis by calculating variance of the fold change values and common statistical values for the gene lists of the individual donors.

● DAVID (https://david.ncifcrf.gov/) is an online bioinformatics resource used to do Cluster Analysis and Gene Ontology Classifications. This was used to find the genes involved in functions associated with PTSD.

● The Gene Ontology Knowledgebase (http://geneontology.org/) is a resource used to delve further on the function of genes. The common genes obtained from the Venny 2.1 results were used to form three Panther Pie Charts covering the three domains of gene ontology: cellular component, molecular function and biological process.

● The STRING Database (https://string-db.org/) was used to create protein interaction networks based on interactions from genetic experimental data to find candidate genes for PTSD. It was also used to identify other genes of interest.

Results1. Gene Expression Profiling

The heat map is a visualization of the microarray values for the gene expression profiles of the four donors mentioned. Each column of the heat map represents a tissue sample. Heatmap color represents the z-score over a probe ranging from green (z-score of -3 and below) through black (z-score of 0) to red (z-score of +3 and above).

The Gene count varied among the donors. Donor H0351.1009 had 292 genes with a fold change greater than 3 while Donor H0351.1015, H0351.1016, H0351.2001 had 275, 286 and 300 genes respectively.

Above each heat map are two bars. The top bar indicates which donor has been selected. The bar below indicates the various brain regions denoted in the heatmap. The genes which were used for statistical analysis had a fold change value greater than 3.

2. Correlating Gene Expression Patterns Among Donors

The venn diagram shows the common genes expressed in the amygdala between the four individual donors.

42 genes (10.5%) were common amongst the four donors.

3. Top 20 Genes with the Highest Fold Change Values

The graphs show the top 20 genes with the highest fold change values from the four individual donors. The x-axis displays the name of the gene while the y-axis displays the fold change. From the four graphs, only one gene (KCTD4) appeared in all lists. Some genes which appeared in 3 graphs include PDYN, MYB, PTGER3, UTS2D and DCSTAMP.

4. Statistical Analysis

The mean fold change ranges from 3.9 to 6.8. The donor with the lowest mean fold change tends to have a lower maximum fold change when compared to the maximum fold change of the other donors. The minimum fold change amongst the donors are relatively similar between 2.9 to 4.4. Variance differs from donor to donor; it extends from a range of 1.3 to 8.5. Standard deviation is very similar to each other all between 2.2 to 2.9 except for Donor H0351.2001 whose genes have a standard deviation of 1.1

5. Finding Genes of Interest through DAVID

In this analysis, all the common genes from the four individual donors were used for determining possible candidate genes for PTSD. The Functional Annotation Table was used to find genes that were involved in functions related to PTSD through the help of a keyword search. Using Gene Ontology classification, certain genes were found which were repeatedly associated with the relevant brain processes. These genes include HTR2C, CCK, CARTPT, NPY, NR2F2, PTGER3, TRPC4, CD36 and TG. These genes of interest tend to be associated with stress hormones such as epinephrine and norepinephrine.They are also related to neurotransmitters, feelings of fear. In some cases, the genes of interest lead to negative regulation on appetite.

7. Protein Interaction Networks in STRING

Protein interaction networks were constructed based on experimental evidence in STRING. STRING shows protein-protein interactions and is useful for identifying genes of interest interacting with other potential candidates. This may help find more candidate genes for PTSD. The interactions of the candidate genes with other genes of potential interest led to some direct and indirect correlations to symptoms and behavior of patients with PTSD.

One gene with a direct relation to both PTSD and a candidate gene is CCKBR. It is said to occur throughout the nervous system where it modulates anxiety, neuroleptic activity and arousal. Another gene with a direct relation is ITCH which controls inflammatory signaling pathways. One gene with an murky correlation is NR2F6 which is said to modulate hormonal responses. However, it is not clear which hormones it modulates.

In this analysis, fold-change-1 represents Donor H0351.1009 while fold-change-2, fold-change-3 and fold-change-4 represent Donor H0351.1015, Donor H0351.1016 and Donor H0351.2001 respectively.

6. Gene Ontology Panther Pie Charts

The second most common molecular function performed by these genes is ‘catalytic activity’.

Cellular Component refers to cellular structures where genes perform a certain function. Most of the genes fall in the category ‘cell’ where they have a function in generalized cell parts. 12 genes (29.3%) were associated with ‘cell’. Most of the genes in ‘cell’ have some relation to PTSD which was detected through DAVID.

Biological Processes refers to the larger processes accomplished by multiple molecular activities. In BP, the most common category is ‘cellular process’. 16 genes (39%) were associated with cellular process In this category, ‘signal transduction’ and ‘cellular response to stimulus’ dominate. These functions have a connection to what PTSD does.

In clockwise order from top left: H0351.1009, H0351.1015, H0351.1016, H0351.2001

Conclusion● The purpose of this study was to identify possible candidate genes for PTSD by

elucidating the connection between PTSD and genetics.● A bioinformatics approach was used to analyze gene expression profiles in order to

identify candidate genes that have an effect on PTSD.● A variety of databases such as the Human Brain Atlas, DAVID and STRING were used in

this study. ● The main recurring genes of interest based on a variety of factors such as fold-change,

relevancy to disorder and relation of functions of these genes to the symptoms of PTSD were HTR2C, CCK, CARTPT, PTGER3 and NPY

● These genes tend to have a high fold-change and are common to all four donors.● Of all these genes, only HTR2C has a known correlation with PTSD. The other genes have

no known correlation but they have functions that are similar to symptoms of PTSD. They tend to interact with other genes and pathways that have a correlation to PTSD.

● These genes are often associated to stress hormones such as epinephrine, serotonin and corticotropin. They are also associated to circadian rhythms, fear regulation and even appetite.

● However, like other mental disorders, PTSD can be hard to diagnose; it can also be misdiagnosed as depression or aggression since it is quite similar. This makes it harder to find a connection between PTSD and genomics.

● Overall, the results indicate that HTR2C, CCK, PTGER3, CARTPT and NPY have a strong connection to PTSD. While this has not been proven in studies yet, they should be studied in order to discover their role in PTSD. Along with this, other genes, receptors and pathways such as CCKBR and ITCH should be investigated to find further links to PTSD.

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Disorder?. Front Psychiatry. 2017;8:222. Published 2017 Nov 6.

CCK HTR2C TRPC4

Molecular Function refers to activities performed by gene products at a molecular-level. GO molecular function terms represent activities. In MF, most of the genes belong in the category ‘binding’. 14 genes (34.1%) were associated with binding.