ABSTRACT Hereditary ataxias are complex, rare autosomal recessive diseases that receive limited funding and public attention. While it is known that the most common types of hereditary ataxia are caused by mutations in a single gene, the extent to which molecular pathways, like DNA repair, are changed remains largely unknown. In order to learn more about how the five main DNA repair mechanisms are altered in the disease state and what changes are common across hereditary ataxia, four microarray dataset representing Friedreich’s Ataxia, Ataxia Telangiectasia, and Spinocerebellar Ataxia Type 2 were obtained from NCBI. Using R and three Bioconductor annotation packages, the fold change level of each probe was calculated and mapped to corresponding gene symbols. Discriminative motif finding was performed on promoter regions of genes of interest, which represented possible transcription factor binding sites. In order to understand the protein interactions of each DNA repair pathway, the STRING database tool was employed, and the connections established here were combined with all other results to produce an informative network image for each DNA repair pathway. Our findings showed that DNA repair mechanisms in each form of ataxia shared three similarities, but that each disease had unique differences that may have implications for the differences in disease presentation. In all ataxias investigated in this study, OGG1 and RAD50 are underexpressed, while PMS1 is overexpressed. Furthermore, each ataxia has at least one form of DNA ligase that is underexpressed, which likely hinders the ability to fully fix DNA breakage. These results have the potential to be used by future researchers as targets for therapy or in 1 the development of diagnostic tests. We conclude that there is shared differential expression of key DNA repair genes among hereditary ataxias, and these similarities may help us understand why the presentation of these diseases are so similar. AUTHOR SUMMARY Orphan diseases, including hereditary ataxia, are so rare that they are not often studied. As a result, very little is understood about the underlying changes in the biological mechanism. The rarity of these diseases also complicates the ability for quick, affordable and direct diagnosis of the disease. Many hereditary ataxias have an age of onset during childhood, and the average time to obtain a correct diagnosis is approximately 18 years. My hope for this study was to shed light on the differences in the ataxic and healthy DNA repair pathways, as well as any similarities between ataxias, in order to uncover any informative changes that could be researched further to develop possible therapies or to simplify the diagnosis of these diseases. If nothing else, my research aims to spread awareness and generate public interest in regards to these diseases so that they may gain more funding to promote future research and to provide hope to the families that are stricken with these diseases. This research found that the genes in the disease repair pathways were generally underexpressed compared to wildtype repair pathways, and it was found that there are similarities in the disregulation of certain DNA repair genes across all three forms of ataxia investigated in this study. 2 ACKNOWLEDGEMENTS The motivation for my research of ataxia stems from learning about the local McCollister family that has two children affected by a rare hereditary ataxia called Ataxia Oculomotor Apraxia Type 1 (AOA1). Their story, strength, and hope inspired me to learn more about hereditary ataxia, with a goal of uncovering new information about the disease, as well as spreading awareness of orphan diseases, like AOA1. It was my desire to find similarities and differences between ataxias that could be used by researchers as targets for future studies so that families like the McCollisters will have more options to heal their children and improve their quality of life. I would like to thank the McCollister family for their support and insight, Veronica Liang, Robert Schmidt, and Rami Al-Ouran for their expertise regarding bioinformatic techniques, Lorie LaPierre for her contribution to biological relevance of my findings, and Lonnie Welch for being my thesis mentor, Soichi Tanda for his constant encouragement and guidance throughout the last four years. Without the help out these men and women, this research would not have been possible. 3 TABLE OF CONTENTS Introduction…………………………………………………………………………6 I. Central Dogma……………………………………………………………..6 II. DNA Repair and Disease………………………………………………….6 III. What is Ataxia?…………………………………………………………...8 IV. DNA Repair………………………………………………………………9 V. Hereditary Ataxia………………………………………………………….10 VI. What is Bioinformatics?………………………………………………….16 VII. Ataxia and Bioinformatics………………………………………………18 VIII. Gathering of Data………………………………………………………20 IX. R Techniques……………………………………………………………..21 X. DNA Microarray………………………………………………………….23 XI. Motif Finding…………………………………………………………….25 XII. STRING Database………………………………………………………27 XIII. Methodology Pipeline………………………………………………….32 XIV. Network Map Generation………………………………………………33 Materials and Methods……………………………………………………………...34 Results………………………………………………………………………………..35 Discussion……………………………………………………………………………63 I. Hypothesis One…………………………………………………………….63 II. Hypothesis Two……………………………………………………………64 Conclusions………………………………………………………………………….67 4 Significance of Work………………………………………………………………..68 Future Directions……………………………………………………………………69 Limitations…………………………………………………………………………..70 Bibliography…………………………………………………………………………72 5 INTRODUCTION All walks of life are made from blueprints, which come in the form of genetic material. For humans, this material is deoxyribonucleic acid, commonly known as DNA. If one thinks of DNA as letters, genes would be considered the words made up of these letters. The genome is the story of a person, strung together with the genetic words. However, these stories are not read from left to right and start to finish, as one normally reads. The reading of our individual genetic stories is controlled by genetic regulatory networks, which dictate when, where, and for how long a gene will be turned on. When these networks don’t work correctly, the results can be genetic disease. I. Central Dogma A pivotal concept in molecular biology is the central dogma, which details how genetic information is used within a biological system. This idea was proposed by Francis Crick and states that DNA is used as the template for RNA, which is translated into a series of amino acids that fold to become proteins.[1] When studying mutations in proteins, it is most important to examine this process of protein creation starting from the source, DNA. II. DNA Repair and Disease Mistakes in DNA replication occur frequently, and every person has them in their genome. Mistakes are made at an approximate rate of 1 incorrect nucleotide in every 100,000 nucleotides synthesized. This may seem like a small number, but there are over 6,000,000,000 nucleotides making up the human genome in a single diploid 6 cell, each of which is synthesized when a cell divides. This means that around 120,000 errors are made in the DNA every time a cell replicates.[2] These errors are called mutations if they go unfixed. Most people never develop a disease due to these mutations, but there are mutations that can be highly deleterious and lead to disease. Because the life of the organism can depend on the DNA being replicated and maintained properly, several biological mechanisms exist to fix DNA mistakes. These are known as DNA repair pathways, of which there are five.[3] The first of these is the base excision repair (BER) pathway, which removes a single, non-bulky damaged or incorrect base from single strand DNA that could hinder the structural integrity of the strand during replication. The nucleotide excision repair (NER) pathway fixes single stranded DNA by removing bases damaged by ultraviolet light. UV damage causes bulky lesions, and the NER pathway detects these, removes the damaged bases, and replaces them with the correct ones. The third mechanism to repair single strand DNA is the mismatch repair (MMR) pathway. The MMR pathway is responsible for detecting and fixing incorrect insertions and deletions along the newly synthesized DNA strand. Homologous recombination (HR) is a pathway that fixes errors in double stranded DNA. HR fixes the DNA by using the undamaged sister chromatid to correct the mistakes. Non-homologous end-joining (NHEJ) is a pathway that is designed to ligate the damaged ends of double stranded DNA molecules. These five repair pathways are crucial to maintaining a functioning, healthy organism. What if the genes involved in these critical repair pathways are mutated themselves? There are two outcomes, depending on the time the mutation occurs. If 7 the gene is mutated later in life, it is possible that organism will develop cancer, as there are key oncogenes, like TP53 and BRCA1, in the DNA repair pathways. Should the organism be born with the mutation, they run more risks than just cancer, but additional diseases. When an organism is born with a deleterious inheritable mutation, their ailment is known as a hereditary disease, one that is passed from the parents to their offspring.[4] These genetic diseases are congenital and can be dominant or recessive, meaning a dominant disorder will always present itself if the mutation is present, while a recessive one will only occur if two copies of the mutation exist. Additionally, some diseases will