Doctoral program in Biomedicine (B11.56.1) Genetic Contribution to Chronic Tinnitus in Patients with Meniere’s Disease and Tinnitus Extreme Phenotype (MD-TEP) Contribución genética al acúfeno crónico en pacientes de enfermedad de Meniere con fenotipo extremo de acúfeno (MD-TEP) International PhD Thesis Tesis Doctoral con Mencion International Sana Amanat Ali May, 2021 Granada Editor: Universidad de Granada. Tesis Doctorales Autor: Sana Amanat Ali ISBN: 978-84-1306-904-3 URI: http://hdl.handle.net/10481/69097 2 Acknowledgements It was not possible to carry out this thesis without the help and collaboration of many people. Thanks to my family especially my parents and sisters who supported my decision of moving abroad to pursue my PhD. Thanks for your constant support, I will never forget. Off course, I can't. This decision seems to have paid off now. Decisions and coincidences that changed my life, Undergraduation in Computer science, Master in Computer science and then Doctorate in Biomedicine as a Bioinformatician. I would like to take this opportunity to thanks Antonio for yout constant support, my supervisor. Thank you for trusting me and giving me the opportunity to be a part of this prestigious group. I was not very clear when I started but you have made sure that it was as straight as possible. Having little or no knowledge of exome sequencing, you taught me very well from basics to advance topics. It is time to thank all the colleagues with whom I have been lucky enough to work in GENyO. I would like to thanks Teresa Requena and Lidia Frejo for your kind support; though we spent very few months together but I will never forget. I am grateful to Álvaro Gallego for your help whenever i have questions and doubts. You helped me a lot to build my understanding about biological interpretation of data. I would like to thanks Pablo for his constant help. I would like to thanks Olga, you have been a great support for me in all administrative tasks related to my contract, even before my arrival. Thanks for churros con chocolate and funny moments we shared. Thank you very much Marisa, for making my stay exciting and cheerful. Thanks for the good and quality time we spent. I still remember few of Portuguese words, thanks for it. Thank you Estrella for the good time we spent, I will remember your name as Star. Alba, thanks for your help whenever I needed, you were always willing to help me. I would like to thanks all Master students for the discussion we had. It is time to say a big thanks to everyone from ESIT. My ESIT colleagues and friends have been a great support for me throughout this journey. Thanks for the amazing trainings, workshops and courses. It was the best time. Thanks to Christopher for organizing my online stay at Karolinska Institute, and arranging amazing activities during this stay. I would like to extend my thanks to Meniere society, UK for hosting my stay. Many thanks to Juanma, Patricia and all clinicians for your participation in this study. Thanks for providing us the samples and clinical data to made this study possible. I would like to thanks Patrick and Joseph for your amazing contribution in this study. I would like to say thanks to all the patients and their families for their support. I hope that together we can find the solution one day. I don't want to forget anyone, Thanks to all those who are in my heart and silently supporting me. Thank to everyone even if I haven't mentioned the name. Thanks to Ammi, Abu, Nida, Farah, Ghazala, Madeha, Farzana, Saeed, Moid, Zamna, Rehana, Mubarak, Ammar and Tayyaba. Thank a lot. 5 6 Contents Funding ................................................................................................................................................................ 9 Resumen ............................................................................................................................................................ 10 Abstract ............................................................................................................................................................. 11 List of abbreviations .......................................................................................................................................... 13 1. Introduction ............................................................................................................................................... 14 1.1. Tinnitus: a complex trait .................................................................................................................... 14 1.2. Inner ear and the peripheral auditory system ..................................................................................... 15 1.2.1. Anatomy of cochlea and its function ......................................................................................... 16 1.2.2. Anatomy of vestibular labyrinth and its function ...................................................................... 17 1.2.3. The auditory central nervous system and tinnitus mechanism .................................................. 18 1.3. Meniere disease ................................................................................................................................. 20 1.3.1. Vertigo ....................................................................................................................................... 20 1.3.2. Sensorineural hearing loss ......................................................................................................... 21 1.3.3. Tinnitus ...................................................................................................................................... 21 1.3.4. Aural fullness ............................................................................................................................. 21 1.4. Extreme phenotype ............................................................................................................................ 21 1.5. Diagnosis and classification .............................................................................................................. 24 1.6. Genetic variants ................................................................................................................................. 25 1.7. Pathogenicity of rare variants ............................................................................................................ 27 1.8. Heritability and genetics contribution to tinnitus .............................................................................. 29 2. Hypothesis ................................................................................................................................................. 31 3. Objectives .................................................................................................................................................. 32 3.1. Main objective ................................................................................................................................... 32 3.2. Specific objectives ............................................................................................................................. 32 4. Methods ..................................................................................................................................................... 33 4.1. Systematic review of extreme phenotype strategies .......................................................................... 33 4.1.1. Search strategy ........................................................................................................................... 33 4.1.2. Research Question and Selection Criteria ................................................................................. 33 4.1.3. Quality assessment of selected studies ...................................................................................... 34 4.1.4. Data Extraction and Synthesis ................................................................................................... 35 4.2. Subjects and definition of phenotype ................................................................................................ 35 4.3. Whole-exome sequencing ................................................................................................................. 36 4.4. Bioinformatics analysis ..................................................................................................................... 36 7 4.5. Gene Ontology and gene-set enrichment analysis ............................................................................. 39 4.6. Gene expression analysis using the Allen Brain Atlas ...................................................................... 39 4.7. Statistical analysis ............................................................................................................................. 40 5. Results ....................................................................................................................................................... 41 5.1. Selection and Characteristics of EP Studies ...................................................................................... 41 5.2. Synthesized Findings of EP Studies .................................................................................................. 42 5.3. Rare missense variants in synaptic genes associated with tinnitus extreme phenotype in MD patients. .........................................................................................................................................................