A Molecular Analysis of 22q11.2 Deletion Syndrome A thesis submitted for the Degree of Doctor of Philosophy at the School of Medicine, Cardiff University By Afnan Salah Salaka MRC Centre for Neuropsychiatric Genetics and Genomics Division of Psychological Medicine and Clinical Neuroscience School of Medicine Cardiff University 2017 Dedication My father, who passed away while I was doing this PhD and he could not see this dream come true. This thesis is dedicated to his soul. Daddy you were, are, and will always be around for me! i Acknowledgements First, I would like to thank Almighty Allah for the perseverance that he has bestowed upon me during this research project and indeed throughout my life. I believe that every single effort I’ve made, would not have happened without his mercy and blessing. I wish to express my sincere gratitude to my supervisor, Prof Nigel Williams, for his invaluable guidance, unfailing patience, and encouragement throughout the PhD. I owe my utmost gratitude to Prof Williams for considering me worthy of this opportunity and for constantly motivating me when I felt down and out. A special thank to my supervisory team, Prof Micelle Owen and Prof Marianne van den Bree for providing me with the permission to use the ECHO study samples to achieve this PhD. I would also like to thank the ECHO study team with special thanks to Miss Hayley Moss, Dr Maria Niarchou, and Dr Samuel Chawner for their great help in obtaining the biological samples and the supporting data for the participants. I am also extremely grateful to Dr Kiran Mantripragada and the lab core team in the MRC Centre for Neuropsychiatric Genetics and Genomics for their valuable support during my lab work. I would also thank the bioinformatics team in the department, Dr Antonio Pardinas, Dr Elliott Rees, Dr Leon Hubbard, and Dr Valentina Escott-Price, for their tremendous help in the bioinformatics analyses. Last but not least, I would like to thank the patients who provided the samples and without whose cooperation this project would not have been possible. I would like to thank my sponsor Umm Al-Qura University with special thanks to Dr Ahmed Asshi, Dr Mohammed Baslamah and the members of the Faculty of Applied Medical Sciences for their continues support. I will not forget to thank the Ministry of the Higher Education of Saudi Arabia and the Saudi Cultural Bureau who sponsored me for this great opportunity to learn, experience, and gain qualifications. I would like to express a special word of thanks with love to my friends especially Dr, Nada Almazrooa, Dr Rania Al-Otabi, Dr Amal Al Salamah, Dr Noa Carrera, Dr Isla Humphreys, and Dr Marisa Taylor, for your valuable friendship and your emotional support. You made my stay in Cardiff enjoyable and I hope to treasure your friendship forever. I am most grateful to my mother, Aunty Faizah, my sisters, and brothers for having a constant faith in me and for your continuous support in all of my life’s aspects. Your love and prayers are a big source of support during this PhD. I cannot thank my husband, Homam, enough for supporting me throughout doing my PhD, and for always being there whenever I needed him, and for being patient during my busy years of the PhD. I am greatly thankful to my father, who passed away while I was abroad for doing this PhD. I will always remember your supportive words of encouragement to continue my journey to learn and will always be praying for you. I love you daddy and I know that you are always around for me. ii Declaration This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. Signed ……….………....………………..…………………Date ……………………. Statement 1 This thesis is being submitted in partial fulfillment of the requirements for the degree of PhD. Signed ……….………....………………..…………………Date ……………………. Statement 2 This thesis is the result of my own independent work/investigation, except where otherwise stated, and the thesis has not been edited by a third party beyond what is permitted by Cardiff University’s Policy on the Use of Third Party Editors by Research Degree Students. Other sources are acknowledged by explicit references. The views expressed are my own. Signed ……….………....………………..…………………Date ……………………. Statement 3 I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed ……….………....………………..…………………Date ……………………. Statement 4 PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loans after expiry of a bar on access previously approved by the Academic Standards & Quality Committee. Signed ……….………....………………..…………………Date ……………………. iii Table of Contents Contents Dedication ................................................................................................................................. i Acknowledgements .................................................................................................................. ii Declaration .............................................................................................................................. iii Table of Contents .................................................................................................................... iv Abbreviations .......................................................................................................................... xi Summary ............................................................................................................................... xiv Associated Publications and Presentations ............................................................................ xv Chapter 1: General Introduction .............................................................................................. 1 1.1. 22q11.2 Deletion Syndrome ......................................................................................... 1 1.1.1. Incidence of 22q11.2DS ........................................................................................ 2 1.1.2. Molecular genetic of 22q11.2DS........................................................................... 2 1.1.2.1. Causes of 22q11.2DS ...................................................................................... 2 1.1.2.2. Complexity of 22q11.2 region ........................................................................ 3 1.1.2.2.1. Low copy repeats of chromosome 22q11.2 ............................................. 3 1.1.2.2.2. Non-allelic homologous recombination ................................................... 4 1.1.2.3. Genes within these deletions ........................................................................... 6 1.1.2.4. Testing of 22q11.2 deletions ........................................................................... 7 1.1.2.4.1. Cytogenetic testing ................................................................................... 7 1.1.2.4.2. Molecular testing...................................................................................... 7 1.2. Phenotypic diversity of 22q11.2DS ............................................................................ 10 1.2.1. History of 22q11.2DS ......................................................................................... 10 1.2.2. Symptoms of 22q11.2DS .................................................................................... 12 1.2.2.1. Physical symptoms ........................................................................................ 12 1.2.2.1.1. Cardiovascular abnormalities ................................................................. 13 1.2.2.1.2. Craniofacial abnormalities ..................................................................... 13 1.2.2.1.3. Thymic aplasia/hypoplasia and related immune disorders .................... 15 1.2.2.1.4. Cleft palate and related velopharyngeal abnormalities .......................... 15 1.2.2.1.5. Hypocalcemia/Hypoparathyroidism ...................................................... 16 1.2.2.2. Cognitive and behavioural symptoms ........................................................... 16 1.2.2.2.1. Developmental trajectories of developmental, cognitive, and behavioural phenotypes ............................................................................................................. 17 1.3. Identifying 22q11.2 candidate genes for neuropsychiatric phenotypes ...................... 21 1.3.1. 22q11.2DS mice model ....................................................................................... 23 iv 1.3.1.1. Studies on Df1 mice model ........................................................................... 28 1.3.1.1.1. Behavioural and cognitive phenotypes in Df1 mice models .................. 28 1.3.1.1.2. Gene expression in Df1 mice models ..................................................... 29 1.3.1.2. Studies on a single 22q11.2 gene knocked out mice ..................................... 31 1.3.2. Analysis of genotypes on the haploinsufficient chromosome in 22q11.2DS ...... 33 1.3.2.1. COMT ........................................................................................................... 33 1.3.2.2. PRODH ........................................................................................................