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Genetic Analysis of Rare Eye Disorders in Pakistani Families

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Genetic Analysis of Rare Eye Disorders in Pakistani Families GENETIC ANALYSIS OF RARE EYE DISORDERS IN PAKISTANI FAMILIES By MUHAMMAD ARIF NADEEM SAQIB Department of Biochemistry Faculty of Biological Sciences Quaid-i-Azam University Islamabad, Pakistan 2015 GENETIC ANALYSIS OF RARE EYE DISORDERS IN PAKISTANI FAMILIES A thesis Submitted in the partial fulfillment of the Requirements for the degree of DOCTOR OF PHILOSOPHY In BIOCHEMISTRY/MOLECULAR BIOLOGY By MUHAMMAD ARIF NADEEM SAQIB Department of Biochemistry Faculty of Biological Sciences Quaid-i-Azam University Islamabad, Pakistan 2015 CERTIFICATE A thesis submitted in the partial fulfillment of the requirements for the degree of the Doctor of Philosophy. We accept this dissertation as conforming to the required standard. 1 _________________ 2________________ Dr. Muhammad Ansar External Examiner (Supervisor) 3 ____________________ Dr. Muhammad Ansar (Chairperson) Dated: February 09, 2015 DECLARATION I hereby declare that the work presented in the following thesis is my own efforts and that the thesis is my own composition. No part of the thesis has been previously presented for any other degree. Muhammad Arif Nadeem Saqib Dedicated To Muhammad Romman Khan (Late) My Son, who died during the journey of my PhD ACKNOWLEDGEMENT All praise to Allah Almighty, the most beneficent, the most merciful, Who gave me strength and enabled me to undertake and execute this research task. Countless salutations upon the Holy Prophet Hazrat Muhammad (Sallallaho Allaihe Waalahe Wassalum), the city of knowledge for enlightening with the essence of faith in Allah and guiding the mankind, the true path of life. I would like to extend my appreciation to those people, who helped me in one way or another to finish the thesis. It is a pleasure to convey my gratitude in my humblest acknowledgment to those people who contributed to make the thesis. I feel highly privileged in taking the opportunity to express my deep sense of gratitude to the Dr. Muhammad Ansar, Chairperson and Associate Professor, Department of Biochemistry, Faculty of Biological Sciences, QAU, Islamabad, for his scholastic guidance and valuable suggestions throughout the study and presentation of this dissertation. I am thankful to him for his inspiration, reassurance, counseling from time to time and specially a friendly environment. I am thankful to Dr. Carlo Rivolta, Assistant Professor, Department of Medical Genetics, University of Lausanne, Lausanne, Switzerland, who provided me opportunity to work in his lab under International Research Support Initiative Program of Higher Education Commission, Pakistan. He remained very kind and boosted my scientific skills. I am obliged to Dr. Bushra Mirza, Ex-Chairperson, Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University (QAU), Islamabad, for extending the research facilities of the department to accomplish this work. I am also obliged to my all teachers who contributed a lot in my studies, especially to Dr. Azra Khanum, Prof of Biochemistry and Sir Sharif Amjad, Science Teacher, Government High School, Ghangwal. Special thanks to Dr. Huma Qureshi, Executive Director, PMRC, Islamabad, Dr. Arif Munir, Principal Research Officer, PMRC, Islamabad and Ibrar Rafique, Research Officer, PMRC, Islamabad who provided a lot of scientific insights and help in learning the research writing. Heartiest and sincerest acknowledgement also goes to Higher Education Commission of Pakistan for granting me Indigenous as well as IRSIP (for foreign research support) scholarship, without which this achievement was not possible. We would like to particularly thank all members of the families that participated in this study for their invaluable participation and cooperation I am also thankful to my lab fellows Dr. Muzamal Ahmad Khan, Falak Sher, Rani Sara, Noureen Aslam, Waleed Ahmed Khan, Naeem Ashraf, Madiha Amin, Neelum Shah, Jamil Iqbal, Sumaira Anjum, Nikhat Siddique, Muhammad Nawaz, Farah Fatima, Rabia, Mehwish, Benish, Jawad Akhtar, Kinza Makhdoom, Sundus Sajid, Khalid Khan, Afeefa Jarral, Noor Han, Zoma Khan, Zainab Akram, Riffat and Memoona Rasheed, who assist me in various ways during my research work Thanks to all the colleagues in Carlo’s lab, especially Konstantinos Nikopoulos, Giulia Venturini, Hanna Koskiniemikundig, Alessandro DI Gioia, Paola Benaglio, Pietro Farinelli, Rosanna Pescini Gobert, Nicola Bedoni, and Beryl Royer-Bertrand for their support and company. Thanks to my friends Ehsan Ullah, Ahmad Waqas, Muhammad Umair, Younus Khan, Haroon Khan, Safdar Abbas, Waqas Kayani for their company, encouragement, concern, laughters stories and for taking their time to listen to everything that I had to say. Special thanks to Dr. Rizwan Alam, Dr. Khalid Khan, Dr. Muhammad Jadoon Khan for their company and time to visit different cities of Europe. It was really pleasant to visit them and see the beauty of Europe. I would like to acknowledge the clerical staff of the Department of Biochemistry especially to Mr. Tariq, Fayaz, Saeed and other staff members for their support and help. I would also like to thank Mr Saeed Ahmad Shahid, Hafiz Saqib, Junaid. Last but not least all goes to my mother (late), who enlightened me and whose prayers still remained in search of my successes. She will remain always alive in my heart. My loving father, Hafiz Abdul Hameed, who remained a guide through my studies and provided me his best, my elder brother, Hafiz Ahmad Tariq, who gave me the strength and courage and provided all opportunities to achieve this task, my younger brother, Hafiz Atif Naveed, who is always a source of joy and provide his best for my leisure times, my all sisters who deserve special mention for their inseparable support and prayers throughout my life. It wouldn’t have been this bearable if I didn’t have these in my life. Thank you for your unconditional support with my studies. Thank you for giving me a chance to prove and improve myself through all walks of my life. Bundle of thanks to my uncle, Abdul Waheed Zafar, and Mian Mehboob ur Rasool (Late), brother in laws (Bhai Ihsan, Bhai Akhtar, Qamar Bhai, Bhai Sohail, Bhai Waqas, Mustansar, Sohaib and Yasir Din) and cousins for providing their continuous support. Special thanks to my beloved wife for her love, devotion and constantly reminding me to take care of myself. Special thanks to Khans (my sons: Muhammad Somaan Khan and Muhammad Affan Khan and my nephews: Muhammad Sinnan Khan and Muhammad Siban Khan) who provides joy by their naughtiest actions. Finally, I would like to thank everybody who was important to the successful completion of the thesis, as well as expressing my apology that I could not mention personally one by one. Muhammad Arif Nadeem Saqib ______________________________________________________________Contents TABLE OF CONTENTS Pages List of Figures ii List of Tables iv List of Abbreviations vi Abstract xi CHAPTER 1 1 1.0 Introduction 2 1.1 Rare Eye Disorders 2 1.2 Inherited Retinal Disorders 4 1.3 Rod Associated Retinal Disorders 4 1.3.1 Congenital Stationary Night Blindness 7 1.3.1.1 Genetics of CSNB 7 1.3.1.1.1 Genes Responsible for Complete CSNB 8 1.3.1.1.2 Genes Responsible for Incomplete CSNB 9 1.3.2 Retinitis Pigmentosa 10 1.3.2.1 Genetics of Retinitis Pigmentosa 11 1.3.2.1.1 RP Genes of Phototransduction Cascade 11 1.3.2.1.2 RP Genes of Splicing and Transcription 12 1.3.2.1.3 Ciliary Gene 13 1.4 Cone Associated Retinal Disorders 14 ____________________________________________________________________ Genetic Analysis of Rare Eye Disorders in Pakistani Families ______________________________________________________________Contents 1.4.1 Achromatopsia 14 1.4.1.1 Genetics of Achromatopsia 15 1.4.2 Cone and Cone Rod Dystrophies 17 1.4.2.1 Genetics of Cone and Cone Rod Dystrophies 17 1.5 Syndromic Retinal Dystrophies 19 1.5.1 Bardet-Biedl Syndrome 19 1.5.2 Usher Syndrome 20 1.5.3 Cohen Syndrome 20 Genetic Analysis of Retinal Dystrophies in Pakistani 1.6 21 Families 1.7 Objectives 24 CHAPTER 2 25 2.0 Materials and Methods 26 2.1 Families Studied 26 2.1.1 Clinical and Ophthalmologic Examination 26 2.2 Blood Sampling 27 2.3 Extraction of Genomic DNA from Human Blood 27 2.3.1 DNA Extraction by Organic Method 27 2.3.1.1 Composition of Solutions 28 2.3.2 DNA Extraction by Commercially Available Kit 28 ____________________________________________________________________ Genetic Analysis of Rare Eye Disorders in Pakistani Families ______________________________________________________________Contents 2.4 Genetic Analysis of Families with Retinal Dystrophies 29 2.4.1 Homozygosity Mapping Using Microsatellite Markers 29 2.4.1.1 Polymerase Chain Reaction (PCR) and Genotyping 30 2.4.1.2 Composition of 8 % Polyacrylamide Gel (50 mL) 30 2.4.2 Homozygosity Mapping using SNPs 31 2.5 DNA Sequencing 32 2.5.1 Amplification of PCR Product 32 2.5.2 Purification of PCR Product 32 2.5.3 Sequencing PCR 33 2.5.4 Purification of Sequencing Reaction 34 2.6 Mutation Analysis 34 2.6.1 Bioinformatics Analysis of Novel Mutations 35 2.6.2 In vitro Analysis of Splice Site Variant 35 2.6.2.1 Amplification of Targeted Exon and its adjacent Regions 35 2.6.2.2 T/A Cloning 36 2.6.2.3 Transformation 36 2.6.2.4 Plasmid Extraction 37 2.6.2.5 Hela Cell Transfection 37 2.6.2.6 RNA Extraction 38 ____________________________________________________________________ Genetic Analysis of Rare Eye Disorders in Pakistani Families ______________________________________________________________Contents 2.6.2.7 cDNA Synthesis and RT-PCR 39 2.7 Deletion Breakpoint Mapping of VPS13B/COH1 39 CHAPTER 3 55 3.0
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