Development of a functional assay for CHD7, a protein involved in CHARGE syndrome Gara Samara Brajadenta To cite this version: Gara Samara Brajadenta. Development of a functional assay for CHD7, a protein involved in CHARGE syndrome. Human genetics. Université de Poitiers, 2019. English. NNT : 2019POIT1401. tel-02459642 HAL Id: tel-02459642 https://tel.archives-ouvertes.fr/tel-02459642 Submitted on 29 Jan 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THESE Pour l’obtention du Grade de DOCTEUR DE L’UNIVERSITÉ DE POITIERS (Faculté Médecine et Pharmacie) (Diplôme National – Arrêté du 25 mai 2016) École Doctorale : Sciences Biologiques et Santé Secteur de Recherche : Aspects Moléculaires et Cellulaires de la Biologie Présenté par : Gara Samara Brajadenta ****************************** Mise au Point d’un Test Fonctionnel pour la Protéine CHD7 Impliquée dans le Syndrome CHARGE (Development of a Functional Assay for CHD7, a Protein Involved in CHARGE Syndrome) ****************************** Directeur de Thèse : Professeur Alain Kitzis Co-directeur de Thèse : Docteur Vincent Thoreau ****************************** Soutenue le 14 juin 2019 devant la Commission d’Examen ***************************** JURY Professeur Thierry Bergès Université de Poitiers Président Professeur Patrick Vourc’h Université de Tours Rapporteur Docteur Veronique Pingault Université Paris Descartes Rapporteur Professeur Sultana MH Faradz Université Diponegoro Examinateur Professeur Alain Kitzis Université de Poitiers Directeur de thèse Docteur Vincent Thoreau Université de Poitiers Co-Directeur de thèse Preface This thesis is written as partial fulfillment of the requirements to obtain a Ph.D. degree at the Faculty of Medicine and Pharmacy, the University of Poitiers, France. The work was carried out from October 2015 to June 2019 in the laboratory EA-3808 Neurovascular Unit and Cognitive Impairments, University of Poitiers, France under the supervision of Professor Alain Kitzis and Doctor Vincent Thoreau. The work was funded by University of Poitiers and was also supported by Indonesia Endowment Fund for Education (LPDP), Ministry of Finance of the Republic of Indonesia. Gara Samara Brajadenta Poitiers, June 2019 ACKNOWLEDGMENTS Many people have contributed their skills into this research; this work would have been impossible without their help and assistance. I would like to express my sincere gratitude to my supervisor, Prof. Alain Kitzis, MD, PhD, for his guidance, time to teach me and endless encouragement. I am also thankful to my co-supervisor Dr. Vincent Thoreau, for his guidance, knowledge sharing, teaching me basic techniques of the molecular biology, his close supervision in almost all my days in the laboratory, for editing my thesis and for widening my research from various perspectives. I would like to thank all staff of laboratory of genetics and EA-3808 Neurovascular Unit and Cognitive Impairments, Faculty of Medicine and Pharmacy, the University of Poitiers for their cooperation and friendship they have shared over the years. Particularly, I would like to thank Dr. Frédéric Bilan, Dr. Sylvie Patri, and Dr. Montserrat Rodriguez-Ballesteros for their scientific support during my research, especially for teaching me how to interpret the molecular analysis. My sincere gratitude goes to Prof. Patrick Vourc’h from the University of Tours and Dr. Veronique Pingault from the University of Paris Descartes for accepting to evaluate my PhD report. I would like to thank Prof. Sultana MH Faradz, MD, PhD from the Diponegoro University, Semarang, Indonesia and Prof. Thierry Bergès from the University of Poitiers, for honoring me by their presence in the jury of my thesis. My grateful thank also goes to Prof. Guylène Page, who provided me an opportunity to join EA-3808 team, and who gave me access to the laboratory and research facilities. Extraordinary gratitude goes to all down at Indonesian Endowment Fund for Education (LPDP) Ministry of Finance, the Republic of Indonesia for helping and providing the funding for the study and the research. I would like to express my special thanks to Prof. Gérard Mauco, MD, PhD and Prof. Sultana MH Faradz, MD, PhD, who have given me recommendations to continue my PhD at the University of Poitiers. Last but not the least, I would like to thank Dr. Affandi (former dean), Dr. Catur Setiya Sulistiyana, the dean of Faculty of Medicine, and Dr. Mukarto Siswoyo, the rector of Swadaya Gunung Jati University, Cirebon, Indonesia for giving me the golden opportunity to do this excellent PhD thesis in France. TABLE OF CONTENTS Abstract …............................................................................................. i Résumé ……………………………………………………………………... ii List of Abbreviations …………............................................................... iii List of Figures ………………………………………………………………. v List of Tables …………………………………….…………………………. viii List of Publication ................................................................................. ix Synopsis ............................................................................................... x CHAPTER I. INTRODUCTION 1.1 General Introduction ……...……………………………………………. 2 1.2 Background ……………………...……………………………………… 2 1.2.1 Molecular Diagnosis of CHARGE Syndrome ..…………..….….. 2 1.2.2 Challenges in the Development of Functional Assay of CHD7 .. 4 1.3 Objectives ………………………. ……………………………………… 5 CHAPTER II. LITERATUR REVIEW 2.1 Overview of CHARGE Syndrome ……………........…………………. 8 2.1.1 CHARGE: From Association to Syndrome ..…………..….…….. 8 2.1.2 Prevalence and Demographics ........................…………………. 10 2.1.3 Inheritance Pattern ....................................……………………… 10 2.1.4 Variability in CHARGE Clinical Features .……………………….. 11 2.1.5 Clinical Criteria Diagnosis of CHARGE Syndrome .................... 18 2.2 Cloning of CHD7 Gene and Mutations in the CHD7 Gene ………... 20 2.3 Updated Diagnosis Criteria for CHARGE Syndrome ...................… 23 2.4 Genetic Causes of CHARGE Syndrome ...............…………………. 25 2.5 CHD7 Protein and Its Function .............................…………………. 27 2.6 Pathomechanism of CHARGE Syndrome ...................................... 33 2.7 Novel Classification System to Predict the Pathogenicity of CHD7 Missense Variants and Prospective .............................................. 36 CHAPTER III. MATERIALS, PATIENTS, AND METHODS 3.1 Molecular Diagnosis of CHARGE Syndrome .............………….…. 41 3.1.1 Chromosome Analysis ............................................................. 41 3.1.2 DNA Extraction ............................................………….………… 42 3.1.3 Targeted NGS Gene Panel ............................…......…………… 43 3.1.4 Mutation Confirmation by Sequencing Analysis ......…………… 44 3.2 Development of a Functional Assay of CHD7 Variants …............... 45 3.2.1 Patients and Bioinformatic Prediction Tools ............................ 45 3.2.2 Plasmids .....................................................……......…………… 49 3.2.3 Site-Directed Mutagenesis ......................................…………… 49 3.2.4 Confirmation of Mutated Variants by Sequencing Analysis ...… 52 3.2.5 Cell Culture and Transfection ..................................…………… 53 3.2.6 Cell Lysis and Protein Assay ...................................…………… 54 3.2.7 Western blot Analysis ............................................................... 55 3.2.8 Immunofluorescence .................................……......…………… 56 3.2.9 Antibodies .................................……......……………................. 57 3.2.10 RNA Extraction and cDNA Synthesis ..................................… 58 3.2.11 Quantitative RT-PCR .............................................................. 59 3.2.11.1 General Protocol ................................................................ 59 3.2.11.2 Choice of Reporter Genes ................................................. 61 3.2.12 Sensitivity and Specificity Test ............................................… 64 3.3 Genome Modification Technologies ...........................………….…. 65 3.3.1 CRISPR/Cas9 System ............................................................. 66 3.3.2 Stages and Types of the CRISPR/Cas .................................... 67 3.3.3 Application of CRISPR/Cas9 System for Genome Modification in Functional Study .................................................................. 70 3.3.4 DNA Reparation System .......................................................... 72 3.4 Functional Assay of Endogenously Expressed CHD7 Missense Variants using CRISPR/Cas9 System ......................................... 76 3.4.1 Choice of CHD7 Gene Sequence to Target with the CRISPR/Cas9 ......................................................................... 76 3.4.2 Cas9 Nuclease and Single Guide RNA (sgRNA) Constructs ... 76 3.4.3 Repair-Template Design: Single-Stranded DNA Oligonucleotides ssODNs and Double-Stranded Targeting Plasmids ................................................................................. 79 3.4.4 Transfection