The RASopathies Molecular Genetics and Genotype-Phenotype Correlations Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) genehmigt durch die Fakultät für Naturwissenschaften der Otto-von-Guericke-Universität Magdeburg von M. Sc. Christina Antonia Lißewski geboren am 05 April 1983 in Wilhelmshaven Gutachter: Prof. Dr. med Martin Zenker Prof. Dr. rer nat. Andreas Winterpacht eingereicht am: 22. Oktober 2017 verteidigt am: 28. März 2018 TABLE OF CONTENTS ABSTRACT ..................................................................................................................................................... V ZUSAMMENFASSUNG ................................................................................................................................. VI 1. INTRODUCTION ................................................................................................................................... 1 1.1. The RASopathies ......................................................................................................................... 1 1.2. The RAS/MAPK pathway ......................................................................................................... 3 1.3. Molecular causes of RASopathies ............................................................................................. 4 1.4. Clinical diagnosis criteria and clinical signs of RASopathies ................................................. 9 1.5. Molecular diagnosis of RASopathies ...................................................................................... 13 1.6. Aims ............................................................................................................................................. 14 2. METHODS ........................................................................................................................................... 15 2.1. Genotyping ................................................................................................................................. 15 2.1.1. Patient samples and ethical issues ........................................................................................... 15 2.1.2. DNA Preparation ...................................................................................................................... 15 2.1.3. Sanger sequencing ...................................................................................................................... 16 2.1.4. Verification of parentage .......................................................................................................... 20 2.1.5. High Resolution Melting (HRM) analysis .............................................................................. 20 2.1.5.1. ESTABLISHMENT OF EXPERIMENTAL METHOD ............................................................... 22 2.1.5.2. VALIDATION OF THE METHOD .......................................................................................... 23 2.1.5.3. ANALYSIS METHOD .............................................................................................................. 23 2.1.5.4. STANDARD PROTOCOL ........................................................................................................ 26 2.1.6. Next Generation Sequencing ................................................................................................... 27 2.1.6.1. SELECTION PROCESS ............................................................................................................ 27 2.1.6.2. NEXTERA RAPID CAPTURE ENRICHMENT ......................................................................... 28 2.1.6.3. SEQUENCING BY SYNTHESIS ............................................................................................... 28 2.1.6.4. SEQUENCE ANALYSIS ........................................................................................................... 28 2.1.6.5. EXPERIMENTAL PROCEDURE ............................................................................................. 29 2.1.6.6. VALIDATION OF VARIANTS ................................................................................................. 31 2.1.7. Classification of variants ........................................................................................................... 31 2.2. Database ...................................................................................................................................... 32 2.2.1. Consensus process ..................................................................................................................... 33 2.2.2. Structure ...................................................................................................................................... 34 2.2.2.1. DATA SUBMISSION ............................................................................................................... 35 2.2.2.2. DATA BROWSER .................................................................................................................... 37 2.2.3. Ethical issues .............................................................................................................................. 38 2.2.4. Data entry and analysis ............................................................................................................. 38 2.3. Statistics ....................................................................................................................................... 50 3. RESULTS .............................................................................................................................................. 51 3.1. Genotyping ................................................................................................................................. 51 3.1.1. HRM ............................................................................................................................................ 51 3.1.1.1. VALIDATION OF THE METHOD .......................................................................................... 51 3.1.1.2. HRM RESULTS AND FALSE-POSITIVES .............................................................................. 55 3.1.1.3. SEQUENCE-SPECIFICITY OF HRM ..................................................................................... 58 3.1.1.4. MUTATION DISTRIBUTION ................................................................................................. 60 3.1.2. Sanger sequencing ...................................................................................................................... 61 3.1.3. Next Generation Sequencing ................................................................................................... 62 3.1.4. Novel or rarely described variants ........................................................................................... 67 3.2. Database ...................................................................................................................................... 70 3.2.1. SHOC2 ........................................................................................................................................ 79 3.2.2. RIT1 ............................................................................................................................................. 81 3.2.3. NRAS .......................................................................................................................................... 83 3.3. CBL .............................................................................................................................................. 89 3.4. HRAS .......................................................................................................................................... 91 3.5. Copy number changes containing RAS-MAPK genes ......................................................... 92 3.6. Novel genes for RASopathies .................................................................................................. 94 4. DISCUSSION ........................................................................................................................................ 98 4.1. Detection and prevalence of mutations ................................................................................. 98 4.2. The NSEuroNet database and its uses ................................................................................. 101 4.2.1. SHOC2 mutations and Mazzanti syndrome ........................................................................ 103 4.2.2. Genotype phenotype correlations with germline RIT1 mutations................................... 106 4.2.3. Genotype phenotype correlations with germline NRAS mutations ................................ 112 4.3. Prenatal symptoms in CBL syndrome .................................................................................. 117 4.4. Copy number changes containing RAS-MAPK genes ....................................................... 120 4.5. Novel RASopathy genes ......................................................................................................... 123 4.6. Conclusion and outlook .......................................................................................................... 132 5. REFERENCES .................................................................................................................................... 134 5.1. Online Resources ....................................................................................................................