About the impact of altered RAS-MAPK and PI3K-AKT signalling in human developmental disorders 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., Sangamitra Boppudi geb.am 16.Juli 1986 Visakhapatnam, Indien Gutachter: Prof. Dr. med. Martin Zenker Prof. Dr. rer. nat. Frank Kaiser eingereichte am: 21-12-2017 verteidigt am: 25-09-2018 Table of Contents Table of Contents ........................................................................................................................ I List of Figures ........................................................................................................................... III List of Tables ............................................................................................................................. V Zusammenfassung .................................................................................................................... VI 1. Abstract ................................................................................................................................... 1 2. Introduction ............................................................................................................................ 2 2.1 RAS signalling pathway ................................................................................................... 2 2.2 PI3K/AKT/mTOR signalling pathway ............................................................................. 4 2.3 Intellectual disability ........................................................................................................ 5 2.3.1 Genetics of intellectual disability .............................................................................. 5 2.4 RAS signalling pathway and Intellectual disability ......................................................... 7 2.4.1 RASopathies .............................................................................................................. 7 2.4.2 RAS signalling pathway in the nervous system ...................................................... 10 2.5 PI3K/AKT/mTOR signalling pathway in the nervous system ....................................... 12 2.6 Mosaic disorders ............................................................................................................. 13 2.6.1 RAS pathway and mosaicism .................................................................................. 14 2.6.2 PIK3CA-related overgrowth spectrum (PROS) ...................................................... 15 2.7 Next generation sequencing technology ......................................................................... 17 2.7.1 Evolution of NGS .................................................................................................... 18 2.7.2 Applications of NGS ............................................................................................... 21 3. Objectives ............................................................................................................................. 23 3.1 Multigene panel sequencing in patients with ID and Short stature ................................ 23 3.2 Mosaic disorders ............................................................................................................. 25 4. Materials and Methods ......................................................................................................... 26 4.1 Study subjects ................................................................................................................. 26 4.2 DNA isolation and quantification ................................................................................... 29 4.3 454 GS junior sequencing............................................................................................... 31 4.4 Sanger sequencing .......................................................................................................... 33 4.5 Fragment analysis ........................................................................................................... 34 4.6 Target selection: Custom-designed gene panel .............................................................. 34 4.7 Target enrichment sequencing ........................................................................................ 40 4.7.1 Probe design for selected genes ............................................................................... 40 4.7.2 Nextera® library preparation and quantification ..................................................... 40 4.7.3 Cluster generation/sequencing ................................................................................. 45 4.7.4 Data analysis and review ......................................................................................... 47 4.8 Variant detection and filtering ........................................................................................ 49 4.9 Statistical analysis .......................................................................................................... 52 5. Results .................................................................................................................................. 54 5.1 Mosaic disorders ............................................................................................................. 54 5.1.1 PIK3CA mutation spectrum of patients with PROS ............................................... 54 5.1.1. (A) Confirmation of the causal variants by NGS ................................................... 55 5.1.1. (B) Fragment Analysis ........................................................................................... 61 5.1.2 Mosaic KRAS mutations in OES/ECCL patients ................................................... 64 5.2 Multigene panel sequencing in patients with ID and Short stature ................................ 66 5.2.1 Phenotyping of the study cohorts ............................................................................ 66 5.2.2 Classification of custom-designed gene panel ........................................................ 66 5.2.3 Evaluation of the gDNA quality and quantity ......................................................... 68 5.2.4 Library preparation and quantification .................................................................... 70 5.2.5 Data analysis ............................................................................................................ 71 5.2.6 Run statistics I – Overview of the quality of runs ................................................... 71 I 5.2.7 Performance of modified protocol ........................................................................... 76 5.2.8 Run statistics II – Variant identification and classification ..................................... 79 5.2.9 Monogenic disorders - Mutations identified in known ID genes ............................ 81 Patient number- ID-001 ................................................................................................ 82 Patient number- ID-002 ................................................................................................ 82 5.2.10 Unclassified novel/rare variants identified in known dominant ID genes............. 83 5.2.11 Unclassified novel/rare variants identified in known autosomal recessive/ X- linked ID genes ................................................................................................................. 86 5.2.12 Unclassified novel/rare variants identified in genes that only have association findings with ID ................................................................................................................ 86 5.2.13 Potentially disease-causing variants in genes not previously linked to ID ........... 89 5.2.14 Case studies: Patients with Multiple Variants ....................................................... 90 Case Study 01 (CS01): ID-026 ..................................................................................... 90 Case Study 02 (CS02): ID-023 ..................................................................................... 92 Case Study 03 (CS03): ID-038 ..................................................................................... 93 Case Study 04 (CS04): ID-005 ..................................................................................... 94 Case Study 05 (CS05): ID-035 ..................................................................................... 96 6. Discussion ............................................................................................................................. 98 6.1 Next generation sequencing technology ......................................................................... 99 6.2 Mosaic disorders ........................................................................................................... 101 6.2.1 Mosaic KRAS mutations in OES/ECCL patients ................................................. 101 6.2.2 PIK3CA-related overgrowth syndrome (PROS) ................................................... 104 6.3 Multigene panel sequencing in patients with ID and Short stature .............................. 110 6.3.1 Target selection: Custom-designed gene panel ..................................................... 111 6.3.2 Sample quality and quantity checks ...................................................................... 112 6.3.3 Performance of modified Nextera® Rapid Capture