5. Additional Genome Studies
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עבודת גמר )תזה( לתואר Thesis for the degree Doctor of Philosophy דוקטור לפילוסופיה מוגשת למועצה המדעית של Submitted to the Scientific Council of the מכון ויצמן למדע Weizmann Institute of Science רחובות, ישראל Rehovot, Israel מאת By דנית עוז לוי Danit Oz Levi זיהוי גנומי של מחלות חד-גניות לא מפוענחות בריצוף של הדור החדש Next Generation Genomic discovery of undeciphered monogenic diseases מנחה: :Advisor פרופ' דורון לנצט Prof. Doron Lancet Month and Year חודש ושנה עבריים March 2016 אדר תשע"ו 1 LIST OF ABBREVIATIONS NGS Next Generation Sequencing HSP Hereditary Spastic Paraparesis IDIS Intractable Diarrhea of Infancy Syndrome THES Trichohepatoenteric Syndrome CLS Capillary Leak Syndrome SNP Single Nucleotide Polymorphism SNV Single Nucleotide Variant InDel Insertion-Deletion VUS Variant of Uncertain Significance WGS Whole genome sequencing CNV Copy Number Variation VCF Variant Call Format MAF Minor Allele Frequency LOF Loss Of Function SPG Spastic Paraplegia SCA Spinocerebellar Ataxia EEC Enteroendocrine Cells TPN Total Parenteral Nutrition TF Transcription Factor RNAPII RNA polymerase 2 SD Syndromic Diarrhea MGUS Monoclonal Gammopathy of Undetermined Significance IVIG Intravenous Immunoglobulins BWA Burrows-Wheeler Alignment GATK Genome Analysis Toolkit SVA Sequence Variant Analyzer NHLBI National Heart Lung and Blood Institute ExAc The Exome Aggregation Consortium 2 CHGV Center Human Genome Variation DMEM Dulbecco's modified eagle medium FBS Fetal Bovine Serum EBSS Earle's balanced salt solution TEM Transition Electron Microscopy ERDS Estimation by read depth with single- nucleotide variants EEG Electroencephalogram EMG Electromyography WD Triptophan-Aspertate repeat LOD Logarithm Of Odds ICR Intestinal Critical Region ENCODE Encyclopedia of DNA Elements UCSC University of California Santa Cruz CHGA Chromogranin A iPSC Induced Pluripotent Stem Cells HIO Human Intestinal Organoids GTex Genotype-Tissue Expression BLAST Basic Local Alignment search tool ORF Open Reading Frame KO Knockout WT Wild-Type MRI/MRS Magnetic Resonance Imaging/Spectrometry CBC Complete Blood Count CRP C-reactive protein ECM Extracellular Matrix FA Focal Adhesion VEGF Vascular Endothelial Growth Factor GO Gene Ontology 3 DEP Differentially Expressed Proteins FDR False Discovery Rate ALS Amyotrophic Lateral Sclerosis HSAN Hereditary Sensory- Autonomic Neuropathy ER Endoplasmatic Reticulum HUVEC Human umbilical vein endothelial cells CMA Chromosomal microarray 4 ACKNOWLEDGMENTS First and foremost, I express my deepest gratitude to my supervisor Prof. Doron Lancet, who has been and will continue to be my mentor for life. I thank him for his continuous support of my PhD studies and research, for his patience and willingness to teach me, for the motivation, and immense knowledge. During my PhD studies I have been collaborating with experts in the field of genetics and genomics that have all contributed greatly to the success of my work, and from whom I have learned a lot and expanded my knowledge in many aspects. Especially I would like to thank Prof. Elon Pras, head of the genetic institute at Sheba Medical Center for his continuous interest and assistance, for fruitful and intelligent discussions and for the highly appreciated personal support. I thank Prof. Yair Anikster and Prof. Bruria Ben-Zeev from Sheba Medical Center for initiating the highly interesting genetic projects I have been involved in and for helping me make them even greater. I thank Prof. Zvulun Elazar for his collaboration in the autophagy study and to his lab members, especially Dr. Amir Gelman, for welcoming me into their lab as one of them while working on the TECPR2 project. I thank Prof. David Goldstein for a brilliant collaboration and to all his group members for sharing their expertise in NGS analysis and bioinformatics. I thank Prof. Len Pennacchio for his collaboration in the IDIS project and all the excellent scientific work he has done in order to make this story a successful publication. I thank Dr. Tsviya Olender for teaching me and guiding me throughout my PhD. I thank Dr. Anna Alkelai and Dr. Gil Stelzer for the stimulating scientific discussion, a lot of moral support but overall for being wonderful friends. I thank all lab members of the Lancet group throughout my PhD period for the motivating discussions, and for making my time extremely enjoyable. An enormous thank you to my loving and extremely supportive family- my husband Sagi for his encouragement and for being a wonderful husband and father allowing me to grow professionally, to my father who is always encouraging me to think forward and be great, and to my mother that with her incredible grandmother skills enabled me to be where I am today. 5 TABLE OF CONTENTS ABSTRACT ..................................................................................................................... 10 11 ................................................................................................................................. תקציר INTRODUCTION........................................................................................................... 12 Next generation sequencing analysis ............................................................................ 12 Analysis of sequence variants ....................................................................................... 13 Databases of control frequencies ............................................................................... 13 Protein damage prediction ......................................................................................... 14 Variant prioritization ................................................................................................. 15 Potential impact of this thesis........................................................................................ 17 Hereditary Spastic Paraparesis (HSP) ........................................................................... 18 Autophagy and neurodegeneration ............................................................................ 18 Intractable Diarrhea of Infancy Syndrome (IDIS) ........................................................ 19 Enhancer activity and connection to diseases ............................................................ 21 Trichohepatoenteric syndrome (THES) ........................................................................ 22 Capillary Leak Syndrome (CLS)................................................................................... 23 Pathophysiology ........................................................................................................ 24 Treatment ................................................................................................................... 24 METHODS ...................................................................................................................... 26 General methods ............................................................................................................ 26 1.1 Subjects ................................................................................................................ 26 1.2 Exome sequencing and variant identification ...................................................... 26 1.3 Bioinformatics analysis ....................................................................................... 27 Methods for the study of HSP ....................................................................................... 27 2.1 Homozygosity mapping ....................................................................................... 27 2.2 Semi-quantitative RT-PCR .................................................................................. 28 2.3 Cell culture and transfection ................................................................................ 28 2.4 Immunoblots ........................................................................................................ 28 6 2.5 Immunofluorescence analyses ............................................................................. 29 2.6 Transmission Electron Microscopy (TEM) ......................................................... 29 Methods for the study of IDIS....................................................................................... 29 3.1 Whole genome sequencing .................................................................................. 29 3.2 Biopsy collection ................................................................................................. 30 3.3 RNA extraction from biopsies ............................................................................. 30 3.4 RNA sequencing of human samples .................................................................... 30 3.5 Quantitative Real-Time Reverse Transcriptase Polymerase Chain Reaction (qPCR) ....................................................................................................................... 30 3.6 Serum Collection ................................................................................................. 31 3.7 ELISA .................................................................................................................. 31 3.8 Linkage analysis and homozygosity mapping ..................................................... 31 3.9 Deletion analysis.................................................................................................. 32 3.10 Mouse transgenic assays .................................................................................... 32 3.11