Identification of Novel Causative Genes for Colorectal Adenomatous Polyposis Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Sukanya Horpaopan aus Nakhonsawan, Thailand Bonn, 2015 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn Die vorliegende Arbeit wurde am Institut für Humangenetik der Rheinischen Friedrich‐Wilhelms‐Universität zu Bonn angefertigt. 1. Gutachter: Prof. Dr. Stefan Aretz 2. Gutachter: Prof. Dr. Michael Hoch Tag der Promotion: February 9, 2015 Erscheinungsjahr: 2015 TABLE OF CONTENTS Page Acknowledgements ................................................................................................................... VI Declaration ............................................................................................................................... VII List of abbreviations ................................................................................................................ VIII 1. INTRODUCTION .................................................................................................................... 1 2. BASIC PRINCIPLES .............................................................................................................. 3 2.1. Hereditary Colorectal Cancer ........................................................................................... 3 2.1.1. Differential diagnosis ................................................................................................. 4 2.1.2. Adenoma-carcinoma sequence ................................................................................. 5 2.1.3. Knudson two-hit hypothesis ....................................................................................... 6 2.2. Genetics of adenomatous polyposis syndromes .............................................................. 7 2.2.1. Familial adenomatous polyposis (FAP) ...................................................................... 7 2.2.2. MUTYH-associated polyposis (MAP) ......................................................................... 9 2.2.3. Mutation negative adenomatous polyposis .............................................................. 10 2.3. Human genome variation ............................................................................................... 11 2.3.1. Single nucleotide polymorphisms (SNPs) ................................................................ 12 2.3.2. Tandem repeats ...................................................................................................... 12 2.3.3. Structural variations ................................................................................................. 12 2.4. Identification of causative genes in human disease ........................................................ 18 2.4.1. Homozygosity mapping ........................................................................................... 19 2.4.2. Loss of heterozygosity (LOH) analysis ..................................................................... 20 2.4.3. Linkage analysis ...................................................................................................... 21 2.4.4. Genome-wide association study (GWAS) ................................................................ 22 2.4.5. Copy number variation (CNV) analysis .................................................................... 23 2.4.6. High-throughput sequencing .................................................................................... 26 2.5. Validation of functionally relevant candidate genes ........................................................ 27 2.5.1. Recurrent findings ................................................................................................... 27 2.5.2. Segregation analysis ............................................................................................... 27 2.5.3. Gene expression in relevant target tissues .............................................................. 28 2.5.4. Candidate gene approach ....................................................................................... 28 2.5.5. Pathway enrichment analysis/network analysis ....................................................... 28 2.6. Scope of the thesis ........................................................................................................ 30 3. MATERIALS AND METHODS .............................................................................................. 31 I 3.1. Databases...................................................................................................................... 31 3.2. Devices .......................................................................................................................... 32 3.3. Software ........................................................................................................................ 33 3.4. Commercial reagents ..................................................................................................... 34 3.5. Study samples ............................................................................................................... 35 3.5.1. Initial patient cohort ................................................................................................. 35 3.5.2. NGS validation cohort .............................................................................................. 36 3.5.3. Heinz Nixdorf RECALL (HNR) study controls .......................................................... 36 3.5.4. GWAS replication study ........................................................................................... 37 3.6. DNA and RNA preparations ........................................................................................... 37 3.6.1. DNA extraction using desalting method ................................................................... 37 3.6.2. Formalin fixed paraffin embedded (FFPE) tissue DNA isolation ............................... 38 3.6.3. RNA extraction using the PAX gene kit .................................................................... 38 3.6.4. Determination of concentration and quality .............................................................. 39 3.6.5. First-strand cDNA synthesis .................................................................................... 39 3.7. Polymerase Chain Reaction (PCR) ................................................................................ 40 3.7.1. Basic principle ......................................................................................................... 40 3.7.2. Primer design .......................................................................................................... 40 3.7.3. PCR reaction components ....................................................................................... 41 3.7.4. Cycling step ............................................................................................................. 42 3.7.5. Agarose gel electrophoresis .................................................................................... 42 3.7.6. PCR product purification .......................................................................................... 42 3.8. Sanger sequencing ........................................................................................................ 43 3.8.1. Basic principle ......................................................................................................... 43 3.8.2. Reaction components .............................................................................................. 44 3.8.3. Cycling step ............................................................................................................. 44 3.8.4. Cycle sequencing product cleaning ......................................................................... 44 3.8.5. Capillary electrophoresis ......................................................................................... 45 3.9. APC transcript analysis .................................................................................................. 45 3.9.1. Primer design .......................................................................................................... 45 3.9.2. cDNA analysis ......................................................................................................... 46 3.9.3. Sanger sequencing .................................................................................................. 46 3.9.4. Data analysis ........................................................................................................... 46 3.9.5. Genomic DNA analysis ............................................................................................ 47 3.9.6. Haplotype analysis .................................................................................................. 47 3.9.7. In-silico analysis ...................................................................................................... 47 II 3.10. Genome-wide SNP array hybridization ........................................................................ 47 3.10.1. Genotyping based on BeadArray Technology (Illumina®)...................................... 47 3.10.2. Protocol ................................................................................................................. 48