The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgementTown of the source. The thesis is to be used for private study or non- commercial research purposes only. Cape Published by the University ofof Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University ELUCIDATION OF THE OCCURRENCE OF EXTRACOLONIC CANCERS IN LYNCH SYNDROME Kusha Kalideen Town Supervisor: ProfessorCape R. Ramesar of Department of Clinical Laboratory Science Division of Human Genetics University Presented for M.Sc. (Med) In the School of Biomedical Sciences Faculty of Health Sciences University of Cape Town 2008 1 DECLARATION 1. I know that plagiarism is wrong. Plagiarism is to use another’s work and to pretend that it is ones own. 2. I have used the American Journal of Human Genetics convention for citation and referencing. Each significant contribution to, and quotation in, this essay/ report/ project/ from the work, or works, of other people has been attributed, and has cited and referenced. 3. This project is my own work using my Townown words. 4. I have not allowed, and will not allow, anyone to copy my work with the intention of passing if off as his or her own work. Cape of DATE.11 November 2008... STUDENT NAMEKUSHA KALIDEEN.. STUDENTUniversity NUMBER KLDKUS001. SIGNATURE. 2 Table of Contents Acknowledgments ............................................................................................... 7 List of abbrieviations ........................................................................................... 8 List of figures ..................................................................................................... 12 List of tables ...................................................................................................... 13 List of tables ...................................................................................................... 13 Plan of thesis .................................................................................................... 14 ABSTRACT ....................................................................................................... 15 Chapter 1: ......................................................................................................... 17 1.1. Lynch Syndrome .................................................................................... 17 1.2. History of Lynch Syndrome ....................................................................Town 17 1.3. Genetic Basis of Lynch Syndrome ......................................................... 19 1.3.1. DNA MMR system .................................................................................. 19 1.3.2 Defects in the MMR system .....................................................................Cape 22 1.3.2.1. Monoallelic Germline Mutations ........................................................... 22 1.3.2.2. Biallelic germline mutationsof .................................................................. 23 1.3.2.3. Compound heterozygotes .................................................................... 25 1.3.3. Epigenetic silencing of a DNA MMR gene .............................................. 26 1.4. Tumourigenesis in MMR deficient cells ...................................................... 28 1.4.1. Mutator Pathway ..................................................................................... 29 1.4.2. InteractionUniversity between Vogelstein and Mutator Pathways ........................... 31 1.5. Tumour Spectrum in Lynch Syndrome ....................................................... 32 1.5.1. Gynaecologic cancers ............................................................................. 33 1.5.2. Hepatobiliary Cancers ............................................................................. 36 1.5.3. Gastric Cancers ...................................................................................... 37 1.5.4. Small intestinal cancers .......................................................................... 38 1.5.5. Upper Uroepithelial Tract ........................................................................ 39 1.5.6. Brain cancer ............................................................................................ 40 1.5.6.1.Turcot syndrome ................................................................................... 41 3 1.5.7. Muire Torre Syndrome ............................................................................ 41 1.5.8. Genotype – Phenotype correlation .......................................................... 41 1.6. Pathology of Lynch Syndrome Cancers ..................................................... 49 1.7. Research in Lynch Syndrome .................................................................... 50 1.8. Aims and Objectives .................................................................................. 51 Chapter 2: ......................................................................................................... 53 2.1 Introduction ................................................................................................. 53 2.2 Materials and Methods ................................................................................ 55 2.2.1 Cohort selection ....................................................................................... 55 2.2.2. DNA Isolation and Integrity ..................................................................... 56 2.2.3. Identification of Gene Sequence and Primer design ............................... 57 2.2.4. Polymerase Chain Reaction ....................................................................Town 59 2.2.4.1. PCR Optimization ................................................................................. 59 2.2.4.2. Amplification ......................................................................................... 59 2.2.5. Resolution of DNA fragments ..................................................................Cape 60 2.2.6. Restriction Enzyme digestion .................................................................. 61 2.2.7. Direct sequencing ...................................................................................of 63 2.2.8. Statistical analysis ................................................................................... 64 2.3 Results ........................................................................................................ 65 2.3.1. Cohort Analysis ....................................................................................... 65 2.3.2. Integrity gel ............................................................................................. 67 2.3.3. TemperatureUniversity gradient .............................................................................. 67 2.3.4. Restriction Endonuclease digestion ........................................................ 68 2.3.5. Distribution of Genotypes and Alleles within the study cohorts ............... 69 2.3.6. Statistical Analysis .................................................................................. 70 2.4 Discussion ................................................................................................... 73 2.4.1. Limitations of the study ........................................................................... 76 2.4.2. Future prospects ..................................................................................... 77 4 Chapter 3: ......................................................................................................... 78 3.1. Introduction ................................................................................................ 78 3.2. Materials and Methods ............................................................................... 82 3.2.1. Cohort selection ...................................................................................... 82 3.2.2. Selection of microsatellite rich genes ...................................................... 82 3.2.3. DNA Isolation .......................................................................................... 83 3.2.3.1. Germline DNA ...................................................................................... 83 3.2.3.2. Formalin-fixed paraffin embedded tumour tissue ................................. 83 3.2.4. Primer design and Polymerase Chain Reaction (PCR) ........................... 85 3.2.4.1. Multiplex PCR ...................................................................................... 85 3.2.4. Genotyping .............................................................................................. 89 3.2.5. MS - MLPA .............................................................................................. 90 3.3. Results .......................................................................................................Town 93 3.3.1. Cohort selection ...................................................................................... 93 3.3.2. DNA Isolation .......................................................................................... 94 3.3.3. Selection of microsatellite rich genesCape ...................................................... 96 3.3.4. Genotyping .............................................................................................. 99 3.3.5. Methylation Specific Multiplexof Ligation – Dependent Probe Amplification (MS - MLPA) ..................................................................................................