THE GENETIC BASIS OF THE MULTIPLE COLORECTAL ADENOMA PHENOTYPE Oliver Sieber Thesis submitted for the degree of Doctor of Philosophy in the University of London Cancer Research UK January 2004 ProQuest Number: U642895 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U642895 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract Multiple adenoma patients are characterised by 3-100 colorectal adenomas, resulting in an increased risk of colorectal cancer. The disease can be inherited as a Mendelian trait, either autosomal dominant or recessive, or occur sporadically. Approximately 10% of multiple adenoma patients carry a truncating mutation in the APC gene, usually in exons 1-4, exon 9 or the 3’-half of exon 15, producing a diagnosis of AAPC. Two APC missense variants, I1307K and E1317Q, have also been associated with the disease, and some whole-gene deletions have been reported. However, these latter associations have been inconsistent. In contrast, germline mutations in the central region of APC cause classical FAP, a dominant predisposition to hundreds or thousands of colorectal adenomas, resulting in a near 100% risk of cancer. The molecular mechanisms underlying these genotype-phenotype correlations are poorly understood. The genetic basis of multiple adenomas in patients without APC mutation remains to be identified. In this thesis, I have excluded a major role of whole-gene APC deletions in causing AAPC/multiple adenomas. Instead, individuals with such mutations were found to develop classical FAP. I have refined the relationship between germline and somatic mutations at the APC locus in AAPC and classical FAP to show that somatic mutations are constrained by selection for an optimal level of Wnt signalling and that this at least partly determines disease severity. In support of this view, I have provided evidence against a role of truncating APC mutations in causing chromosomal instability. I have shown that the genetic basis underlying multiple adenomas is heterogeneous, by establishing the importance of recessive germline mutations in the base excision repair gene MYH. Some individuals with such mutations were also found to develop classical polyposis. I provide evidence that multiple adenomas are not generally attributable to germline mutations in the Wnt and TGFp/BMP signalling pathways. Acknowledgements I owe thanks to my supervisor Ian Tomlinson for his great support and guidance of my project. He has been of invaluable help in numerous discussions and a continuous source of encouragement. My thanks also go to my collaborators Hanan Lamlum, Karl Heinimann, Lara Lipton, Kelly Woodford-Richens and Michael Crabtree for their intellectual and practical contributions, as well as to my colleagues from the Cancer Research UK Family Cancer Clinic, Colorectal Unit and Polyposis Registry at St Mark's Hospital. I am indebted to the patients for their participation in this study as well as to their respective doctors and pathologists for contributing samples and clinical information. I would also like to thank the staff from the Cancer Research UK Equipment Park, Histopathology and FACS Laboratories for their advice and technical support. I am particularly grateful to the Boehringer Ingelheim Fonds for their financial support. A big thank you goes to all my colleagues in the Molecular and Population Genetics Laboratory who have kept my spirits up during my studies. In particular, I am grateful to Andrew Rowan and Pat Gorman who were readily available for advice and practical help and who also contributed to my work. Lastly, I would especially like to thank my family without whose support and encouragement completion of this thesis would have not been possible. Abbreviations aa amino acid AAPC attenuated adenomatous polyposis coli Alb albumin APC adenomatous polyposis coli BMP bone morphogenic protein BMPRIA bone morphogenic protein receptor type 1A cDNA complementary DNA CGH comparative genomic hybridisation CIN chromosomal instability Co-SMAD common-mediator SMAD C0X2 cyclooxygenase-2 CPKl-a casein kinase 1-a CRC colorectal cancer c^uK CCLWClr ReStOLTclv ttK dHzO distilled water DI DNA index DNA deoxyribonucleic acid Dsh dishevelled ES cell embryonic stem cell FAP familial adenomatous polyposis FCM flow cytometry GSK3-P glycogen synthase kinase 3-p hDLG human homologue of the Drosophila discs large tumour suppressor HMPS hereditary mixed polyposis syndrome HNPCC hereditary nonpolyposis colorectal cancer IPS juvenile polyposis syndrome kb kilo base pairs LOH loss of heterozygosity LRP low-density lipoprotein receptor-related protein mb mega base pairs MCR mutation cluster region MLHl human homologue of E. coli MutL MMR mismatch repair MSH2 human homologue of E. coli MutS MSI microsatellite instability MTHl human homologue of E. coli MutT MYH human homologue of E. coli MutY nm nanometer NSAID nonsteroidal anti-inflammatory drug nt nucleotides OGGI human homologue of E. coli MutM 8-oxo-G 8-oxo-7,8-dihydroxy-2'-deoxyguanosine PCR polymerase chain reaction PI propidium iodide PJS Peutz-Jeghers syndrome PMS2 human homologue of S. cerevisiae postmeiotic segregation increased PPAR peroxisome proliferator-activated receptor PTEN phosphatase and tensin homologue deleted on chromosome 10 PTT protein truncation test RNA ribonucleic acid ROS reactive oxygen species rpm rotations per minute RQM-PCR real-time quantitative multiplex PCR R-SMAD receptor-regulated SMAD SMAD human homologue of Drosophila small mothers against decapentaplegic SSCP single-strand conformation polymorphism TCP T cell factor TGF-p transforming growth factor-^ p-TRCP P-transducin repeat-containing protein All genes have been italicised throughout the text, whereas proteins are shown in plain text. Table of Contents Abstract.............................................................................................................................2 Acknowledgements.......................................................................................................... 3 Abbreviations ...................................................................................................................4 Table of Contents.............................................................................................................6 Table of Figures.............................................................................................................10 Table of Tables...............................................................................................................12 Chapter 1 Introduction to Colorectal Cancer .....................................................14 1.1 The Epidemiology of Colorectal Cancer ...........................................................14 1.2 The Natural History of Colorectal Cancer ....................................................... 17 1.2.1 The Anatomy of the Colorectum ...............................................................17 1.2.2 Adenomatous Polyps ..................................................................................19 1.2.3 Hamartomatous Polyps..............................................................................21 1.2.4 Stages of Colorectal Cancer...................................................................... 22 1.3 The Adenoma-Carcinoma Sequence .................................................................23 1.4 Hereditary Colorectal Cancer Syndromes ........................................................26 1.4.1 Polyposis Syndromes ................................................................................. 27 1.4.1.1 Familial Adenomatous Polyposis ..........................................................27 1.4.1.1.1 The APC Gene and its Functions ...................................................29 1.4.1.1.2 APC and the Regulation of Wnt Signalling .................................. 32 1.4.1.1.3 Germline Mutations in the APC Gene ........................................... 35 1.4.1.1.4 Genotype-phenotype Associations in FAP ....................................36 1.4.1.1.5 Somatic Mutations in the APC Gene ............................................ 37 1.4.1.1.6 Associations between Germline and Somatic APC Mutations.... 38 1.4.1.1.7 Genetic Testing and Clinical Management .................................. 39 1.4.1.2 Attenuated Adenomatous Polyposis Coli ...............................................40 1.4.1.2.1 APC Missense Variants and AAPC ..............................................41 1.4.1.2.2 Clinical Management ..................................................................... 42 1.4.1.3 Juvenile Polyposis ...................................................................................42 1.4.1.3.1 The TGF-(3/BMP Signalling Pathway...........................................44 6 1.4.1.4 Peutz-Jeghers Syndrome ........................................................................ 46 1.4.1.5 Cowden’s Syndrome and Bannayan-Riley-Ruvalcaba Syndrome.... 48 1.4.1.6 Hereditary Mixed Polyposis Syndrome ...............................................