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Identifying Susceptibility Genes for Familial Pancreatic Cancer Using Novel High-Resolution Genome Interrogation Platforms

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Identifying Susceptibility Genes for Familial Pancreatic Cancer Using Novel High-Resolution Genome Interrogation Platforms Identifying Susceptibility Genes for Familial Pancreatic Cancer Using Novel High-Resolution Genome Interrogation Platforms by Wigdan Ridha Al-Sukhni A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Institute of Medical Science University of Toronto © Copyright by Wigdan Ridha Al-Sukhni 2012 Identifying Susceptibility Genes for Familial Pancreatic Cancer Using Novel High-Resolution Genome Interrogation Platforms Wigdan Ridha Al-Sukhni Doctor of Philosophy Institute of Medical Science University of Toronto 2012 Abstract Familial Pancreatic Cancer (FPC) is a cancer syndrome characterized by clustering of pancreatic cancer in families, but most FPC cases do not have a known genetic etiology. Understanding genetic predisposition to pancreatic cancer is important for improving screening as well as treatment. The central aim of this thesis is to identify candidate susceptibility genes for FPC, and I used three approaches of increasing resolution. First, based on a candidate-gene approach, I hypothesized that BRCA1 is inactivated by loss- of-heterozygosity in pancreatic adenocarcinoma of germline mutation carriers. I demonstrated that 5/7 pancreatic tumors from BRCA1-mutation carriers show LOH, compared to only 1/9 sporadic tumors, suggesting that BRCA1 inactivation is involved in tumorigenesis in germline mutation carriers. Second, I hypothesized that the germline genomes of FPC subjects differ in copy-number profile from healthy genomes, and that regions affected by rare deletions or duplications in FPC subjects overlap candidate tumor-suppressors or oncogenes. I found no significant difference in the global copy-number profile of FPC and control genomes, but I identified 93 copy-number variable genomic regions unique to FPC subjects, overlapping 88 genes of which several have functional roles in cancer development. I investigated one duplication to sequence the breakpoints, but I found that this duplication did not segregate with disease in the affected family. Third, I hypothesized that in a family with multiple pancreatic cancer patients, genes containing rare variants shared by the affected members constitute ii susceptibility genes. Using next-generation sequencing to capture most bases in coding regions of the genome, I interrogated the germline exome of three relatives who died of pancreatic cancer and a relative who is healthy at advanced age. I identified a short-list of nine candidate genes with unreported mutations shared by the three affected relatives and absent in the unaffected relative, of which a few had functional relevance to tumorigenesis. I performed Sanger sequencing to screen an unrelated cohort of approximately 70 FPC patients for mutations in the top two candidate genes, but I found no additional rare variants in those genes. In conclusion, I present a list of candidate FPC susceptibility genes for further validation and investigation in future studies. iii Acknowledgments My research would not have been possible without the contribution of the following individuals: A. Borgida, S. Holter, H. Rothenmund, and K. Smith at Ontario Pancreas Cancer Study and Ontario Familial Gastrointestinal Cancer Registry for patient recruitment and selection. T. Selander of Samuel Lunenfel Research Institute Biospecimen Repository for DNA extraction. S. Joe (Gallinger Lab) for script-writing; N. Zwingerman, A. Gropper, and S. Moore (Gallinger Lab) for assistance with qPCR; A. Lionel (Scherer Lab) for computational analysis of Affy6.0 data on Birdsuite and iPattern; Q. Trinh (McPherson Lab) for computational analysis of exome data; R. Grant (Gallinger Lab) for assistance with exome data interpretation; H. Kim and T. McPherson (Gallinger Lab) for assitance with PCR and Sanger validation of exome variants. K. Hay, J. Keating, and S. Levitt (Gallinger Lab) for administrative support; J. McPherson (Ontario Institute for Cancer Research) for exome sequencing data; and C. Marshall, D. Pinto, D. Merico (The Centre for Applied Genomics), A. Shlien and D. Malkin (Malkin Lab) for their advice on my data analysis and manuscript preparations. My sincere gratitude to the Pancreatic Cancer Genetic Epidemiology Consortium (PACGENE) (PI - G Petersen, Mayo) for being an invaluable source of DNA samples and insight into pancreatic cancer genetics. I am very grateful to my Program Advisory Committee (Gary Bader, Steven Narod, Stephen Scherer) for their insightful feedback and advice throughout the five years of my PhD. In particular, their thoughtful review of my manuscripts and thesis was most helpful and deeply appreciated. To my supervisor, Steve Gallinger – I cannot adequately thank you in this crowded page for all that your mentorship has meant to me since I first met you seven years ago. You pushed me when I needed pushing and supported me when I was afraid of falling. You listened patiently to my complaints. You cared about my success. I will always appreciate your open-mindedness, your integrity, and your compassion. I feel most fortunate that I am able to call you my mentor and friend. Thank you for everything. A special thank you to M. Crump for helping me maneuver around some unexpected bumps in the road of my PhD, and for exemplifying the compassionate clinician. iv I dedicate this thesis to my beautiful family: To Mama and Baba – Your love for me has been the greatest gift and blessing in my life, it is the reason for who I am today. Thank you for supporting my aspirations even when you did not always understand where they were taking me. To Eisar, Mayce, Mohammed, and Bann – Thank you for putting up with me in my worst days… I am proud of you all. To my aunts, uncles, and cousins in Iraq and elsewhere – Thank you for keeping me alive in your hearts despite the long years and oceans separating us. You inspire me. I am grateful for the financial support received from the CIHR Vanier Doctoral Research Award, Lustgarten grant, Invest-in-Research grant from Princess Margarte Hospital, Canadian Society for Surgical Oncology grant, Johnson & Johnson research award, American HepatoPancreaticoBiliary Association grant, and the Department of Surgery at the University of Toronto. v Table of Contents Abstract..........................................................................................................................................................ii Acknowledgments.........................................................................................................................................iv List of Tables...............................................................................................................................................vii List of Figures.............................................................................................................................................viii List of Appendices........................................................................................................................................ix Abbreviations................................................................................................................................................xi Chapter 1 Literature Review.........................................................................................................................1 1. Pancreatic Cancer.................................................................................................................1 2. Copy Number Variation.......................................................................................................12 3. Whole-Exome Sequencing..................................................................................................37 Chapter 2 Loss of Heterozygosity at BRCA1 Locus in Pancreatic Adenocarcinoma.................................51 1. Abstract................................................................................................................................51 2. Introduction..........................................................................................................................51 3. Materials & Methods...........................................................................................................52 4. Results..................................................................................................................................55 5. Discussion............................................................................................................................58 Chapter 3 Germline Genomic Copy Number Variation in Familial Pancreatic Cancer.............................63 1. Abstract................................................................................................................................63 2. Introduction..........................................................................................................................63 3. Materials & Methods...........................................................................................................64 4. Results..................................................................................................................................73 5. Discussion............................................................................................................................94 Chapter 4 Exome Sequencing in a Familial Pancreatic Cancer Kindred..................................................100 1. Abstract..............................................................................................................................100
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