Identification of bleomycin and radiation-induced pulmonary fibrosis susceptibility genes in mice Anne-Marie Lemay Department of Human Genetics McGill University, Montréal February 4th, 2010 A thesis submitted to McGill University in partial fulfilment of the requirements of the degree of Doctor of Philosophy © Anne-Marie Lemay 2010 Comme il est profond, ce mystère de l’Invisible ! Nous ne pouvons le sonder avec nos sens misérables, avec nos yeux qui ne savent apercevoir ni le trop petit, ni le trop grand, ni le trop près, ni le trop loin, ni les habitants d’une étoile, ni les habitants d’une goutte d’eau… Guy de Maupassant Le Horla ii Table of contents Table of contents ................................................................................................... iii Abstract...................................................................................................................vi Résumé ................................................................................................................ viii Acknowledgments................................................................................................... x Abbreviations........................................................................................................ xii Original contributions to knowledge...................................................................xiv Author contribution to research...........................................................................xv List of figures ......................................................................................................... 1 List of tables ........................................................................................................... 3 CHAPTER I ........................................................................................................... 4 Introduction............................................................................................................ 4 Pulmonary fibrosis ....................................................................................................... 6 Hypotheses on the development of pulmonary fibrosis.......................................................... 7 Familial pulmonary fibrosis.................................................................................................... 9 Animal models of pulmonary fibrosis....................................................................... 10 Route of administration......................................................................................................... 11 Bleomycin .................................................................................................................... 12 Mode of action...................................................................................................................... 13 Thoracic irradiation ................................................................................................... 15 Identification of susceptibility genes using mouse genetics..................................... 16 Strain dependant fibrosis responses to bleomycin and radiation treatments......................... 16 Mouse crosses for QTL analysis........................................................................................... 21 Confirmation of a QTL ......................................................................................................... 25 Reduction of the size of the QTL.......................................................................................... 27 Positional cloning in mice..................................................................................................... 30 Candidate genes evaluation ....................................................................................... 32 Gene expression analysis ...................................................................................................... 32 Sequence variations in candidate genes ................................................................................ 35 Evaluation of candidate genes............................................................................................... 35 Experimental demonstration of the gene effect on the phenotype ........................................ 36 Aims of this research project..................................................................................... 37 CHAPTER 2......................................................................................................... 39 Bleomycin-induced pulmonary fibrosis susceptibility genes in AcB/BcA recombinant congenic mice................................................................................. 39 Abstract ....................................................................................................................... 40 iii Introduction ................................................................................................................ 41 Materials and Methods .............................................................................................. 43 Mice ...................................................................................................................................... 43 Bleomycin treatment............................................................................................................. 43 Histology and Fibrosis Scoring............................................................................................. 44 QTL Analysis........................................................................................................................ 45 Gene expression.................................................................................................................... 46 Microarray data analysis....................................................................................................... 47 Quantitative real-time PCR................................................................................................... 48 Sequence Comparison........................................................................................................... 51 Results.......................................................................................................................... 51 Fibrosis phenotype of A/J and B6 mice ................................................................................ 51 AcB/BcA fibrosis phenotype ................................................................................................ 52 Mapping of pulmonary fibrosis susceptibility ...................................................................... 55 Candidate Gene Identification .............................................................................................. 57 Gene Expression Studies....................................................................................................... 57 Sequence Variation............................................................................................................... 61 Discussion .................................................................................................................... 63 Chapter transition ...................................................................................................... 68 CHAPTER 3......................................................................................................... 69 Radiation-induced lung response of AcB/BcA recombinant congenic mice..... 69 Abstract ....................................................................................................................... 70 Introduction ................................................................................................................ 71 Materials and Methods .............................................................................................. 73 Mice ...................................................................................................................................... 73 Radiation treatment............................................................................................................... 74 Histology............................................................................................................................... 74 Bronchoalveolar Lavage Fluid (BAL) Analysis ................................................................... 75 Data analysis......................................................................................................................... 76 Results.......................................................................................................................... 76 Parental Strain radiation-induced lung phenotype ................................................................ 76 AcB/BcA radiation-induced lung phenotype........................................................................ 77 RC strains informative for the radiation-induced lung response........................................... 87 Discussion .................................................................................................................... 90 Chapter transition ...................................................................................................... 94 CHAPTER 4......................................................................................................... 95 Bleomycin-induced pulmonary fibrosis candidate gene analysis ...................... 95 Abstract ......................................................................................................................