Elucidation of the functional consequences of NLRP7 mutations By: Ebtihaj Bukhari Department of Human Genetics McGill University, Montreal July 2009 A thesis submitted to McGill University in partial fulfillment of the requirement of the Master of Science degree in Human Genetics © Ebtihaj Bukhari, 2009 TABLE OF CONTENTS ABSTRACT............................................................................................................................... 4 RÉSUMÉ ................................................................................................................................... 5 LIST OF ABBREVIATIONS .................................................................................................. 6 LIST OF FIGURES AND TABLES ....................................................................................... 7 ACKNOWLEDGEMENTS ..................................................................................................... 8 CHAPTER 1.............................................................................................................................. 9 1.1 Introduction and Clinical Manifestations of Hydatidiform Moles .............................. 9 1.1.2 Epidemiology of Hydatidiform Moles....................................................................... 10 1.1.3 Karyotype and Genotype of Moles............................................................................ 11 1.1.4 Imprinting and DNA Methylation in Moles .............................................................. 12 1.2 Identification of NLRP7 and Genotype Phenotype Correlations............................... 16 1.2.1 Expression of NLRP7................................................................................................ 18 1.2.2 The Known Role of NLRP7 ...................................................................................... 20 1.2.3 Inflammation and Pregnancies .................................................................................. 22 1.3 The NLR Family ............................................................................................................ 24 1.3.1 Inflammasome Activation: Mechanism of Action .................................................... 26 1.3.2 NLRP Genes and Autoinflammatory Diseases.......................................................... 29 1.3.2.1 NLRP1................................................................................................................. 30 1.3.2.2 NLRP2................................................................................................................. 32 1.3.2.3 NLRP 3................................................................................................................ 33 1.3.2.4 NLRP5................................................................................................................. 35 1.3.2.5 NLRP12............................................................................................................... 36 2 CHAPTER 2............................................................................................................................ 39 2. Materials and Methods .................................................................................................... 39 2.1 Isolation of Full-length and Mutated NLRP7 cDNA.................................................... 39 2.2 Site-Directed Mutagenesis............................................................................................ 40 2.3 Subcloning of Wild-type NLRP7 cDNA ...................................................................... 42 2.4 Subcloning of Mutated NLRP7 cDNA ......................................................................... 43 2.5 Cell culture and Transient Transfection ....................................................................... 46 2.6 Enzyme-linked Immunosorbent Assay (ELISA).......................................................... 47 2.7 Western Blot Analysis .................................................................................................. 47 2.8 Statistical Analysis........................................................................................................ 48 CHAPTER 3............................................................................................................................ 49 3. Results................................................................................................................................ 49 3.1 Site-Directed Mutagenesis............................................................................................ 49 3.2 Subcloning of Wild-type and Mutated NLRP7 cDNA ................................................. 51 3.3 Optimizing the Transfection Conditions ...................................................................... 51 3.4 NLRP7 Inhibits IL-1β Secretion in HEK293 cells ....................................................... 53 CHAPTER 4............................................................................................................................ 56 Discussion .............................................................................................................................. 56 Conclusions and Future Perspectives ................................................................................. 58 References.............................................................................................................................. 60 APPENDIX A :Published Abstract and Presentations............................................................ 67 APPENDIX B :Published Abstract ........................................................................................ 67 APPENDIX C: Ethics Approval and Certificates ................................................................... 69 3 ABSTRACT Hydatidiform mole (HM) is an abnormal human pregnancy characterized by the absence of, or abnormal, embryonic development and hydropic degeneration of the chorionic villi. In both Canada and the United States, the incidence of HM is 1 in every 1000 pregnancies. Recently, NLRP7 has been found to be responsible for recurrent hydatidiform moles (RHM) after the identification of various mutations in this gene. To investigate the functional consequences of NLRP7 mutations on IL-1β maturation and secretion, I used site-directed mutagenesis to introduce the various mutations found in patients with RHM into WT-NLRP7. HEK293 cell lines were cotransfected with WT- NLRP7, procaspase-1, and pro-IL-1β, as well as with or without Cardinal (CARD-8.). My results demonstrate that WT-NLRP7 inhibits IL-1β secretion in a dose-dependent manner. Furthermore, I found that Cardinal (CARD8), an important component of other inflammasomes, has no impact on the inhibitory effect of WT-NLRP7. My findings strengthen the idea that NLRP7 may function as a feedback regulator of IL-1β secretion. 4 RÉSUMÉ Une môle hydatidiform (MH) est une anomalie de la grossesse caractérisée par soit l'absence de l’embryo ou du développement embryonnaire ainsi que la dégénérescence des villosités choriales. Au Canada et aux États-Unis, l'incidence des MH est 1 sur 1000 naissances. Récemment, NLRP7 a été trouvé responsable des môles hydatidiform à répétition, après l'identification de différentes mutations dans le gène. Pour étudier les conséquences fonctionnelles des mutations de NLRP7 sur la maturation et la sécrétion de IL-1β, j'ai utilisé le site de mutagenèse dirigée pour introduire les différentes mutations trouvées chez les patientes atteintes de môles recurrentes. Des cellules HEK293 ont été cotransfectées avec la copie normale de WT-NLRP7, procaspase-1, pro-IL-1β, avec et sans Cardinal (CARD-8.). Mes résultats démontrent que WT-NLRP7 inhibe la sécrétion de IL-1β d'une manière dose-dépendante. De plus, j'ai trouvé que Cardinal (CARD8), une composante importante d’autres inflammasomes, n'a pas d'effet sur l’inhibition de WT-NLRP7. Mes constatations renforcent l'idée que NLRP7 peut fonctionner comme un régulateur de la sécrétion de l'IL-1β. 5 LIST OF ABBREVIATIONS BiHM: Biparental hydatidiform moles CHM: Complete hydatidiform mole cDNA: Complementary DNA ELISA: Enzyme-linked immunosorbent assay FBS: Fetal bovine serum HM: hydatidiform mole HEK293: Human embryonic kidney 293 cell line IL-1β: Interleukin -1β LB media: Luria-Bertani media NLRP: Nucleotide- binding domain, leucine rich repeat protein PAMP: Pathogen-associated molecular pattern PBS: phosphate buffered saline PHM: Partial hydatidiform mole RHM: Recurrent hydatidiform mole WT: Wild-type 6 LIST OF FIGURES AND TABLES Figure1. NLRP7 structure, domains, and positions of some mutations………………..…….19 Figure 2 NLRP3 inflammasome …………………………………………………………......28 Figure 3 Schematic representation of the protocol used to in molecular cloning…………....45 Figure 4. The amplification of NLRP7 cDNA using four different primers……....................49 Figure5. Site directed mutagenesis showing the sequences of the various mutations…….... 50 Figure 6. Cloning in PcDNA3.1+………………………………………………………….... 51 Figure7. Optimizing the transfection conditions……………………………………………. 52 Figure 8. NLRP7 inhibits IL-1β secretion in HEK293 cells…................................................54 Figure 9. NLRP7 inhibits IL-1β secretion in the presence or absence of Cardinal………..... 55 Table 1. Methylation at imprinted genes in BiHMs ………………………………………... 15 Table 2. NLRP7 mutations identified in males with Normal reproduction ……………….... 18 Table 3. NLR subfamily and associated autoinflammatory human diseases…………….....