GENETICS OF SPONTANEOUS IDIOPATHIC PRETERM BIRTH: EXPLORATION OF MATERNAL AND FETAL GENOMES By Jude James McElroy Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Human Genetics August, 2013 Nashville, Tennessee Approved: Dana C. Crawford, Ph.D. Michael R. DeBaun, M.D., M.P.H. Louis J. Muglia, M.D., Ph.D. Dan M. Roden, M.D. Scott M. Williams, Ph.D. Copyright © 2013 Jude James McElroy All Rights Reserved To all my friends and family without whom I would never have made it this far and A very special thank you to a charming person who saw me better than I saw myself and helped me believe… iii ACKNOWLEDGEMENTS The work presented in this dissertation was supported by the Vanderbilt Medical Scientist Training Program (MSTP) grant T32 GM007347, as well as funding form the March of Dimes (21-FY2011-16 "Genetic Analysis of Human Preterm Birth"). A very heartfelt thank you to all the individuals who participated in these studies. All of the work presented here would have not been possible without you. The work presented here was guided by the input from my thesis committee: Dana Crawford (committee chair), Michael DeBaun, Louis Muglia, Dan Roden and Scott Williams. Special acknowledgements are needed for my mentor, Louis Muglia. Lou has helped give me the skills required to complete my dissertation and allowed me grow to become an excellent scientist, but even more importantly was an excellent role model for how to be an outstanding person. Thanks to the students of the Program in Human Genetics for their discussions and support. Special thanks to Will Bush and Stephen Turner for their excellent genetics blog, “Getting Genetics Done”, and especially for their R code which was used for the QQ and Manhattan plots presented here and used throughout my training. I would also like to thank all of the staff of the DNA Resources Core especially Holli Dilks, Cara Sutcliffe and Paxton Baker. Additionally, Christian iv Shafer was extremely helpful with our whole-exome sequencing and answering all of my computational questions. Modern human genetics requires a team, and all of the people above have been instrumental in helping me complete my dissertation and made the journey more enjoyable. v TABLE OF CONTENTS Page DEDICATION ........................................................................................................... iii ACKNOWLEDGEMENTS ....................................................................................... iv LIST OF TABLES ....................................................................................................... ix LIST OF FIGURES ..................................................................................................... xi LIST OF APPENDICES .......................................................................................... xiii Chapter I. INTRODUCTION .............................................................................................1 Classification of preterm birth ............................................................. 2 Pathways hypothesized to be involved in preterm birth ................ 3 Epidemiological risk factors ................................................................. 5 Evidence of a genetic component ........................................................ 6 Summary and objectives of dissertation ............................................ 9 II. MATERNAL GENOME-WIDE ASSOCIATION FOR PRETERM BIRTH RISK AND GESTATION LENGTH .............................................11 Introduction ....................................................................................................... 11 Results ................................................................................................................. 13 Study population characteristics ....................................................... 13 Finnish mothers preterm birth chi-square association ................... 14 Box-Cox transformation of gestational ages .................................... 15 Box-Cox transformed gestational age unadjusted linear regression association analysis of Finnish mothers genotyped on the Affymetrix 6.0 SNP array .................................................................... 16 Discussion .......................................................................................................... 18 Materials and methods ..................................................................................... 24 Sample collection ................................................................................. 24 Sample inclusion and exclusion criteria ........................................... 24 Finnish mothers Affymetrix Genome-Wide Human SNP Array 6.0 ................................................................................................................. 25 Statistical analysis ................................................................................ 26 vi III. FETAL VARIANTS IN THE INF2 REGION ARE ASSOCIATED WITH GESTATIONAL AGE AND PRETERM BIRTH ..........................27 Introduction ....................................................................................................... 27 Results ................................................................................................................. 28 Study population characteristics ....................................................... 28 Preterm birth additive logistic regression adjusted for BMI, gravidity and smoking status association analysis in Helsinki infants on the Illumina Omni2.5 BeadChip ..................................... 29 Preterm birth genotypic logistic regression adjusted for BMI, gravidity and smoking status association analysis in Helsinki infants on the Illumina Omni2.5 BeadChip ..................................... 30 Box-Cox transformation of gestational age in Helsinki infants .... 31 Box-Cox transformed gestational age adjusted for BMI, gravidity and smoking status linear regression in Helsinki infants .............. 33 TaqMan genotyping of rs7153053 in an Oulu infant cohort .......... 35 Discussion .......................................................................................................... 38 Materials and methods ..................................................................................... 41 Sample collection ................................................................................. 41 Sample inclusion and exclusion criteria ........................................... 41 Helsinki infant Illumina Omni2.5 BeadChips .................................. 42 Applied Biosystems TaqMan genotyping ........................................ 42 Statistical analysis ................................................................................ 42 IV. FETAL CODING REGION VARIANTS IN ADAM METALLOPEPTIDASE DOMAIN 29 (ADAM29) ARE ASSOCIATED WITH INCREASED BIRTH WEIGHT Z-SCORES ..................................44 Introduction ....................................................................................................... 44 Results ................................................................................................................. 46 Study population characteristics ....................................................... 46 Birth weight z-score linear regression adjusted for BMI, gravidity and smoking status in Helsinki infants genotyped on Illumina Omni2.5 BeadChips ............................................................................. 47 TaqMan genotyping of ADAM29 SNPs in Oulu infants ............... 50 Discussion .......................................................................................................... 53 Materials and methods ..................................................................................... 57 Sample collection ................................................................................. 57 Sample inclusion and exclusion criteria ........................................... 57 Helsinki infant Illumina Omni2.5 BeadChips .................................. 58 Applied Biosystems TaqMan genotyping ........................................ 58 Statistical analysis ................................................................................ 59 V. MATERNAL CODING REGION VARIANTS IN COMPLEMENT RECEPTOR 1 INCREASE RISK FOR SPONTANEOUS IDIOPATHIC PRETERM BIRTH ..........................................................................................60 Introduction ....................................................................................................... 60 vii Results ................................................................................................................. 61 Analysis of shared variants in mother-daughter pairs ................... 61 Pathway analysis of the most significant genes in mother- daughter pairs ...................................................................................... 63 Examination of complement and coagulation cascades in our other PTB exomes ................................................................................. 66 Interrogation of the complement and coagulation cascade in nuclear PTB mothers ........................................................................... 68 Discussion .......................................................................................................... 71 Materials and methods ....................................................................................