UNIVERSITÀ DEGLI STUDI DI TRIESTE XXX CICLO DEL DOTTORATO DI RICERCA IN Scienze della Riproduzione e dello Sviluppo IDENTIFICATION OF GENETIC VARIANTS REGULATING FEMALE FERTILITY Settore scientifico-disciplinare: MED/03 DOTTORANDA: Caterina Maria Barbieri COORDINATORE: PROF. Paolo Gasparini SUPERVISORE DI TESI: PROF.SSA Daniela Toniolo PROF. Paolo Gasparini ANNO ACCADEMICO 2016/2017 UNIVERSITÀ DEGLI STUDI DI TRIESTE XXX CICLO DEL DOTTORATO DI RICERCA IN Scienze della Riproduzione e dello Sviluppo IDENTIFICATION OF GENETIC VARIANTS REGULATING FEMALE FERTILITY Settore scientifico-disciplinare: MED/03 DOTTORANDA: Caterina Maria Barbieri COORDINATORE: PROF. Paolo Gasparini SUPERVISORE DI TESI: PROF.SSA Daniela Toniolo PROF. Paolo Gasparini ANNO ACCADEMICO 2016/201 CONTENTS CONTENTS ............................................................................................................................ 4 LIST OF ABBREVIATION .................................................................................................. 6 INTRODUCTION .................................................................................................................. 7 Women fertility ........................................................................................................................... 8 Follicle development and ovarian reserve ...................................................................... 10 Age-related decline of female fertility .............................................................................. 12 Anti Mϋllerian Hormone ........................................................................................................ 13 A Biological role for AMH ................................................................................................................... 16 Genetic variation in reproductive ageing ........................................................................ 17 The resource of isolated cohorts ........................................................................................ 20 Val Borbera project .............................................................................................................................. 22 Friuli Venezia Giulia genetic park .................................................................................................. 24 Carlantino project ................................................................................................................................. 25 GWAS ............................................................................................................................................ 25 Statistical power limitation and the contribution of large consortia ..................... 28 Human knockout ...................................................................................................................... 29 ANALYSIS AND RESULTS ............................................................................................... 31 1. Rare coding variants and X-linked loci associated with age at menarche. .. 35 GWAS on X-chromosome ............................................................................................................................... 37 Gene expression data ...................................................................................................................................... 38 2. Genomic analyses identify hundreds of variants associated with age at menarche and support a role for puberty timing in cancer risk. ............................ 41 Implicated genes and tissue ......................................................................................................................... 42 Transcription factor binding enrichment .............................................................................................. 44 Pathway analyses .............................................................................................................................................. 44 Imprinted genes and parent-of-origin effects. ..................................................................................... 45 Disproportionate genetic effects on early or late puBerty timing. .............................................. 45 Effects of puberty timing on cancer risk ................................................................................................. 46 3. Large-scale genomic analyses link reproductive aging to hypothalamic signaling, breast cancer susceptibility and BRCA1-mediated DNA repair. .......... 47 ANM SNPs strongly enriched in DNA damage response pathways ............................................ 49 ANM SNPs enriched in known POI genes and correlation with other traits and disease . 50 4. Genome-wide analysis identifies 12 loci influencing human reproductive behavior. ..................................................................................................................................... 52 Causal variants ................................................................................................................................................... 54 eQTL and meQTL analyses ............................................................................................................................ 54 Functional network and polygenic prediction ..................................................................................... 55 Signal associated with related traits and disease ............................................................................... 55 5. WGS INGI data ................................................................................................................... 58 Loss of function and human knockouts ............................................................................ 60 GnRH2 knockout .................................................................................................................................... 61 6. Quantitative AMH GWAS with WGS INGI samples ................................................. 63 7. Fertility preservation in endometriosis patients: is AMH a reliable marker of the ovarian follicle density? ............................................................................................. 69 MATERIALS AND METHODS ........................................................................................ 72 INGI genotyping and imputation ......................................................................................... 73 CONTENTS Val Borbera samples ............................................................................................................................ 73 Friuli Venezia Giulia samples ........................................................................................................... 73 Carlantino samples ............................................................................................................................... 73 1. Rare coding variants and X-linked loci associated with age at menarche. (Lunetta et al., 2015) .................................................................................................................. 74 GWAS age at menarche on exome array ..................................................................................... 74 GWAS age at menarche on X chromosome ................................................................................ 74 2. Genomic analyses identify hundreds of variants associated with age at menarche and support a role for puberty timing in cancer risk. (Day et al., 2017) 75 GWAS age at menarche imputed to 1000G ................................................................................ 75 Parent-of-origin-specific associations and variance .............................................................. 75 Mendelian randomization analyses .............................................................................................. 75 Pathway analyses .................................................................................................................................. 76 Gene expression data integration .................................................................................................. 76 3. Large-scale genomic analyses link reproductive aging to hypothalamic signaling, breast cancer susceptibility and BRCA1-mediated DNA repair. (Day et al., 2015) ....................................................................................................................................... 77 GWAS age at menopause on all genome ...................................................................................... 77 GWAS age at menopause on exome chip data .......................................................................... 77 Conditional analysis ............................................................................................................................. 78 Causative gene identification ........................................................................................................... 78 Pathway identification ........................................................................................................................ 78 Estimating variance .............................................................................................................................. 78 4. Genome-wide analysis identifies 12 loci influencing human reproductive behavior. (Barban et al., 2016) ............................................................................................... 79 GWAS reproductive