UNIVERSITA’ DEGLI STUDI DI MILANO Scuola di Dottorato in Scienze Biomediche Cliniche e Sperimentali Corso di Dottorato in Biotecnologie Applicate alle Scienze Mediche Ciclo XXIV Tesi di dottorato di ricerca DNA METHYLATION AS A PREDISPOSITION FACTOR IN THE PATHOGENESIS OF CONGENITAL HYPOTHYROIDISM IN PRETERM INFANTS Facoltà di Medicina e Chirurgia Settore Scientifico Disciplinare Bio13 Coordinatore: Prof. Enrico GINELLI Tutor: Prof. Luca PERSANI Dottorando: Federica MARELLI Matricola n. R08135 Anno Accademico 2011-2012 Table of contests TABLE OF CONTESTS ................................................................................................................... 2 ABSTRACT ................................................................................................................................... 5 INTRODUCTION-CHAPTER I ......................................................................................................... 7 1. THYROID GLAND DEVELOPMENT AND DEFECTS ...................................................................... 7 1.1 FETAL THYROID DEVELOPMENT: A BRIEF OVERVIEW ............................................................ 7 1.1.1 MORPHOLOGICAL ASPECTS ................................................................................................ 7 1.1.2 GENETIC COMPONENT ON THYROID DEVELOPMENT ....................................................... 10 1.2 THYROID HORMONE METABOLISM AND ACTION ............................................................... 11 1.3 CONGENITAL HYPOTHYROIDISM ......................................................................................... 15 1.3.1 THYROID DYSGENESIS ...................................................................................................... 15 1.3.2 DYSORMONOGENESIS ...................................................................................................... 17 1.3.3 INCIDENCES OF CONGENITAL HYPOTHYROIDISM ............................................................. 18 INTRODUCTION-CHAPTER II ...................................................................................................... 22 2. EPIGENETIC MECHANISMS .................................................................................................... 22 2.1 A BRIEF OVERVIEW ............................................................................................................. 22 2.2 EPIGENETIC COMPONENTS ................................................................................................. 23 2.2.1 HISTONES MODIFICATIONS .............................................................................................. 23 2.2.2 MICRORNAS ..................................................................................................................... 24 2.3 THE DNA METHYLATION ..................................................................................................... 26 2.3.1 INTRODUCTION OF DNA METHYLATION........................................................................... 26 2.3.2 5′-METHYLCYTOSINE AND THE CPG DINUCLEOTIDE .......................................................... 28 2.3.3 GENOMIC DISTRIBUTION OF 5′-METHYLCYTOSINE ........................................................... 31 2.3.4 CPG SITES HELP DETERMINE GENE EXPRESSION STATE .................................................... 34 2.4 ROLES OF DNA METHYLATION ............................................................................................ 35 2.4.1 TRANSPOSON SUPPRESSION-DNA METHYLATION AS A GENETIC DEFENSE ...................... 35 2.4.2 DNA METHYLATION AND DEVELOPMENT ........................................................................ 36 2.4.3 X CHROMOSOME INACTIVATION ..................................................................................... 38 2.4.4 IMPRINTING ..................................................................................................................... 39 INTRODUCTION-CHAPTER III ..................................................................................................... 41 3. DNA METHYLATION AND HUMAN DISEASES ......................................................................... 41 3.1 EPIGENETIC DYSREGULATION IN HUMAN DISEASES ............................................................ 41 3.1.1 DNA METHYLATION AND CANCER .................................................................................... 41 2 3.1.2 DNA METHYLATION AND REPRODUCTION ....................................................................... 42 3.1.3 DNA METHYLATION AND CARDIOVASCULAR DISEASE ...................................................... 43 3.1.4 DNA METHYLATION AND TYPE 2 DIABETES ...................................................................... 44 3.1.5 DNA METHYLATION AND OBESITY .................................................................................... 45 3.2. DEVELOPMENTAL ORIGIN OF HEALTH AND DISEASE HYPOTHESIS (DOHAD) ...................... 46 3.2.1 FETAL PROGRAMMING ..................................................................................................... 46 3.2.2 RETROSPECTIVE STUDIES .................................................................................................. 50 3.2.3 EXPERIMENTAL STUDIES ................................................................................................... 51 CHAPTER IV ............................................................................................................................... 53 4. AIMS OF PHD PROJECT .......................................................................................................... 54 CHAPTER V ................................................................................................................................ 55 5. MATERIALS AND METHODS ................................................................................................... 55 5.1 SAMPLES COLLECTION AND DNA EXTRACTION .................................................................... 56 5.1.1 RECRUITMENT OF PATIENTS ............................................................................................. 56 5.1.2 DNA ISOLATION FROM PERIPHERAL BLOOD SAMPLES ..................................................... 56 5.1.3 DNA QUALITY AND QUANTITY .......................................................................................... 58 5.2 DNA METHYLATION ANALYSIS............................................................................................. 59 5.2.1 DNA BISULFITE TREATMENT ............................................................................................. 59 5.2.2 INFINIUM ASSAY FOR METHYLATION ............................................................................... 60 5.3 ANALYSIS OF DNA METHYLATION DATA .............................................................................. 63 5.3.1 EXPERIMENTAL DESIGN AND QUALITY CONTROL ............................................................. 63 5.3.2 PRINCIPAL COMPONENT ANALYSIS .................................................................................. 64 5.3.3 DETERMINATION OF DIFFERENTIALLY METHYLATED CPG SITES........................................ 64 5.4 BIOLOGICAL PROCESSES AND PATHWAY ANALYSIS ............................................................. 66 5.4.1 FUNCTIONAL ANNOTATION OF GENE LISTS ...................................................................... 66 5.4.2 INDIVIDUAL GENE INFORMATION .................................................................................... 66 5.5 ANALYSIS OF MTHFR POLYMORPHISMS .............................................................................. 67 CHAPTER VI ............................................................................................................................... 68 6. RESULTS ................................................................................................................................ 68 6.1 DESIGN OF DNA METHYLATION STUDY ............................................................................... 68 6.1.1 STATISTICAL METHODS .................................................................................................... 68 6.1.2 COLLECTIONS OF CH AND CONTROL SUBJECTS ................................................................. 69 6.1.3 DESIGN OF DNA METHYLATION STUDIES .......................................................................... 71 6.1.4 CATEGORIZATION OF SUBJECTS ACCORDING TO THEIR CLINICAL DATA ........................... 74 3 6.2 ANOVA STATISTICAL ANALYSIS ........................................................................................... 78 6.3 CLUSTERING OF SUBJECTS ................................................................................................... 80 6.4 GLOBAL DNA METHYLATION ANALYSIS ............................................................................... 82 6.4.1 REPRESENTATION OF DNA METHYLATION TRENDS .......................................................... 82 6.4.2 COMPARISON OF MEAN-AVGBETA VALUES ..................................................................... 84 6.4.3 SUMMARY OF GLOBAL METHYLATION ANALYSIS RESULTS .............................................. 87 6.5 GENE-SPECIFIC DNA METHYLATION ANALYSIS .................................................................... 88 6.5.1 SELECTION OF DIFFERENTIALLY METHYLATED GENES (DMGS) ......................................... 88 6.5.2 SELECTION OF “COMMON GENES” ................................................................................... 90 6.5.3
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