Genetic studies on recurrent miscarriage

____________________________________________________________ 9 Introduction ________________________________________________________ 11 Review of the literature ________________________________________________ 12 1 Early human embryonic development _______________________________ 12 1.1 Fertilization __________________________________________________ 12 1.2 Implantation __________________________________________________ 12 1.3 Placentation __________________________________________________ 14 1.4 Gastrulation and organogenesis ___________________________________ 14 2 Recurrent miscarriage ____________________________________________ 15 2.1 Identifiable causes of miscarriage _________________________________ 16 2.1.1 Risk factors for miscarriage __________________________________ 19 2.2 Genetic factors causing miscarriage _______________________________ 19 2.2.1 Chromosomal abnormalities __________________________________ 20 2.2.2 Skewed X chromosome inactivation ___________________________ 21 2.2.3 Y chromosome microdeletions ________________________________ 21 2.3. 4 Gene mutations and polymorphisms ___________________________ 22 2.2.5 Aberrant placental gene expression ____________________________ 24 2.2.6 Epigenetic mechanisms _____________________________________ 25 3 Identification of genetic causes underlying RM ________________________ 26 3.1 Identification of disease genes ____________________________________ 27 3.1.1 The Human Genome Project _________________________________ 27 3.1.2 Traditional identification of disease genes _______________________ 28 3.1.3 The candidate gene approach _________________________________ 28 3.1.4 Mutation screening _________________________________________ 29 3.1. 5 Confirming the nature of a new genetic variation _________________ 30 3.2 Mouse as a model organism _____________________________________ 30 3.3 Candidate genes for RM ________________________________________ 32 3.3.1 Amnionless _______________________________________________ 32 3.3.2 Thrombomodulin and Endothelial Protein C Receptor _____________ 35 3.3.3 p53 _____________________________________________________ 38 3.3. 4 The mitochondrial genome ___________________________________ 39 Aims _______________________________________________________________ 43 Materials and methods ________________________________________________ 44 1 Study subjects ___________________________________________________ 44 1.1 Patients (I, II, III, IV, V) ______________________________________ 44 1.2 Controls (I, II, III, IV, V) ______________________________________ 44 1.3 Placental samples (III, IV) _____________________________________ 45 1.4 Ethical issues (I, II, III, IV, V) __________________________________ 45 2 Methods ________________________________________________________ 45 2.1 Genealogic analysis (I) _______________________________________ 45 2.2 DNA extraction (I, III) ________________________________________ 45 2.3 Candidate gene analysis _______________________________________ 46 2.4 Immunohistochemical analysis of p53 (III) ________________________ 48 2.5 X chromosome inactivation analysis (V) __________________________ 48 2.6 Y chromosome microdeletion analysis (V) ________________________ 48 2.7 Statistical analysis (I, II, III, IV, V) ______________________________ 49 Results and discussion ________________________________________________ 50 1 Genealogic studies (I) _____________________________________________ 50 2 Candidate gene analysis (I, II, III, IV) _______________________________ 52 2.1 Amnionless (I) ________________________________________________ 52 2.1.1 Variations in the Amnionless gene _____________________________ 52 2.2 Thrombomodulin and Endothelial Protein C Receptor (II, unpublished) ___ 54 2.2.1 Variations in the TM gene ___________________________________ 54 2.2.2 Variations in the EPCR gene _________________________________ 55 2.2.3 TM/EPCR variations and outcome of heparin treatment ____________ 56 2.3 p53 (III, unpublished) __________________________________________ 57 2.3.1 Variations in the p53 gene ___________________________________ 57 2.3.2 p53 expression in RM placenta ________________________________ 58 2.4 Mitochondrial genes (IV) ________________________________________ 59 2.4.1 Variations in the mitochondrial genome _________________________ 60 2.4.2 Analysis of placental samples _________________________________ 62 3 The impact of polymorphisms on phenotype __________________________ 63 3.1 Variations in the nuclear genome (I, II, III, unpublished) _______________ 63 3.1.1 Possible splice site variations _________________________________ 65 3.1.2 Couple analysis _____________________________________________ 66 3.2 Variations in the mitochondrial genome (IV) ________________________ 67 4 Analysis of sex chromosome characteristics (V) _______________________ 68 4.1 X chromosome inactivation analysis _______________________________ 68 4.2 Y chromosome microdeletion analysis _____________________________ 71 5 Limitations of the study ___________________________________________ 73 Conclusions _________________________________________________________ 74 Acknowledgements ___________________________________________________ 77 References __________________________________________________________ 79