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Chapter 1: Introduction A Clinical and Molecular Study of the Growth Disorder 3-M Syndrome A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Medical and Human Sciences 2011 Philip George Murray School of Medicine Faculty of Medicine and Human Sciences Table of Contents List of Figures ................................................................................................ 6 List of Tables ................................................................................................. 11 Abstract ........................................................................................................ 16 Declaration ................................................................................................... 17 Copyright Statement ..................................................................................... 18 Acknowledgements ...................................................................................... 19 Attributed Work ........................................................................................... 19 Chapter 1: Introduction ................................................................................ 20 1.1 Overview ............................................................................................... 21 1.2 Normal Growth and Short Stature ................................................................. 22 1.3 The Small for Gestational Age Infant ............................................................. 23 1.3.1 Definition ....................................................................................................... 23 1.3.2 Hormonal Control of Intra-uterine Growth ................................................... 24 1.3.3 Outcomes for the SGA child .......................................................................... 25 1.3.4 Management of the SGA Child ...................................................................... 26 1.4 3-M syndrome .............................................................................................. 27 1.4.1 Differential diagnosis of 3-M syndrome ........................................................ 34 1.5 Identification of mutations in CUL7 in 3-M syndrome .................................... 43 1.5.1 CUL7 gene and Mouse knockout models ...................................................... 44 1.5.2 The Ubiquitination-Proteasomal System ...................................................... 47 1.5.4 CUL7, apoptosis and p53 ............................................................................... 53 1.5.5 CUL7, the cell cycle and IGF-I signaling ......................................................... 55 1.5.6 Expression and methylation of CUL7 in IUGR placental tissue ..................... 56 1.6 OBSL1........................................................................................................... 60 1.7 Aims ............................................................................................................. 64 1.8 Key points .................................................................................................... 65 Chapter 2: Materials and Methods ................................................................ 66 2.1 Introduction ................................................................................................. 67 2.2 Clinical data .................................................................................................. 67 2.3 Statistical analysis ........................................................................................ 67 2.4 Tissue and Cell Culture Procedures ................................................................ 68 2.4.1 Skin biopsy technique and establishing fibroblast cell lines ......................... 68 2.4.2 Routine growth culture and passaging ......................................................... 68 2.4.3 Long term storage of cells ............................................................................. 69 2.4.4 Cell counting .................................................................................................. 69 2.4.5 Generation of Cell Lysates ............................................................................. 69 2.4.6 GH and IGF-1 stimulation .............................................................................. 70 2.4.7 Generation of cell culture conditioned media .............................................. 70 2.4.8 WST-8 cell growth assay ................................................................................ 70 2.4.9 5-ethynyl-2'-deoxyuridine (EdU) incorporation ............................................ 70 2.4.10 Terminal dUTP nick end labelling (TUNEL) staining .................................... 71 2.5 Protein based methods ................................................................................. 71 2.5.1 Bradford Assay of protein concentration ...................................................... 71 2.5.2 Precipitation of protein from Conditioned cell culture media ...................... 72 2 2.5.3 SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western Immunoblotting ..................................................................................................... 72 2.5.4 Stripping of Western Blots ............................................................................ 73 2.5.5 Measurement of IGF-2 in cell culture medium ............................................. 73 2.5.6 Measurement of serum IGF-2 and IGFBP-3 .................................................. 74 2.5.7 Immunoflourescence microscopy ................................................................. 74 2.5.8 Cleaved Caspase-3 ELISA ............................................................................... 74 2.6 Nucleic Acid based techniques ...................................................................... 75 2.6.1 Extraction of DNA .......................................................................................... 75 2.6.2 Extraction of RNA .......................................................................................... 75 2.6.3 Measurement of DNA and RNA concentration ............................................. 75 2.6.4 Reverse Transcription .................................................................................... 75 2.6.5 Polymerase chain reaction ............................................................................ 75 2.6.6 Agarose gel electrophoresis .......................................................................... 76 2.6.7 Purification of DNA from PCR products ........................................................ 76 2.6.8 DNA sequencing ............................................................................................ 76 2.6.9 Quantitative Reverse Transcription Polymerase Chain Reaction (QPCR) ..... 77 2.7 Gene Expression Microarrays ........................................................................ 79 2.7.1 Preparation of RNA and hybridization to Affymetrix HU-133 plus 2.0 chip .. 79 2.7.2 Software and statistical analysis of gene expression microarrays ................ 79 2.8 Cellular Metabolomic Studies ....................................................................... 80 2.8.1 Metabolite Extraction ................................................................................... 80 2.8.3 Metabolome Statistical Analysis ................................................................... 80 2.9 Fertilisation of Xenopus tropicalis oocytes and microinjection of Morpholino Oligonucleotides ................................................................................................ 81 2.9.1 Obtaining Oocytes ......................................................................................... 81 2.9.2 Obtaining Sperm............................................................................................ 81 2.9.3 Fertilisation and Preparation for Microinjection .......................................... 82 2.9.4 Microinjection ............................................................................................... 82 2.9.5 Embryo selection and husbandry .................................................................. 83 2.10 Suppliers .................................................................................................... 83 2.10.1 Antibodies Used .......................................................................................... 84 2.11 Buffers and Solutions .................................................................................. 84 Chapter 3: Patients ....................................................................................... 87 3.1 Introduction and Aims .................................................................................. 88 3.2 Patients with Cullin 7 mutations ................................................................... 88 3.3 Patients with Obscurin-Like 1 mutations ....................................................... 94 3.4 Patients with no identified mutation in OBSL1 or CUL7 ................................ 107 3.5 Phenotype-Genotype comparison ............................................................... 111 3.6 Response to treatment with recombinant human growth hormone ............. 119 3.7 IGFBP3 and IGF-I levels in 3-M syndrome patients ....................................... 121 3.9 Key Points .................................................................................................. 122 Chapter 4: Functional studies on patient
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