Table of contents 1 INTRODUCTION........................................................................................................................................... 1 1.1 MEDICAL GENETICSAND THE GENOMIC REVOLUTION.............................................................1 1.2 GENETIC DISORDERS: AN OVERVIEW............................................................................................1 1.3 CONVENTIONAL DIAGNOSTICS AND THE DIAGNOSTIC ODYSSEY....................................... 2 1.4 SANGER AND THE HUMAN GENOME PROJECT...........................................................................3 1.5 THE NEXT-GENERATION-SEQUENCING TECHNOLOGY REVOLUTION................................4 1.6 NGS MODALITIES USED IN THIS STUDY........................................................................................ 5 1.6.1 Whole-Exome Sequencing (WES)..................................................................................................... 5 1.6.2 TruSight™ One Sequencing Panel: “The Mendeliome” ....................................................................6 1.7 AN OVERVIEW OF SYNDROMIC SHORT STATURE SYNDROMES........................................... 7 1.8 THE SKELETAL DYSPLASIAS............................................................................................................8 1.8.1 Osteogenesis Imperfecta.....................................................................................................................9 1.8.1.1 Type I Collagen biosynthesis..................................................................................................10 1.8.1.2 Defects in osteoblast development and the WNT-signalling................................... pathway 11 1.8.2 Undiagnosed Skeletal Dysplasias......................................................................................................14 1.9 SELECTED SHORT STATURE SYNDROMES..................................................................................14 1.9.1 Dubowitz Syndrome......................................................................................................................... 14 1.9.1.1 C.elegam as a model organism to study DNA damage response........................................... 18 1.9.2 Undiagnosed Short Stature................................................................................................................18 2 AIMS ............................................................................................................................................................19 3 METHODS AND MATERIALS................................................................................................................. 20 3.1 PATIENT RECRUITMENT AND INFORMED CONSENT..............................................................20 3.1.1 Ethics approval..................................................................................................................................20 3.1.2 Patients and Families.........................................................................................................................20 3.2 MATERIALS........................................................................................................................................20 3.2.1 Reagents/Buffers/Enzymes...............................................................................................................20 3.2.2 Kits....................................................................................................................................................21 3.2.3 Antibodies.........................................................................................................................................21 3.2.4 Primers.............................................................................................................................................. 21 3.2.5 Web resources...................................................................................................................................22 3.3 METHODS............................................................................................................................................22 3.3.1 Nucleic acid analysis.........................................................................................................................22 3.3.1.1 DNA extraction........................................................................................................................22 3.3.1.2 Polymerase Chain Reaction (PCR)..........................................................................................22 3.3.1.3 Agarose gel electrophoresis and gel extraction....................................................................... 23 3.3.1.4 Sanger sequencing.................................................................................................................... 23 3.3.2 Molecular cloning.............................................................................................................................23 3.3.2.1 Exon trapping..........................................................................................................................23 3.3.2.2 GenerationMESDC2 of mutant constructs............................................................................. 23 3.3.3 NGS technologies.............................................................................................................................24 3.3.3.1 Whole-Exome Sequencing (WES)...........................................................................................24 3.3.3.2 TruSight™ One Panel: Mendeliome...................................................................................... 24 3.3.3.3 NGS Data Analysis..................................................................................................................24 3.3.3.4 NGS Data Filtering strategy................................................................................................... 25 3.3.3.5 Variant validation and curation.............................................................................................. 25 3.3.4 Cell culture........................................................................................................................................25 3.3.4.1 Cell proliferation......................................................................................................................25 3.3.4.2 USP20-deletion mutant HAP1 cell-line.................................................................................25 3.3.4.3 Cell transfection.......................................................................................................................26 3.3.4.4 Cell treatment..........................................................................................................................26 3.3.5 Immunological Methods...................................................................................................................26 3.3.5.1 Western blot............................................................................................................................26 3.3.5.2 Immunofluorescent (IF) staining............................................................................................ 27 3.3.6 C.elegans- specific methods............................................................................................................ 27 3.3.6.1 Solutions / media / strains........................................................................................................27 3.3.6.2 Synchronization of worm cultures...........................................................................................27 3.3.6.3 Single worm lysis for genotyping............................................................................................28 3.3.6.4 Ionizing radiation and UV radiation........................................................................................ 28 3.3.6.5 HU treatment............................................................................................................................ 28 3.3.6.6 Embryonic survival assay.........................................................................................................28 3.4 STATISTICS.......................................................................................................................................... 28 4 RESULTS........................................................................................................................................................29 4.1 MOLECULAR GENETIC TESTING...................................................................................................29 4.2 SKELETAL DYSPLASIAS.................................................................................................................. 30 4.2.1 Osteogenesis imperfecta: Novel Genes........................................................................................... 30 4.2.1.1 Autosomal recessive mutationsMESDC2 in cause osteogenesis imperfecta....................... 30 4.2.1.2 MutationsSEC24D in cause non-syndromic 01 ...................................................................37 4.2.2 Osteogenesis imperfecta: Novel Candidate Gene............................................................................ 39 4.2.2.1 HYAL4 - a novel gene underlying autosomal recessive................................................. 01? 39 4.2.3 Skeletal dysplasias: Novel Candidate Genes..................................................................................