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 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...... 40 4.2.3.1 ERI1: A candidate gene for a novel autosomal recessive SEMD syndrome associated with cutaneous syndactyly and early lethality...... 40 4.2.3.2 MEGF8: A candidate gene for a autosomal recessive SEMD resembling pseudoachondroplasia...... 42 4.2.3.3 KDM5C: A novel candidate gene for Angel-Shaped-Phalango-Epiphyseal .....Dysplasia?43 4.2.4 Skeletal Dysplasias: Novel mutations and further delineation of the clinical spectrum...... 45 4.2.4.1 PAM16: A novel mutation in a patient with a milder phenotype and longer...... survival45 4.2.4.2 IARS2: Confirmation of CAGSSS syndrome and the proposal of novel bone dysplasia family related to mitochondrial dysfunction...... 48 4.2.4.3 Metatarsal bony syndactyly in two fetuses with Smith-Lemli-Opitz syndrome: an under­ recognized part of the clinical spectrum...... 50 4.2.4.4 Severe spondylar dysplasia in a patientCOL10A1 with a mutation ...... 53 4.2.4.5 A novelCOL10A1 mutation underlies SMD comer fracture...... type 54 4.2.4.6 A novel homozygousNPR2 mutation in an Algerian patient thought to have achondroplasia 56 4.2.4.7 The complex phenotypesB3GAL of 76-associated disorders: the long road to a definitive diagnosis...... 57 4.3 SYNDROMIC SHORT STATURE SYNDROMES...... 59 4.3.1 Novel Genes Underlying Syndromic Short Stature...... 59 4.3.1.1 MutationsCKAP2L in cause Filippi syndrome...... 59 4.3.2 Novel Candidate Genes For Dubowitz Syndrome...... 61 4.3.2.1 First evidence implicatingde novoa mutation inUSP20 in Dubowitz syndrome...... 61 4.3.2.2 Probable digenic inheritance: 2 novel candidate genes to explain the severe immunological phenotype and the Dubowitz syndrome phenotype in a single patient...... 70 4.3.2.3 KREMEN1: another player in autosomal recessively inherited Dubowitz syndrome?...... 72 4.3.2.4 A novel mutationDYRK1A in in a patient with a clinical diagnosis of Dubowitz syndrome...... 72 4.3.3 Syndromic Short Stature Syndromes: Novel Mutations and Further Delineation of The Clinical Spectrum...... 73 4.3.3.1 Novel mutationTELÖ2 in : severe expression of You-Hoover-Fong syndrome...... 73 4.3.3.2 Novel mutationCDK5RAP2 in causes severe intra-uterine growth retardation and ...... 77 4.3.3.3 Novel mutationVPS13B in in an Algerian family and intra-familial variability in Cohen syndrome...... 78 4.4 THE MENDELIOME STUDY...... 79 4.4.1 Mendeliome-Testing Process...... 79 4.4.2 Mutation Mining And The “MM-Team” Workflow...... 80 4.4.3 Patients and Mutations...... 81 4.4.4 Novel Genes...... 84 4.4.4.1 AP3B2: A novel gene underlying microcephaly...... 85 4.4.4.2 PRMT7 : A novel gene underlying syndromic intellectual...... disability 85 4.4.4.3 RNF213: A novel gene underlying systemic sclerosis...... 85 4.4.5 Novel Phenotypes...... 86 4.4.5.1 USP9X: X-linked dominant Syndromic Intellectual Disability...... 87 4.4.5.2 BMP1: Syndromic short stature with deficiency...... 88 4.4.5.3 PMM2: Micro-lissencephaly...... 88 4.4.S.4 RARS2: Severe ID with cerebral atrophy...... 89 4.4.6 Microdeletion Syndromes...... 89 4.4.6.1 M12: Microdeletion 18...... 89 4.4.6.2 M l3: Microdeletion Chromosome 7 ...... 89 4.4.6.3 M14: Microdeletion Chromosome(ACSL4) X ...... 90 4.4.7 A Family with 2 Children with Different Genetic Disorders...... 91 4.4.8 Results from the Beta Test of the Updated Mendeliome...... 92 4.4.9 Novel Mutations...... 92 5 DISCUSSION...... 93 5.1 NGS ENDS THE DIAGNOSTIC ODYSSEY...... 93 5.2 NOVEL GENES FOR OSTEOGENESIS IMPERFECTA...... 93 5.2.1 Autosomal Recessive MutationsMESDC2 In Cause Osteogenesis Imperfecta...... 94 5.2.2 SEC24D: A Novel Autosomal Recessive 01 Gene...... 97 5.2.3 HYAL4: An Interesting Novel Candidate Gene For Autosomal Recessive...... OI 99 5.3 SKELETAL DYSPLASIAS...... 99 5.3.1 ER11: A Novel Mechanism Implicated In Skeletal Dysplasia...... 99 5.3.2 MEGF8: A Novel Candidate For An Autosomal Recessive SEMD Resembling Pseudoachondroplasia...... 100 5.3.3 Implications of De Novo VariantKDM5C in - A Novel ASPED Gene?...... 101 5.3.4 Patient with a Novel MutationPAM16 in Shows That SMDMDM Is Not Always Associated With Early Demise...... 102 5.3.1 Confirmation of CAGSSS Syndrome and a New Mitochondrial Bone Dysplasia...... 103 Family 5.3.2 Confirmation of Bony Syndactyly as an Under-Recognized Part of the SLOS Clinical Spectrum...... 107 5.3.3 Novel Findings In Patients With MutationsCOL10A1 In ...... 108 5.3.1 AMDM: Correct Diagnosis Facilitates Accurate Genetic Counselling...... 109 5.3.2 B3GALT6: Ending The 15 Year Diagnostic Odyssey...... 109 5.4 SHORT STATURE SYNDROMES...... 110 5.4.1 Mutations CKAP2LIn Cause Filippi Syndrome...... 110 5.4.2 Dubowitz Syndrome: Genetic Heterogeneity The Rule Rather Than The Exception...... 111 5.4.2.1 USP20: A novel player in DNA damage repair...... I ll 5.4.2.2 FAT1/FYB: Digenic inheritance may explain complex phenotype...... 113 5.4.2.3 KREMEN1: A further underling cause for Dubowitz syndrome?...... 114 5.4.2.4 DYRK1A: A novel mutation in a patient with a clinical diagnosis of Dubowitz syndrome 115 5.4.3 Novel MutationsTEL02 in and expanding the YHFS clinical spectrum...... 116 5.4.4 Further Novel Mutations for Syndromic Short...... Stature 117 5.5 THE MENDELIOME STUDY...... 118 5.6 AFTER GENE IDENTIFICATION - NEXT STEPS?...... 120 6 CONCLUSION AND OUTLOOK______121 7 REFERENCES...... 124 8 ACKNOWLEDGEMENTS______132 9 APPENDICES______135 9.1 APPENDIX 1: TABLE A 1...... 135 9.2 APPENDIX 2: TABLE A 2...... 141 9.3 APPENDIX 3: SCIENTIFIC PRESENTATIONS BASED ON THIS PHD...... 148 9.4 CURRICULUM VITAE/LEBENSLAUF ...... Error! Bookmark not defined. 9.5 ERKLÄRUNG...... 149