UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New Series No. 1259 ISSN 0346-6612 ISBN 978-91-7264-753-4 Clinical and Genetic Studies of Three Inherited Skeletal Disorders Eva-Lena Stattin From the Department of Medical Biosciences Medical and Clinical Genetics Umeå University SE-901 85 Umeå Sweden 2009 Copyright© 2009 by Eva-Lena Stattin New Series No. 1259 ISBN: 978-91-7264-753-4 ISSN: 0346-6612 Cover: Pedigree, familial osteochondritis dissecans Printed by: Hemströms tryckeri, Härnösand, Sweden 2009 To Pär, Kalle and Oskar 3 Contents CONTENTS .......................................................................................................................4 PUBLICATIONS...............................................................................................................6 THESIS AT A GLANCE ..................................................................................................7 ABSTRACT .......................................................................................................................8 ABBREVIATIONS............................................................................................................9 M0B EDICAL GENETICS ............................................................................................ 11 Human28B genome................................................................................................................. 11 DNA29B structure...................................................................................................................11 Characteristics30B of Mendelian inheritance.......................................................................... 11 Inter31B individual variations................................................................................................. 12 INTRODUCTION ...........................................................................................................14 The15B Skeleton................................................................................................ 14 Extracellula16B r matrix ...................................................................................... 14 Cartila17B ge....................................................................................................... 15 C1B OMPONENTS OF THE EXTRACELLULAR MATRIX NETWORK.................................... 16 The18B large aggregating Proteoglycans.......................................................... 16 ACAN-gene19B and Aggrecan .......................................................................... 16 Molecular32B gene structure, organization and function........................................................ 16 C2B HONDRODYSPLASIAS DUE TO AGGRECAN DEFECTS............................................. 18 C3B HONDRODYSPLASIAS DUE TO AGGRECAN DEFECTS - ANIMAL MODELS ................. 19 Cartilage3B matrix deficiency............................................................................................... 19 Cartilage34B matrix deficiency-Bc......................................................................................... 19 Nanomelia......................................................................................................................35B ... 19 Bulldog36B dwarfism ............................................................................................................. 19 C4B HONDRODYSPLASIAS DUE TO AGGRECAN DEFECTS – HUMAN DISORDERS............ 20 Spondyloepiphyseal37B dysplasia type Kimberley (SEDK)................................................... 20 Spondyloepimetaphyseal38B dysplasia (SEMD-aggrecan-type) ............................................ 20 Familial39B osteochondritis dissecans.................................................................................... 20 Disorders associated with defective sulfation of proteoglycans .................. 21 Degenerative20B diseases involving aggrecan ................................................. 21 VNTR-region40B of the ACAN-gene and OA ....................................................................... 21 Age-related41B changes of aggrecan...................................................................................... 22 C5B OMPONENTS OF THE EXTRACELLULAR MATRIX NETWORK.................................... 22 The42B Collagens...................................................................................................................22 CHONDRODYSPLASIA DUE TO DEFECTS IN ECM-PROTEINS ................................... 23 Multiple43B epiphyseal dysplasia........................................................................................... 23 SKELETONENESIS ............................................................................................... 23 The2B Indian Hedgehog (IHH)-gene; an important gene for skeletal development................................................................................................. 24 Brachydactyly4B type A1 ..................................................................................................... 25 AIMS OF THE THESIS .................................................................................................26 4 METHODOLOGY ..........................................................................................................27 Ethics.........................................................................................................................45B ....... 27 Introduction23B to field work .............................................................................. 27 Collecting46B clinical material............................................................................................... 27 Evaluation47B of suspected skeletal dysplasias...................................................................... 27 Field24B work..................................................................................................... 27 Study48B I and II (Familial OCD).......................................................................................... 27 Study49B III (MED) ............................................................................................................... 28 Study50B IV (BDA1).............................................................................................................. 28 Methods25B ....................................................................................................... 29 Statistical51B analysis............................................................................................................. 29 Linkage52B analysis ............................................................................................................... 29 Molecular26B genetics methods ........................................................................ 29 DNA53B extraction................................................................................................................. 29 PCR .................................................................................................................................. 29 Genome-wide54B scan ........................................................................................................... 30 Sequencing/Candidate5B gene analysis ................................................................................ 30 Interpretation56B of identified variations ............................................................................... 31 DHPLC..........................................................................................................................57B ... 31 Restriction58B endonuclease digestion of the PCR product ................................................... 32 Protein59B interaction analysis............................................................................................... 32 Mass60B spectrometry............................................................................................................ 33 RESULTS AND DISCUSSION ......................................................................................34 P7B APER I ............................................................................................................. 34 P8B APER II ............................................................................................................ 36 P9B APER III ........................................................................................................... 39 P10B APER IV ........................................................................................................... 41 Concluding27B remarks and future perspectives .............................................. 43 POPULÄRVETENSKAPLIG SAMMANFATTNING PÅ SVENSKA ......................44 I1B NTRODUKTION ................................................................................................... 44 M12B ANUSKRIPT I & II .............................................................................................. 44 M13B ANUSKRIPT III .................................................................................................. 45 M14B ANUSKRIPT IV.................................................................................................. 46 ACKNOWLEDGEMENTS ............................................................................................47 REFERENCES ................................................................................................................49 5 Publications This thesis is based on the following original publications, which will be referred to by their roman numerals: