Dymeclin, the Gene Underlying Dyggve-Melchior-Clausen
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Dymeclin, the gene underlying Dyggve-Melchior-Clausen syndrome, encodes a protein integral to extracellular matrix and Golgi organisation and is associated with protein secretion pathways critical in bone development. Celine Denais, Carolyn Dent, Laura Southgate, Jacqueline Hoyle, Dimitra Dafou, Richard Charles Trembath, Rajiv David Machado To cite this version: Celine Denais, Carolyn Dent, Laura Southgate, Jacqueline Hoyle, Dimitra Dafou, et al.. Dymeclin, the gene underlying Dyggve-Melchior-Clausen syndrome, encodes a protein integral to extracellular matrix and Golgi organisation and is associated with protein secretion pathways critical in bone development.. Human Mutation, Wiley, 2011, 32 (2), pp.231. 10.1002/humu.21413. hal-00612011 HAL Id: hal-00612011 https://hal.archives-ouvertes.fr/hal-00612011 Submitted on 28 Jul 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Human Mutation Dymeclin, the gene underlying Dyggve-Melchior-Clausen syndrome, encodes a protein integral to extracellular matrix and Golgi organisation and is associated with protein secretionFor pathways Peer critical Review in bone development. Journal: Human Mutation Manuscript ID: humu-2010-0467.R1 Wiley - Manuscript type: Research Article Date Submitted by the 25-Oct-2010 Author: Complete List of Authors: Denais, Celine; King's College London, Medical and Molecular Genetics Dent, Carolyn; King's College London, Medical and Molecular Genetics Southgate, Laura; King's College London, Medical and Molecular Genetics Hoyle, Jacqueline; King's College London, Medical and Molecular Genetics Dafou, Dimitra; King's College London, Medical and Molecular Genetics Trembath, Richard; King's College London, Medical and Molecular Genetics Machado, Rajiv; King's College London, Medical and molecular genetics Key Words: Dymeclin, Skeletal dysplasia, secretion, chondrogenesis, Golgi John Wiley & Sons, Inc. Page 1 of 32 Human Mutation 1 2 Title: Dymeclin, the gene underlying Dyggve-Melchior-Clausen syndrome, encodes 3 4 a protein integral to extracellular matrix and Golgi organisation and is associated 5 6 with protein secretion pathways critical in bone development. 7 8 9 10 1 1 1 1 11 Authors: Celine Denais , Carolyn L. Dent , Laura Southgate , Jacqueline Hoyle , 12 1 1* 1 13 Dimitra Dafou , Richard C. Trembath , Rajiv D. Machado . 14 15 16 17 Author’s affiliations: 18 1 For Peer Review 19 King’s College London, Department of Medical & Molecular Genetics, School of 20 21 Medicine, Guy’s Hospital, London, UK. 22 23 24 25 *Corresponding author: 26 27 Professor Richard C. Trembath, Professor of Medical Genetics, King’s College London, 28 Department of Medical & Molecular Genetics, School of Medicine, Floor 8 Tower Wing, 29 30 Guy’s Hospital, London SE1 9RT, United Kingdom. Tel: +44(0)2071887993; Fax: 31 32 +44(0)2071882585; Email: [email protected] 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 2 of 32 1 2 ABSTRACT 3 4 Dyggve-Melchior-Clausen syndrome (DMC), a severe autosomal recessive skeletal 5 6 disorder with mental retardation, is caused by mutation of the gene encoding Dymeclin. 7 8 Employing patient fibroblasts with mutations characterized at the genomic and, for the 9 10 11 first time, transcript level, we identified profound disruption of Golgi organization as a 12 13 pathogenic feature, resolved by transfection of heterologous wild-type Dymeclin. 14 15 Collagen targeting appeared defective in DMC cells leading to near complete absence of 16 17 cell surface collagen fibres. DMC cells have an elevated apoptotic index ( p<0.01) likely 18 For Peer Review 19 due to a stress response contingent upon Golgi-related trafficking defects. We performed 20 21 spatio-temporal mapping of Dymeclin expression in zebrafish embryos and identified 22 23 high levels of transcript in brain and cartilage during early development. Finally, in a 24 25 chondrocyte cDNA library, we identified two novel secretion pathway proteins as 26 Deleted : The tightly regulated cleavage and secretion of the Golgi-associated 27 Dymeclin interacting partners, GOLM1 and PPIB. Together these data identify the role of protein GOLM1 by furin, a cycling 28 protease, is severely¶ dysregulated in DMC. PPIB null 29 Dymeclin in secretory pathways essential to endochondral bone formation during early mutations lead to the skeletal dysplasia 30 osteogenesis imperfecta, characterized by delayed export of pro-collagen to the 31 development. Golgi apparatus. 32 33 34 35 KEY WORDS 36 37 Dymeclin, Skeletal dysplasia, secretion, chondrogenesis, Golgi. 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 3 of 32 Human Mutation 1 2 INTRODUCTION 3 4 The osteochondroplasias are a heterogeneous group of skeletal disorders, the result of 5 6 inherited defects in bone formation, growth and maintenance [Ortega et al., 2004; 7 Formatted: 8 Font color: Orange Superti-Furga and Unger, 2007 ]. To date, some 250 distinct clinical phenotypes have 9 10 11 been reported with an incidence of 1 in 4000 across European populations [Stoll et al., 12 13 1989]. Dyggve-Melchior-Clausen syndrome (DMC) is part of a sub-group of 14 15 osteochondroplasias known as the spondyloepimetaphyseal dysplasias (SEMDs) 16 17 characterised by malformations of the vertebrae, with abnormal ossification of long bone 18 For Peer Review 19 epiphyses and metaphyses. DMC is a progressive SEMD with additional clinical features 20 21 that include microcephaly, facial dysmorphism and variable mental retardation [Dyggve 22 23 et al., 1962; Toledo et al., 1979; Beighton, 1990]. Classical radiological features of DMC 24 25 include the appearance of lacy iliac crests, consistent with widespread bone abnormalities 26 27 of development and growth [Spranger et al., 1975], driven by deficient chondrocyte 28 29 organisation and differentiation with columnar structures that contain populations of 30 31 degenerating cells [Horton and Scott, 1982; Nakamura et al., 1997]. 32 33 34 35 DMC is a rare recessive disorder predominantly due to loss-of-function mutations in the 36 37 DYM gene (MIM ID: 607461) located at chromosome 18q12 [Cohn et al., 2003; El 38 39 Ghouzzi et al., 2003]. Defects in this gene also result in Smith-McCourt dysplasia 40 41 (SMC), an allelic variant of DMC without mental retardation [Ehtesham et al., 2002; 42 43 Cohn et al., 2003]. The ubiquitously expressed DYM transcript encodes Dymeclin, a 44 45 novel 669 amino acid protein under tight evolutionary conservation, which does not 46 Deleted : network 47 belong to a recognised protein family and has limited characteristics for functional 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 4 of 32 1 2 motifs. Bioinformatic analyses predict the presence of an N-myristoylation site, several 3 4 transmembrane loops and dileucine motifs but, to date, these have not been 5 Deleted : unambiguously 6 experimentally validated [Cohn et al., 2003; El Ghouzzi et al., 2003]. In short, the cellular 7 8 and molecular function of Dymeclin remains unclear, confounding efforts to better 9 10 11 elucidate the processes critical in the genesis of DMC and SMC. 12 13 14 15 In the present study, the cellular functions of Dymeclin have been investigated by a 16 17 combinatorial approach. This report is the first to delineate, in human patient cell lines 18 For Peer Review 19 characterised for Dymeclin loss-of-function mutations at the genomic and transcript 20 21 level, the profound consequences for Golgi and extracellular matrix organisation. Using a 22 23 zebrafish model, we demonstrate prominent head and cartilage DYM expression during 24 25 early development. By means of two yeast two-hybrid screens of protein-protein 26 27 interactions, we identified novel partners that provide insight into Dymeclin-associated 28 29 cellular function, likely to be critical for chondrocyte development and maintenance. 30 31 Together, these data reveal Dymeclin driven processes central to bone development 32 33 pathways, including Golgi organisation, Golgi-coupled protein secretion, and collagen 34 35 deposition in the extracellular matrix, thereby illuminating the likely molecular 36 37 mechanisms underlying DMC pathogenesis. 38 39 40 41 MATERIALS AND METHODS 42 43 Cell culture and transfection 44 45 Primary human wild-type (WT) fibroblasts were harvested from skin biopsies obtained 46 47 under ethics committee approval. All three mutant fibroblast lines were obtained from the 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 5 of 32 Human Mutation 1 2 Coriell Institute ( www.coriell.org ), (Table 1). Primary lines, HeLa and U2OS cells were 3 4 cultured in Dulbecco’s modified Eagle’s medium with Glutamax (DMEM, GIBCO life 5 6 technologies) supplemented with 10% foetal bovine serum at 37 °C and 1% 7 8 penicillin/streptomycin. Transient transfections of HeLa and U2OS cells were performed 9 10 11 using Lipofectamine 2000 (Invitrogen) and FuGENE (Roche) respectively in accordance 12 13 with manufacturers’ instructions. 14 15 16 17 DNA, RNA isolation and RT-PCR 18 For Peer Review 19 DNA and total RNA was isolated from fibroblast cell lines and whole zebrafish embryos 20 21 using TRI Reagent (Sigma Aldrich) according to manufacturer’s instructions. cDNA was 22 23 generated by priming with oligo dT using the First strand synthesis SuperScript II kit 24 25 (Invitrogen).