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D 911 OULU 2007 D 911

UNIVERSITY OF OULU P.O. Box 7500 FI-90014 UNIVERSITY OF OULU FINLAND ACTA UNIVERSITATIS OULUENSIS ACTA UNIVERSITATIS OULUENSIS ACTA D SERIES EDITORS Marja Majava MEDICA MarjaMajava ASCIENTIAE RERUM NATURALIUM Professor Mikko Siponen MOLECULAR GENETICS OF BHUMANIORA STICKLER AND MARSHALL Professor Harri Mantila SYNDROMES, AND THE ROLE CTECHNICA Professor Juha Kostamovaara OF COLLAGEN II AND OTHER DMEDICA Professor Olli Vuolteenaho CANDIDATE PROTEINS IN HIGH MYOPIA AND ESCIENTIAE RERUM SOCIALIUM Senior Assistant Timo Latomaa IMPAIRED HEARING FSCRIPTA ACADEMICA Communications Officer Elna Stjerna

GOECONOMICA Senior Lecturer Seppo Eriksson

EDITOR IN CHIEF Professor Olli Vuolteenaho EDITORIAL SECRETARY Publications Editor Kirsti Nurkkala FACULTY OF MEDICINE, DEPARTMENT OF MEDICAL BIOCHEMISTRY AND MOLECULAR BIOLOGY, COLLAGEN RESEARCH UNIT, ISBN 978-951-42-8361-1 (Paperback) BIOCENTER OULU, ISBN 978-951-42-8362-8 (PDF) UNIVERSITY OF OULU ISSN 0355-3221 (Print) ISSN 1796-2234 (Online)

ACTA UNIVERSITATIS OULUENSIS D Medica 911

MARJA MAJAVA

MOLECULAR GENETICS OF STICKLER AND MARSHALL SYNDROMES, AND THE ROLE OF COLLAGEN II AND OTHER CANDIDATE PROTEINS IN HIGH MYOPIA AND IMPAIRED HEARING

Academic dissertation to be presented, with the assent of the Faculty of Medicine of the University of Oulu, for public defence in the Auditorium of the Medipolis Research Center (Kiviharjuntie 11), on February 23rd, 2007, at 10 a.m.

OULUN YLIOPISTO, OULU 2007 Copyright © 2007 Acta Univ. Oul. D 911, 2007

Supervised by Professor Leena Ala-Kokko Doctor Minna Männikkö

Reviewed by Docent Sirpa Kivirikko Docent Leena Pulkkinen

ISBN 978-951-42-8361-1 (Paperback) ISBN 978-951-42-8362-8 (PDF) http://herkules.oulu.fi/isbn9789514283628/ ISSN 0355-3221 (Printed) ISSN 1796-2234 (Online) http://herkules.oulu.fi/issn03553221/

Cover design Raimo Ahonen

OULU UNIVERSITY PRESS OULU 2007 Majava, Marja, Molecular genetics of Stickler and Marshall syndromes, and the role of collagen II and other candidate proteins in high myopia and impaired hearing Faculty of Medicine, Department of Medical Biochemistry and Molecular Biology, Collagen Research Unit, Biocenter Oulu, University of Oulu, P.O. Box 5000, FI-90014 University of Oulu, Finland Acta Univ. Oul. D 911, 2007 Oulu, Finland

Abstract Stickler and Marshall syndromes are genetic disorders both inherited in an autosomal dominant manner. The genotype-phenotype correlation was performed in ten Stickler/Marshall syndrome patients with mutations in the COL11A1 gene. Four patients had a phenotype classified as Marshall syndrome based on early-onset severe hearing loss and characteristic facial dysmorphism. A splice site mutation in intron 50 of COL11A1 was found in these patients, while the remaining six patients had an overlapping Marshall-Stickler phenotype with a mutation elsewhere in the gene. These results indicate exon 50 as a hot spot for splice site mutations leading to a phenotype of Marshall syndrome rather than Stickler syndrome. Collagen II (COL2A1) precursor mRNA undergoes alternative splicing resulting in two different isoforms, IIA including exon 2 and IIB excluding exon 2. Recent evidence indicates that premature termination codon mutations in exon 2 cause Stickler syndrome with no or minimal extraocular manifestations. Two mutations were observed in this study: Cys64Stop, and a novel structural mutation, Cys57Tyr. Results from the COL2A1 mini-gene studies suggested that both mutations altered positive cis elements for splicing resulting in a lower IIA:IIB ratio. The results further emphasize the importance of exon 2 in the development and normal function of the eye. In addition, patients displaying eye phenotypes in the absence of extraocular manifestations should be analyzed first for exon 2 mutations. Linkage analysis identified a new locus for autosomal recessive nonsyndromic hearing loss (DFNB32) on chromosome 1p13.3-22.1 in a Tunisian family with congenital profound autosomal recessive deafness. The COL11A1 gene is located in this region and was analyzed as a candidate gene. No disease causing sequence variation was observed. The analysis of 85 English and 40 Finnish subjects with high myopia resulted in the identification 23 sequence variations in the SLRP genes LUM, FMOD, PRELP, and OPTC. The two intronic variations and seven amino acid changes, one synonymous and six non-synonymous, were not found in the 308 controls analyzed. Five changes were detected in opticin, and all but one were shown to co-segregate with high myopia in families with incomplete penetrance. The results suggested that sequence variations in the SLRP genes expressed in the eye are genetic risk factors underlying the pathogenesis of high myopia.

Keywords: autosomal recessive nonsyndromic hearing loss, high myopia, predominantly ocular variant, Stickler syndrome

“Don’t be afraid of the space between your dreams and reality. If you can dream it, you can make it so.” Belva Davis

Acknowledgements

This work was carried out at the Collagen Research Unit of the Department of Medical Biochemistry and Molecular Biology, University of Oulu, and Biocenter Oulu, during the years 2001-2007. I wish to express my deepest gratitude to my supervisor, Professor Leena Ala-Kokko, for her continuing encouragement and optimism throughout these years. It has been a privilege to have the opportunity to work in such a great research group under your guidance. I am most grateful to my other supervisor Minna Männikkö, Ph.D. for introducing me to the world of science and for her patience and encouragement. During these years, you have always been available when I needed help with my research. Thank you for teaching me the basics of lab work at the start of this research project, for the company during scientific journeys abroad, and for your friendship over the years. I wish to express my respect for Professors Taina Pihlajaniemi, Peppi Karppinen, Kari I. Kivirikko, and Ilmo Hassinen for providing excellent research facilities and an inspiring scientific environment at the department. Professor Johanna Myllyharju deserves my appreciation for her positive attitude and example at work. Docents Sirpa Kivirikko and Leena Pulkkinen are acknowledged for their scientific expertise and valuable comments on the thesis. I wish to thank Sandra Hänninen, M.Sc. for her careful and expert revision of the language of this thesis. I would like to thank all my co-authors for giving me the great opportunity to work with all the specialists in their fields, Professors Geert Mortier, Paul N. Bishop, and Pasi Hägg, MD, Ph.D. Colleagues at the Department of Medical Biochemistry and Molecular Biology, University of Oulu, deserve my warmest thanks. Marja-Leena Kivelä, Auli Kinnunen, Risto Helminen, Seppo Lähdesmäki, and Pertti Vuokila are acknowledged for all their patient help with the practical matters. I wish to thank Minta Lumme, Aira Harju, Helena Satulehto, and Satu Koljonen for their excellent technical assistance. Special gratitude goes to the past and present members of group Ala-Kokko for many enjoyable and supporting discussions and the practical assistance at work. I would like to thank Iita Daavittila for her contribution to the practical arrangement of the dissertation and organization of the party. I warmly thank my dear friends and colleagues Heini Savolainen, Minna Jääskeläinen, Erja Mustonen, and Maria Haanpää for their friendship over the years during and after medical school. The numerous relaxing moments and intense study sessions have very special meaning in my life. My special hugs go to Heini and Hannaleena for always being there for me, through laughter and tears. I wish to never lose my friendship with Hannaleena and Mikko, Heini and Jarno, and Annina and Saku, no matter where life takes us. I wish to express my warmest gratitude to my parents, Hilkka and Heikki, for always believing in me no matter what I was planning to do. The loving support from my whole family, four sisters and a brother, and their dear spouses; Johanna and Jape, Kaisa and Miika, Antti and Eevamaria, Anna and Jussi, and Henrika has carried me over the difficulties. All of my four nephews and five nieces have brought so much real joy into my life! Above all, I owe my deepest respect to Jani, being my very best friend, company, and colleague. Your love, support, and effort in everyday life have made it possible to finish this work. I admire your attitude to life and work and your never-ending optimism. Thank you for sharing the future plans with me. The Evald and Hilda Nissi Foundation and the Farmos Research Foundation have financially supported this work. Rovaniemi, January 2007 Abbreviations

ACPTA air-conduction pure-tone average ARNSHL autosomal recessive nonsyndromic hearing loss C carboxy Cx26 Connexin 26 CHO Chinese hamster ovary cells CSGE conformation-sensitive gel electrophoresis D diopters DNF X-linked nonsyndromic deafness DFNA dominant nonsyndromic deafness DFNB recessive nonsyndromic deafness ECM extracellular matrix EDS Ehlers-Danlos syndrome FACIT fibril-associated collagens with interrupted triple helices FMOD fibromodulin GAG glycosaminoglycan GJB2 gene encoding the Connexin 26 ILL inner limiting lamina ILM internal limiting lamina IOP intraocular pressure kb kilobase kDa kilodalton LPIN2 lipin 2 LRR leucine-rich repeat LUM lumican MED multiple epiphyseal dysplasia mRNA messenger RNA N amino NAS nonsense mediated altered splicing NLD no locus designation NMD nonsense mediated mRNA decay nt nucleotide OA osteoarthritis OI osteogenesis imperfecta OMIM online Mendelian inheritance in man OPTC opticin OSMED otospondylomegaepiphyseal dysplasia PARP proline-arginine rich protein PCR polymerase chain reaction PRELP proline arginine-rich end leucine-rich repeat protein PTC premature termination codon RPRD radial perivascular retinal degeneration RT-PCR reverse transcription PCR SED spondyloepiphyseal dysplasia SLRP small leucine-rich repeat protein SNHL sensorineural hearing loss SNP single nucleotide polymorphism STL1 Stickler syndrome type I STL2 Stickler syndrome type II STL3 Stickler syndrome type III TG1F TGF-β-induced factor VR variable region

List of original articles

This thesis is based on the following articles, which are referred to in the text by their Roman numerals: I Majava M, Hoornaert KP, Bartholdi D, Bouma MC, Bouman K, Carrera M, Devriendt K, Hurst J, Kitsos G, Niedrist D, Petersen MB, Shears D, Stolte-Dijkstra I, Van Hagen JM, Ala-Kokko L, Männikkö M & Mortier GR (2006) A Report on 10 New Patients with Heterozygous Mutations in the COL11A1 Gene and a Review of Genotype-Phenotype Correlations in Type XI Collagenopathies. Am J Med Genet, In press. II Majava M, McAlinden A, Bishop PN, Perveen R, Black G, Pierpoint MEM, Ala- Kokko L & Männikkö M (2006) Missense and nonsense mutations in the alternatively spliced exon 2 of COL2A1 cause the ocular variant of Stickler syndrome. (Manuscript) III Masmoudi S, Tlili A, Majava M, Ghorbel AM, Hardenoux S, Lemainque A, Zina ZB, Moala J, Männikkö M, Weil D, Lathrop M, Ala-Kokko L, Drira M, Petit C & Ayadi H (2003) Mapping of a new autosomal recessive nonsyndromic hearing loss locus (DFNB32) to chromosome 1p13.3-22.1. Eur J Hum Genet 11: 185-188. IV Majava M, Bishop PN, Hägg N, Scott PG, Rice A, Inglehearn C, Hammond CJ, Spector TD, Ala-Kokko L & Männikkö M (2006) Novel Mutations in the Small Leucine-Rich Repeat Protein/Proteoglycan (SLRP) Genes in High Myopia. Hum Mut, In press.

Contents

Abstract Acknowledgements Abbreviations List of original articles Contents 1 Introduction ...... 17 2 Review of the literature ...... 19 2.1 Extracellular matrix ...... 19 2.1.1 Collagens...... 19 2.1.1.1 Collagen biosynthesis ...... 19 2.1.1.2 Collagen II...... 20 2.1.1.3 Collagen XI ...... 21 2.1.2 SLRP proteins...... 21 2.1.2.1 Opticin ...... 21 2.1.2.2 Lumican...... 22 2.1.2.3 Fibromodulin ...... 23 2.1.2.4 PRELP ...... 23 2.2 Cartilage ...... 24 2.2.1 Collagenous components of cartilage...... 24 2.2.2 Noncollagenous components of cartilage...... 26 2.2.3 Animal models...... 26 2.2.3.1 Animal models for cartilage collagens II and XI ...... 26 2.2.3.2 Animal models for SLRP proteins ...... 28 2.3 Type II and XI collagenopathies...... 29 2.3.1 Achondrogenesis type II, hypochondrogenesis ...... 29 2.3.2 Spondyloepiphyseal dysplasia...... 29 2.3.3 Kniest dysplasia...... 30 2.3.4 Stickler and Marshall syndromes ...... 30 2.3.4.1 Stickler syndrome type I and II...... 31 2.3.4.2 Predominantly ocular Stickler syndrome...... 32 2.3.4.3 Stickler syndrome type III ...... 32 2.3.5 Otospondylomegaepiphyseal dysplasia...... 33 2.4 Genetic factors in hearing loss...... 34 2.4.1 Nonsyndromic hearing loss ...... 35 2.5 Eye...... 36 2.5.1 Anatomy and function ...... 36 2.5.2 Proteins expressed in the eye...... 38 2.6 Myopia...... 39 2.6.1 Clinical diagnosis and classification of myopia ...... 39 2.6.2 Prevalence of myopia ...... 39 2.6.3 Pathogenesis of myopia...... 39 2.6.3.1 Genetic factors in myopia...... 40 3 Outlines of the present research...... 42 4 Materials and methods...... 44 4.1 Patients and controls (I-IV) ...... 44 4.2 CSGE (I, III, IV)...... 45 4.3 Sequencing (I-IV)...... 45 4.4 Minigene analyses (II)...... 46 4.4.1 COL2A1 constructs (II) ...... 46 4.4.2 Cell culture and transfections (II)...... 46 4.4.3 RT-PCR (II) ...... 47 4.5 Genotyping and linkage analyses (III)...... 47 4.6 PolyPhen, Ensembl, and statistical analyses (IV)...... 48 4.7 Homology model for the opticin LRR domain (IV) ...... 48 5 Results ...... 49 5.1 Analyses of Stickler and Marshall patients (I)...... 49 5.1.1 COL11A1 sequence variations (I)...... 49 5.1.2 Genotype-phenotype correlation (I) ...... 50 5.2 Analyses of patients with predominant ocular findings typical for Stickler syndrome (II)...... 50 5.2.1 Analyses of COL2A1 sequence variations and RT-PCR (II)...... 50 5.2.2 Minigene analyses for COL2A1 (II) ...... 51 5.3 Analyses of patients with congenital profound autosomal recessive deafness (III)...... 57 5.4 Linkage analyses (III)...... 57 5.4.1 Analyses of the COL11A1 gene (III) ...... 59 5.5 Candidate gene analysis of patients with myopia (IV) ...... 59 5.5.1 OPTC, LUM, FMOD and PRELP sequence variations...... 59 5.5.2 Family studies...... 60 5.5.3 PolyPhen, Ensembl and statistical analyses...... 61 6 Discussion ...... 62 6.1 Role of the COL11A1 gene in patients with Stickler and Marshall syndromes...... 62 6.2 Structural and premature termination codon mutations in the alternatively spliced exon 2 of COL2A1 causing an ocular variant of Stickler syndrome...... 65 6.3 DFNB32 locus associated with recessively inherited nonsyndromic hearing loss and the role of the COL11A1 gene...... 66 6.4 Role of the four SLRP genes, OPTC, LUM, FMOD, and PRELP, in the development of high myopia ...... 67 7 Conclusion...... 69 References Original articles

1 Introduction

The extracellular matrix (ECM) is a complex tissue containing structural proteins such as collagen, elastin and fibrillin, and proteoglycans as the major classes of biomolecules. Molecules of the ECM play an important role in the development and maintenance of the structure of many tissues. Therefore, the ECM is also involved in many pathological processes. Cartilage collagens II and XI are minor but important components of the ECM. They are mainly expressed in hyaline cartilage and in the vitreous of the eye. For this reason, mutations in the COL2A1 and COL11A1 genes result in similar phenotypes. These genes have been shown to be responsible for chondrodysplasias such as Stickler and Marshall syndromes, but due to their role as fibril-forming collagens in the ECM they are excellent candidate genes for chondrodysplasias, including nonsyndromic phenotypes such as hearing loss. The ECM small leucine-rich repeat proteoglycans (SLRP) are a family of extracellular matrix molecules to which a number of functions have been described, such as regulation of the diameter and spacing of collagen fibrils, inhibition of transforming growth factor- β, and control of cell proliferation. Opticin, fibromodulin, lumican, and proline-arginine- rich end leucine-rich repeat protein are members of the SLRP family that are expressed in the eye in addition to other tissues. The ocular SLRPs are believed to have a role in regulating eye shape and the axial length of the eye through a variety of mechanisms. Myopia is the most common eye condition with a wide variability in the prevalence between ethnic groups, from 10-20 % in Africa, 30-40 % in Europe and the USA, to as high as 70-90 % in Asia. It is a common cause of blindness due to complications such as rhegmatogeneous retinal detachment and choroidal neovascularisation. Several syndromic diseases, such as Stickler syndrome types I (STL1) and II (STL2) and Marshall syndrome have high myopia as a common feature, and mutations in the ECM genes have been associated with them. Genetic heterogeneity is also seen in nonsyndromic high myopia since ten chromosomal loci have been identified so far. Stickler syndrome is a genetically heterogeneous disorder resulting in different phenotypes with facial, orofacial, auditory, articular, and ocular manifestations. STL1 and STL2 are caused by mutations in COL2A1 and COL11A1, respectively. Clinically, patients with STL1 and STL2 have a variable amount of typical ocular findings such as 18 radial perivascular retinal degeneration (RPRD), congenital synergetic vitreous, high rate of retinal detachment, high myopia, cataract, and glaucoma, together with extraocular findings including joint degeneration and hypermobility, cleft palate, orofacial abnormalities, and hearing loss. Although hearing loss is a common feature in several syndromic diseases, such as Stickler and Marshall syndromes, approximately 80 % of the hereditary hearing loss is nonsyndromic. Patients suffering from these syndromes have been shown to have mutations in the COL2A1 and COL11A1 genes. COL11A1 mutations have also been suggested to associate with nonsyndromic hearing loss. The goal of this thesis is to increase our knowledge of pathological processes caused by mutations in the genes coding for the ECM proteins. Firstly, to obtain new information on type II and XI collagenopathies, Stickler and Marshall syndromes, and to analyze the correlation between genotype and phenotype in order to improve the differential diagnostics within and between the different diagnostic entities. In addition, the role of COL11A1 in nonsyndromic hearing loss at locus DFNB32 was studied. The four SLRPs proteins of the ECM, opticin, fibromodulin, lumican, and PRELP, that are expressed in the eye were analyzed as candidate genes for high myopia. We show here for the first time that these proteins may have a role in the development of high myopia. 2 Review of the literature

2.1 Extracellular matrix

The extracellular matrix (ECM) is a complex of biomolecules that surrounds most mammalian cells. The three major classes of molecules of the ECM are structural proteins, including collagen, elastin, and fibrillin; specialized proteins such as fibrillin, fibronectin, and laminin; and proteoglycans. The ECM is involved in many developmental and normal processes of tissues. The ECM is also involved in many pathological processes in tissues, since its components participate in several biomolecular pathways. Collagens form a family of extracellular matrix proteins that have a role in maintaining the structure of various tissues, but they also play other important functions in the interplay between cells and regulating tissue remodelling during growth, differentiation, and morphogenesis. (Olberg et al. 1990, Kielty & Shuttleworth 1993, see reviews Prockop & Kivirikko 1995, Myllyharju & Kivirikko 2004)

2.1.1 Collagens

2.1.1.1 Collagen biosynthesis

Collagens are a superfamily containing at least 28 proteins that are formally defined as collagens (see review Myllyharju & Kivirikko 2004, Veit et al. 2006). Collagens are divided into two major classes depending on their supramolecular structures. Fibril- forming collagens contain continuous triple helices, whereas non-fibril-forming collagens are more heterogeneous and further classified by structural matters (see review Vuorio & Crombrugghe 1990). Synthesis of collagen is a complex event including a number of intracellular and extracellular steps (see review Prockop & Kivirikko 1995). Fibril-forming collagens are synthesized as procollagens. They are large precursor molecules with N- and C-terminal polypeptide extensions. Intracellular steps of the biosynthesis include cleavage of the signal peptide, hydroxylation of proline and lysine 20 residues to 4-hydroxyproline and hydroxylysine, and hydroxylation of a few proline residues to 3-hydroxyproline. The biosynthesis also includes glycosylation of the procollagen chain hydroxylysine residues, requiring the activity of two enzymes, hydroxylysyl galactosyl transferase and galactosyl hydroxylysyl glucosyl tranferase (Kielty et al. 1993). While the N-terminal extension of procollagen forms only intrachain disulfide bonds, C-terminal extension of procollagen forms both intrachain and interchain disulfide bonds. After association of the C-terminal propeptides, the triple helix forms from the C-terminal region. (Kielty et al. 1993, see review Prockop & Kivirikko 1995). Secretion of procollagen from cells is followed by the cleavage of N-propeptides and C-propeptides by procollagen N-proteinase and C-proteinase. After self-assembling into fibrils, lysine and hydroxylysine residues of collagens are converted by lysyl oxidase into reactive aldehydes to finally form a complex of lysine-derived and stable covalent cross- links. (see review Prockop & Kivirikko 1995). Non-fibrillar collagens go through mainly the same processing and assembling steps, although there are notable exceptions. The enzymatic cleavage of the procollagen N- and C-terminal propeptides is characteristic for the fibril-forming collagens. (Kielty & Grant 2002).

2.1.1.2 Collagen II

Collagen II belongs to the class of fibril-forming collagens. It is a homotrimer of three identical α1(II) chains that are encoded by the COL2A1 gene (Cheah et al. 1989). The gene consists of 54 exons and is about 31 kb in size (Ala-Kokko & Prockop 1990). The COL2A1 gene is located on chromosome 12q13.11-q13.12 (Takahashi et al. 1990). Collagen II is a major component of hyaline cartilage and also predominant in human vitreous. It is also expressed in the intervertebral disc and inner ear. (Vikkula et al. 1992, Oganesian et al. 1997). Collagen II is initially synthesized as a procollagen consisting of the N- and C- terminal propeptides, which both are extracellularly removed by N- and C-proteases (Vuorio & Crombrugghe 1990). Procollagen II undergoes alternative splicing, resulting in two different forms, type IIA (long form) including exon 2 and type IIB (short form) with exon 2 spliced out (Ryan & Sandell 1990). The short form of COL2A1 is mainly expressed by differentiated chondrocytes in adult cartilage tissue, whereas the long form is preferentially expressed in the vitreous of the eye. The embryonic expression differs so that splicing variant IIA is expressed in embryonic spinal ganglion and also a number of non-cartilaginous fetal tissues, whereas variant IIB is expressed in the embryonic vitreous. (Sandell et al. 1991, Bishop et al. 1994). Since collagen II is mainly expressed in hyaline cartilage and vitreous, its decreased amount or disturbed structure is associated with spinal, epiphyseal, hearing, and ocular abnormalities. Collagen II has a critical role in forming the backbone of cartilage heteropolymeric fibrils. Collagen IX molecules are covalently bound to the surface of the fibrils, whereas collagen XI is located inside the fibrils (Cremer et al. 1998). Mutations in the COL2A1 gene result in disorders ranging in severity from lethal to mild chondrodysplasias (Horton et al. 1989, see review Spranger et al. 1994). 21 2.1.1.3 Collagen XI

Collagen XI is a heterotrimeric fibril-forming collagen composed of three different α chains. The α1(XI), α2(XI), and α3(XI) chains are encoded by the COL11A1, COL11A2, and COL2A1 genes, respectively (Eyre & Wu 1987, Henry et al. 1988, Bernard et al. 1988, Kimura et al. 1989, Takahashi et al. 1990). The COL11A1 gene consists of 68 exons, is over 150 kb in size, and is located on chromosome 1p21 (Henry et al. 1988, Annunen et al. 1999b). The COL11A2 gene is located on 6p21.2, and it is 28 kb in size consisting of 66 exons (Kimura et al. 1989, Vuoristo et al. 1995). The gene coding the α3(XI) chain of collagen XI is a IIB splicing variant of the COL2A1 gene (Ryan & Sandell 1990). Collagen XI is mainly found in tissues that express collagen II, such as hyaline cartilage and vitreous, but it is also expressed in a wide variety of non- cartilaginous tissues including brain, tendon, heart valve, skin, calvaria, and endochondral bone (Mendler et al. 1989, Nah et al. 1992). The α1(XI) and α2(XI) chains contain similar large, N-terminal domains. Consisting of a PARP (proline-arginine rich protein) or a PARP-like domain, a variable region, and a minor triple helix (Gregory et al. 2000). All three collagen XI genes undergo alternative splicing in the N-propeptide coding domain. The α1(XI) procollagen has been shown to undergo alternative splicing within the variable region (VR) of the N-propeptide, which is encoded by exons 5-9. The α2(XI) chain undergoes alternative splicing in exon 7. (Tsumaki et al. 1995, Zhidkova et al. 1995, Lui et al. 1996). Collagen XI is essential for the assembly, organization, and development of cartilage (Eyre & Wu 1987, Blaschke et al. 2000, Gregory et al. 2000). In cartilage it assembles with collagens II and IX, forming a thin network of collagen fibrils (Mendler et al. 1989, Nah et al. 1992). In non-cartilaginous tissues collagen XI has been shown to interact with the chains of collagen V, which is highly homologous to collagen XI (Mayne et al. 1993, Fichard et al. 1994, Gregory et al. 2000).

2.1.2 SLRP proteins

2.1.2.1 Opticin

Opticin (OPTC) belongs to a family of small leucine-rich repeat proteins/proteoglycans (SLRP) that are a group of extracellular matrix molecules (Hobby et al. 2000). A number of functions for SLRP proteins have been described, including the regulation of the diameter and spacing of collagen fibrils, inhibition of transforming growth factor-β, and control of cell proliferation (see minireviews Iozzo 1999, Ameye 2002). SLRP proteins are non-covalently bound to the surface of the collagen fibrils of the ECM and create a dilute meshwork to associate with them (see review Hocking et al. 1998, Bishop 2000). The OPTC gene is composed of eight exons and encodes a 1.6 kb transcript. The gene is located at q31-q32 on chromosome 1 (Hobby et al. 2000, Friedman et al. 2002). One immunolocalization study localized opticin specifically to the vitreous humor (Ramesh et al. 2004), but others found a wider distribution in the eye including the iris, choroids, 22 ciliary body, and retina (Hobby et al. 2000, Reardon et al. 2000, Friedman et al. 2002). The human vitreous contains a dilute network of thin collagen fibrils (types II, IX, and V/XI) and opticin is involved in fibrillogenesis of the collagen molecules to form the gel- like structure of the vitreous (Bishop 2000). Due to the eye-specific expression of opticin it has been suggested to be a good candidate gene for hereditary eye diseases associated with the same region on chromosome 1 (Table 1) (Hobby et al. 2000), although opticin has also been detected in human skin (Reardon et al. 2000). OPTC belongs to class III SLRP proteins that have a unique cysteine-rich region (CX2CXCX6C) containing eight leucine-rich repeats (LRRs) (see minireview Iozzo 1999, McEwan et al. 2006), including one recently found long LRR referred as the “ear repeat” (Scott et al. 2004). LRRs form curved solenoid structures with a β-sheet on their inner surface and variable topology on their outer surfaces (Scott et al. 2004). The C-terminal region contains LRR repeat containing a disulphide bonded loop with the ear repeat, whereas the N-terminal region contains a cluster of potential O-glycosylation sites. This is an exception from the other LRR proteins of the ECM, which are generally substituted with chondroitin sulphate or keratan sulphate glycosaminoglycan (GAG) chains. (Bishop 2000, McEwan et al. 2006). OPTC is one of the two members of the SLRP family that forms stable dimers through interactions between the LRR domains (Scott et al. 2003, Le Goff et al. 2003, Scott et al. 2004).

2.1.2.2 Lumican

Lumican (LUM) is a member of the class II SLRP family together with fibromodulin (FMOD), keratocan, and proline-arginine-rich end leucine-rich repeat protein (PRELP) (see minireview Hocking et al. 1998). It is 1.8 kb in size and consists of three exons that are separated by introns of 2.2 and 3.5 kb. The gene is located on chromosome 12q22 (Grover et al. 1995) (Table 1). Lumican expression was originally identified in cornea, but it has also been detected from sclera, tendon, cartilage, and skin (Blochberger et al. 1992, Chakravarti et al. 1998, Säämänen et al. 2001, Dunlevy et al. 2004). Lumican has been shown to participate in the regulation of collagen fibril formation and organization (Chakravarti et al. 1998, Ezura et al. 2000). Class II SLRP proteins have twelve LRRs in the central region between the flanking disulfide-bonded domains, including also the ear repeat in the C-terminal region (McEwan et al. 2006). The cysteine-rich consensus sequence of class II SLRP is CX3CXCX9C. The N-terminal region contains a cluster of tyrosine-sulphate residues that are believed to contribute to the polyanionic nature of the proteoglycan. Lumican, as a class II SLRP protein, is primarily substituted with keratan sulphate GAG chains (see minireview Iozzo 1999). Because glycoproteins share common structural motifs, they have been suggested to have similar functional properties, such as the interactions with the fibrillar collagens. Since the binding site on the collagen fibrils for each proteoglycan appears to be different, they are also suggested to have specific roles to fulfill. (Hedbom & Heinegård 1993, Grover et al. 1995). Lumican has been shown to interact with aggrecan molecules in the ECM of the human sclera and to form an increasing amount of 23 high molecular weight complexes with aging. These complexes are suggested to have a role in age-related changes of scleral ECM. (Dunlevy & Rada 2004).

2.1.2.3 Fibromodulin

Fibromodulin (FMOD) is a member of class II SLRP proteins having the typical sequence of this group (Oldberg et al. 1989, see minireview Hocking et al. 1998, ). The chromosomal location for FMOD is at 1q32 in the same region with the OPTC and PRELP genes (Table 1). The coding region is approximately 3 kb in size and contains two exons (Sztrolovics et al. 1994). Fibromodulin is a keratan sulphate proteoglycan originally isolated from cartilage (Heinegård et al. 1986), but it is also present in several tissues such as sclera, tendon and cornea (Oldberg et al. 1989). It contains four keratan sulphate sites in the central domain, of which one or two is normally distributed with such chains. In the N-terminal region of fibromodulin, there are also sulphated tyrosine-residues (Anttonson et al. 1991, Hedbom & Heinegård 1993). Together with a structurally related glycoprotein named decorin, fibromodulin has binding sites for collagens I and II. Interactions between these individual ECM macromolecules are supposedly involved in organization at the supramolecular level. A well-organized matrix is important for tissue function and collagen assembly. (Heinegård et al. 1986, Oldberg et al. 1989, Hedbom & Heinegård 1993).

2.1.2.4 PRELP

Proline-arginine-rich end leucine-rich repeat protein (PRELP) is one of the five members of class II SLRP proteins sharing a consensus cysteine-rich region and twelve LRRs. The gene is about 16 kb in size, including an approximately 6.7 kb transcript within three exons (Grover et al. 1996). The FMOD, PRELP, and OPTC genes form a cluster on chromosome 1q31-32 (Sztrolovics et al. 1994, Grover et al. 1996, Hobby et al. 2000). In addition to sclera and cornea, PRELP is expressed in a variety of tissues such as kidney, aorta, liver, skeletal muscle, skin, and tendon (Heinegård et al. 1986, Grover et al. 1996, Bengtsson et al. 2002, Johnson et al. 2006) (Table 1). It has been proposed to function as an anchoring molecule connecting the basement membrane to the underlying connective tissue (Bengtsson et al. 2002). Despite the identical structural motifs of class II SLRP proteins, there are a number of structural features that distinguish PRELP from the others. The long ear repeat includes 39 amino acids in PRELP whereas fibromodulin and lumican have 31 amino acids in the ear repeat (McEwan et al. 2006). The N-terminal region of PRELP differs from fibromodulin and lumican by being extremely rich in proline and arginine. It also contains two unique N-linked substitution sites for oligosaccharides compared to the four in lumican and five in fibromodulin (Bengtsson et al. 1995, Grover et al. 1996). 24 Table 1. Four SLRP proteins gene localization, expression in eye and the eye phenotype of the mouse models.

Gene Localization Cornea Ciliary Iris Vitreous Retina Choroid Sclera Mouse body model eye phenotype OPTC 1q31-q32 + + + + + - FMOD 1q32 + + Abnormal and less numerous fibrils of collagens. PRELP 1q31-q32 + + - LUM 12q22 + + Cloudy and thinner cornea.

2.2 Cartilage

Cartilage is a specialized form of connective tissue that is unique in its physical and molecular properties. Cartilage provides permanent flexible components for skeleton while also being a temporary template for skeletal structures during development. The three different types of cartilage are hyaline cartilage, fibrocartilage, and elastic cartilage. Hyaline cartilage is the most common cartilage type in humans and is mainly found in the developing skeleton, while fibrocartilage is mainly found in the annulus fibrosus of the intervertebral disc and elastic cartilage in the ears, epiglottis, and larynx (Sciller 1994, see review Cremer et al. 1998). Chondrocytes are specialized cells whose primary function is to produce and maintain the ECM of cartilage (see review Muir 1995). Articular cartilage is a type of hyaline cartilage that is relatively acellular with elasticity and low-friction surface as the typical characteristics. About two-thirds of the dry weight of human adult articular cartilage consists of collagens. Another key component of the cartilage matrix is hyaluron aggrecan, whose gel-like structure is essential for its function as a general stabilizing component in the matrix of cartilage (see review Muir 1995, Eyre 2001).

2.2.1 Collagenous components of cartilage

The collagenous matrix of articular cartilage is a highly complex assemblage of multiple gene products. The tissue’s material strength depends on the extensive cross-linking of the different collagen molecules of cartilage and characteristic fibrillar organization that changes with tissue depth. (Eyre 2001, Eyre 2004). Collagen is crucially important for the biomechanical function of cartilage. The network of developing cartilage is a copolymer 25 of collagens II, IX, X and XI, so far identified in remarkable amounts in articular cartilage tissue (Muir 1995, Eyre 2004). Other collagen molecules including types III, VI, XII, and XIV are only found as proteins in small amounts in extracts of articular cartilage (Eyre 2004). Covalently cross-linked collagens form a heterotypic fibrillar network which interacts with a variable amount of other extracellular matrix components (Bateman 2001). Collagen II is the major collagen secreted by chondrocytes. In addtition to hyaline cartilage, it is also expressed in the intervertebral disc and vitreous, providing tensile strength and stiffness to the tissues. The splicing variant IIB lacking exon 2 is predominantly expressed in cartilage tissue, whereas splicing variant IIA is predominantly expressed in the eye. Collagen IX is a heterotrimer consisting of three different α chains (Eyre & Wu 1987). It belongs to the class of fibril-associated collagens with interrupted triple helices (FACITs). Collagen IX decorates the surface of collagen II fibrils by covalent binding, particularly the thin fibrils that are important for the network surrounding chondrocytes. (see review Muir 1995, Bateman 2001). The main function of collagen IX molecules in the cartilage matrix is to give lateral strength with flexibility to the fibrillar network (see review Muir 1995). Collagen XI is another minor heterotrimeric collagen associated with the cartilage collagen network. The molecules are primarily cross-linked to each other, forming the inner core of the template and restraining the lateral growth of collagen II fibrils. As cartilage has a crucial role in the development of the skeleton, mutations in the genes coding for cartilage-specific proteins often produce developmental abnormalities. Many groups are actively searching for variations among the cartilage collagen genes as causative mutations for inherited disorders. (see review Muir 1995, Aszódi et al. 2000, Superti-Furga et al. 2001). Collagen II mutations cause a wide spectrum of diseases from lethal in utero phenotypes to early-onset osteoarthritis and minimal skeletal dysplasia (Myllyharju & Kivirikko 2001). Mutations in the collagen IX genes have been shown to cause multiple epiphyseal dysplasia (MED), but are not the major cause (Unger et al. 2001, Jakkula et al. 2004). Collagen IX defects have also been shown to be associated with intervertebral disc disease (Annunen et al. 1999, Paassilta et al. 2001, Noponen- Hietala et al. 2003, Jim et al. 2005), and recently a homozygous PTC mutation in COL9A1 has been shown to cause a new autosomal recessive form of Stickler syndrome (Van Camp et al. 2006). COL11A1 and COL2A1 mutations cause forms of Stickler and Marshall syndromes with similar and overlapping phenotypes of eye and cartilage manifestations (Griffith et al. 1998, Eyre 2001). Besides the expression in the cartilage, collagens are significant structural components of the inner ear, the ocular vitreous body, and the intervertebral discs. Collagen II has been found in numerous tissues in the inner ear such as the tectorial, basilar, and vestibular membranes (Thalmann 1986). Collagens V, IX, and XI are also expressed in the tectorial membrane and cochlea in the inner ear (Thalmann 1993). Hearing impairment is a common feature of cartilage collagen diseases like Stickler and Marshall syndromes (Griffith et al. 1998). COL11A1 has been suggested to be a candidate gene for nonsyndromic hearing loss due to its tissue-specific expression as well as the common hearing loss impairment in syndromic diseases and a mutant mouse model producing a hearing impairment (Cho et al. 1991, Thalmann 1993, Richards et al. 1996, Griffith et al. 1998). 26 2.2.2 Noncollagenous components of cartilage

Besides the collagenous components of cartilage, the noncollagenous components are also important for the functional and biomechanical structure. The predominant components of cartilage are aggrecan, a range of other matrix proteins, and glycoproteins/proteoglycans including decorin, biglycan, fibromodulin, lumican, and PRELP. (Grover et al. 1995, Bengtsson et al. 1995, Bateman 2001). Aggrecan and other proteoglycans are highly hydrated by the GAG side chains. Aggrecan consists of a multidomain core protein of over 200 kDa and also over a hundred chondroitin and keratan sulphate side chains, whereas the other cartilage glycoproteins, SLRP proteins, have relatively small core proteins with only a few GAGs. These molecules provide the swelling and hydrating capacity of cartilage, while also regulating collagen fibrillogenesis, cell adhesion, and migration. (Bengtsson et al. 1995, Grover et al. 1995, see review Muir 1995, Bateman 2001).

2.2.3 Animal models

2.2.3.1 Animal models for cartilage collagens II and XI

A continuous triple helix is important particularly for the fibril-forming collagens, so mutations in the triple-helical glycine residues usually result in severe phenotypical consequences (see review Myllyharju & Kivirikko 2004). Several mouse models with spontaneous mutations, or transgenic or knockout models for cartilage collagens have been reported (Tables 2 and 3). Col2a1 knockout mice develop a severe chondrodysplasia and the mice die either before or shortly after birth (Li S-W et al. 1995, Aszódi et al. 1998). Heterozygous mice with one inactive allele develop a mild phenotype with minimal changes in cartilaginous structures and structural defects in the eye (Kaarniranta et al. 2006). Transgenic mice with structural mutations show a phenotype resembling human chondrodysplasias (Vanderberg et al. 1991, Gaiser et al. 2002, Donahue et al. 2003, Kaarniranta et al. 2006). Clinical features of mutated mice included dwarfism, a short snout, cranial changes, a cleft palate, and destroyed bone mineralization. The ocular phenotype of targeted heterozygous inactivation of the Col2a1 gene showed structural changes in the ciliary body and changes in the extracellular matrix of the lens and vitreous (Kaarniranta et al. 2006). 27 Table 2. Mouse models of Col2a1 mutations

Defect in Col2a1 Mouse model Reference 3-bp del in C-terminal region Spontaneous mutation Pace et al. 1997 Expression of human COL2A1 Transgenic Vanderberg et al. 1991, Helminen with large internal deletion et al. 1993 G85C mutation in exon 11a Transgenic Garofalo et al. 1991 150-bp del of exon 7 and intron 7 Transgenic Metsäranta et al. 1992, Säämänen et al. 2000 Targeted inactivation Knockout Li S-W et al. 1995, Aszódi et al. 1998, Kaarniranta et al. 2006 G574S mutation in exon 24 Transgenic Maddox et al. 1998 G904C mutation in exon 39 Transgenic So et al. 2001 R789C mutation in exon 34 Transgenic Gaiser et al. 2002 R519C mutation in exon 22 Transgenic Arita et al. 2002, Sahlman et al. 2004 R1417C mutation in exon 51 Spontaneous mutation Donahue et al. 2003 Del of exon 48 Transgenic Barbieri et al. 2003 a Indicates a residue from the beginning of the triple-helical domain. Fibrillar collagen XI has been shown to be essential for skeletal morphogenesis. Homozygous mice with one bp deletion about 570 nt downstream of the translation initiation codon in Col11a1 develop chodrodysplasia (cho) and die at birth having abnormal cartilage formation. The mutation causes a frameshift and premature termination codon and results in a severely truncated α1(XI) collagen polypeptide, absence of α1(XI) collagen in the ECM, and abnormally thicker collagen fibrils in the cartilage due to overexpression of collagen II. Particularly the cartilage of the limbs, ribs, mandible, and trachea is disturbed. Cleft palate is a common finding with mice carrying the cho mutation. (Li Y et al. 1995, Lavrin et al. 2001, Szymko-Bennet et al. 2003, Rodriquez et al. 2004). The heterozygous loss-of-function mutation in Col11a1 leads to a less severe phenotype with the development of OA in cho/+ mice. The morphological and biochemical evidence of OA appears to precede significant mechanical changes, suggesting that the mechanism of development of OA does not involve initial mechanical factors in cho/+ mice. (Xu et al. 2003).

Table 3. Mouse models of Col11a1 mutations

Defect in Col11a1 Mouse model Reference 1 bp deletion (cho) Spontaneous mutation Li Y et al. 1995, Lavrin et al. 2001, Szymko-Bennet et al. 2003, Xu et al. 2003, Rodriquez et al. 2004 Col2a1 mutations seem to have more severe consequences than mutations in the Col11a1 gene. Premature termination codon mutations usually cause a less severe phenotype than glycine substitutions in the triple-helical region. For example, although homozygous cho mice die at birth having abnormal cartilage formation, Col2a1 knockout mice have a 28 more severe phenotype. Heterozygous Col2a1 knockout mice develop minimal cartilage changes with an ocular phenotype, while heterozygous cho mice also have a less severe phenotype with development of OA and no ocular findings.

2.2.3.2 Animal models for SLRP proteins

Knockout mouse models for SLRP genes exist for decorin, lumican, and fibromodulin, but not for opticin and PRELP (Danielson et al. 1997, Chakravarti et al. 1998, Svensson et al. 1999). Irregular forms of fibrils primarily in the dermis, tendon, and cornea have been detected in the knockout mice. These knockout models support the hypothesis that members of the SLRP protein family bind to collagen fibrils through their leucine-rich repeat domains to regulate collagen fibril diameter and spacing (Danielson et al. 1997, Chakravarti et al. 1998, Svensson et al. 1999). Although a mouse model for OPTC has not been made yet, it has been hypothesized to have similar consequences as those seen in the fibromodulin and lumican mice (Takanosu et al. 2001). Lumican null homozygous mutant mice have abnormally thick collagen fibrils particularly in the skin and cornea. The observed skin laxity and fragility resembled certain types of Ehlers-Danlos syndrome (EDS). The loss of lumican also disturbs corneal transparency by interrupting the maintenance of the collagen architecture and assembly, but also by causing corneal opacification (Chakravarti et al. 1998). Further analyses indicated that the defect in the corneal transparency is due to the increased diameter of collagen fibrils in the posterior stroma of the cornea, while the comparable fibrils in the anterior stroma had normal diameter and packing (Chakravarti et al. 2000). The consequence of the loss of lumican was cloudy and thinner corneas. The significant defects in scleral collagen fibril formation could also lead to alterations in ocular shape and size and affect vision. (Chakravarti et al. 2000, Austin et al. 2002). Fibromodulin-deficient mice were reported to be viable, but showed abnormal and less numerous fibrils of collagen particularly in tendons where the expression of fibromodulin is relatively abundant. Also, the cross sections of the collagen fibrils were shown to be irregular (Ameye et al. 2002a). The ratio of lumican to fibromodulin is estimated to be 1:3, at least in the tendons of wild-type mice (Svensson et al. 1999). Although fibromodulin is absent, the amount of lumican in tendons increases rapidly. Therefore, it has been suggested that these two proteins compete for binding sites on the collagen molecules. (Svensson et al. 2000, see minireview Ameye & Young 2002). To understand the unique and also overlapping functions of the lumican and fibromodulin proteins, double knockout mice were developed by intercrossing the single- null mutants. Due to the eye-specific expression of these proteins, ocular and scleral alterations in particular were analyzed. The proteins were shown to have unique and complementary functions in regulating the axial length of the eye, the structure of the sclera, and also the ultrastructure of the scleral collagen fibrils. Double-null mice had a significantly higher ocular axial length compared to the wild-types. Also, thinning of the sclera was observed in all null mutant mice, including both the single and double-null mice. The latter also had areas in the posterior sclera showing retinal detachment, which was also observed in the lumican null mice, although less frequently. These eye findings 29 are features of a high myopic eye and suggest that these proteins may have a role in the development of high myopia. (Chakravarti et al. 2003).

2.3 Type II and XI collagenopathies

2.3.1 Achondrogenesis type II, hypochondrogenesis

Achondrogenesis type II (MIM #200610), also known as Langer-Saldino type achondrogenesis, is inherited in an autosomal dominant manner and is caused by mutations in the COL2A1 gene (Table 4). Patients with achondrogenesis have a short trunk and very short extremities. Skeletal abnormalities such as very short tubular bones and an underossified axial skeleton are characteristic findings for this syndrome. Fetal disruption is also likely to occur in prematurity and fetal hydrops leading to a stillborn baby or death shortly after birth. Glycine substitutions in the the α1(II) chain have been identified from patients with achondrogenesis type II, leading to abnormal triple helix formation and a decreased amount of collagen II in the ECM of the patient. (Godfrey & Hollister 1988, see review Spranger et al. 1994, Körkkö et al. 2000). Hypochondrogenesis is also an autosomal dominantly inherited disorder clinically similar to achondrogenesis type II. It is caused by mutations in the COL2A1 gene, and glycine substitutions have also been detected in these cases (Table 4). Patients suffer from short stature, oval and flattened face, and widely spaced eyes, and cleft palate often occurs. In the most severe cases, respiratory distress is common and these patients die shortly after birth. The difference between these two distinct entities is the less severely retarded ossification of vertebral bones and longer tubular bones in patients with hypochondrogenesis (Maroteaux et al. 1983, see review Spanger et al. 1994).

2.3.2 Spondyloepiphyseal dysplasia

Spondyloepiphyseal dysplasia (SED, MIM # 183900) is a chondrodysplasia caused by mutations in the COL2A1 gene. Characteristic findings include abnormal epiphyses and flattened vertebral bodies (Table 4). Myopia may occur with or without retinal detachment. Indeed, SED belongs to a continuous spectrum of type II collagenopathies with similar clinical and radiographic findings compared to achondrogenesis II and hypochondrogenesis. This disorder has been reported to result from in-frame deletions, duplications in COL2A1 and mutations leading to glycine substitutions in the protein. The severity of the clinical manifestations has been shown to correlate with the location of the disease- causing mutation. The closer the mutation is located to the C-terminal portion end of the triple helix, the more severely affected the patient usually is. (Murray et al. 1989, Lee et al. 1989, see review Spranger et al. 1994). 30 2.3.3 Kniest dysplasia

Kniest dysplasia (MIM #156550) is a severe autosomal dominantly inherited disease characterized by short trunk dwarfism, kyphoscoliosis, enlarged joints, and craniofacial manifestations such as flat midface, cleft palate, myopia, and hearing loss. Abnormalities of the vertebra, epiphyses, and metaphyses are also common findings in patients with Kniest dysplasia. Kniest dysplasia is caused by mutations in the COL2A1 gene. Most of the mutations are in-frame deletions located between exons 12 and 24 (Table 4). Thus, the mRNA encoded by the mutant allele leads to a shorter α1(II) chain (Spranger et al 1994, Wilkin et al. 2000). Stickler syndrome shares a similar phenotype with Kniest dysplasia, and the gene responsible for these diseases is also the same. Patients with Stickler syndrome, however, usually have normal stature and milder skeletal changes. (Kniest & Leiber 1977, see review Spranger et al. 1994, Wilkin et al. 2000).

2.3.4 Stickler and Marshall syndromes

Stickler syndrome (MIM #108300, #604841, #184840) is an autosomal dominant disorder resulting from mutations in at least three different genes, COL2A1, COL11A1, and COL11A2. Characteristic findings for Stickler syndrome are typical ophthalmological and orofacial features, hearing loss, and arthritis (Stickler et al. 1965, Stickler et al. 2001) (Table 4). Patients usually develop moderate to severe myopia in early childhood and are at risk for spontaneous retinal detachment (see review Snead & Yates 1999, Donoso et al. 2003). Extraocular manifestations characteristic for Stickler syndrome are joint degeneration, joint hypermobility, facial dysmorphism, and hearing impairment (Baitner et al. 2000, Stickler et al. 2001). Stickler syndrome is a clinically and genetically heterogeneous condition with at least three different subtypes. Some studies have suggested that the subtypes can be differentiated from each other based on the vitreoretinal phenotype. Patients with a congenital membranous type 1 vitreous seem to often have a mutation in the COL2A1 gene, whereas patients with beaded vitreous seem to have a mutation in the COL11A1 gene. Mutations in the COL11A2 gene cause non-ocular Stickler syndrome type III without any ocular findings. (Brunner et al. 1994, Vikkula et al. 1995, Sirko-Osada et al. 1998). Recently Van Camp et al. reported a homozygous R295X mutation in the COL9A1 gene in a Moroccan family with four patients having characteristic symptoms for Stickler syndrome. In contrast to all previously described Stickler mutations inherited in an autosomal dominant way, this family showed an autosomal recessive inheritance pattern. (Van Camp et al. 2006). Marshall syndrome (MIM #154780) is also an arthro-opthalmopathy with orofacial, auditory, articular, and ocular manifestations. It is inherited in an autosomal dominant manner and caused by mutations in the COL11A1 gene, and the clinical phenotype overlaps with Stickler syndrome (Marshall et al. 1958, Annunen et al. 1999). Splicing 31 mutations mainly in the 54 bp exons coding for the 3’-terminal half of the triple helix, particularly exon 50, are common in patients with the Marshall syndrome phenotype (Annunen et al. 1999). There has been discussion whether Stickler and Marshall syndromes are two separate entities or just one phenotypic spectrum (Baraitser 1982, Winter et al. 1983, Ayme & Preus 1984, Annunen et al. 1999). Patients with Marshall syndrome more often have short stature, less severe eye findings, early-onset hearing loss, and more pronounced maxillary hypoplasia than patients with Stickler syndrome (Griffith et al. 1998, Annunen et al. 1999, Griffith et al. 2000, Poulson et al. 2004).

2.3.4.1 Stickler syndrome type I and II

The most common Stickler syndrome-causing mutations are found in the COL2A1 gene. They lead to Stickler syndrome type I (STL1). The mutations are spread throughout the gene. Stickler syndrome type II (STL2) is a subtype of this syndrome caused by mutations in the COL11A1 gene. The similarity of the STL1 and STL2 phenotypes is due to the similar structure and function of the disease causing genes, COL2A1 and COL11A1 (Annunen et al. 1999). Patients with STL1 have both extraocular and ocular manifestations. Extraocular manifestations characteristic for Stickler syndrome are joint degeneration, joint hypermobility, cleft palate, flat face, small chin, and different ranges of hearing impairment (Baitner et al. 2000, Stickler et al. 2001). Typical ocular findings include radial perivascular retinal degeneration (RPRD), congenital synergetic vitreous, a high rate of retinal detachment, high myopia, presenile cataract development, and glaucoma (see review Snead & Yates 1999, Donoso et al. 2003). There are two different vitreoretinal phenotypes in Stickler syndrome. Patients with mutations in the COL2A1 gene are likely to have the congenital membranous vitreous (see review Snead & Yates 1999, Donoso et al. 2003). The type II vitreous, also known as the beaded vitreous type, is likely to occur in patients with COL11A1 mutations. Unlike the membranous vitreous, beaded vitreous is not congenital. Ocular findings of STL1 are often more serious compared to STL2. (see review Snead & Yates 1999). Also, other ocular manifestations typical for STL1 tend to be less severe in STL2, particularly vitreoretinal degeneration, cataract, and retinal detachment. The other main difference between these two entities is hearing impairment. Patients with STL2 more often have moderate-to-severe hearing impairment that is usually congenital or diagnosed in childhood, whereas patients with STL1 may have normal hearing or develop a minor hearing loss later in life. (Annunen et al. 1999). STL1 patients have been shown to have null-allele mutations in COL2A1, but some structural mutations have also been detected (Brown et al. 1992, Ritvaniemi et al. 1993, Ahmad et al. 1995, Ballo et al. 1998, see review Snead & Yates 1999, Hoornaert et al. 2005). STL2 patients have mutations in the triple-helical coding region of the COL11A1 gene. Splice site mutations outside the region of exon 50, missense mutations, in-frame deletions, and glycine substitutions have been shown to associate with STL2 (Richards et al., 1996, Annunen et al. 1999, see review Snead & Yates 1999). 32 2.3.4.2 Predominantly ocular Stickler syndrome

Some studies have recently reported cases of Stickler syndrome with predominant ocular findings and without clinically apparent systemic findings, including articular, hearing, and facial manifestations. These patients have mutations in exon 2 of COL2A1 that are mostly premature termination codon (PTC) mutations, but also deletions and duplications leading to premature termination codons (Richards et al. 2000, Donoso et al. 2002, Parma et al. 2002, Donoso et al. 2003) (Table 4). It has been shown that exon 2 of the COL2A1 gene undergoes alternative splicing and has tissue-specific expression. The long form of COL2A1 including exon 2 is mostly expressed in the vitreous body of the eye, but both procollagen types, IIA and IIB, are expressed in the early stage of development (Sandell et al. 1991, Bishop et al. 1994). Hence, mutations present in exon 2 may cause a Stickler syndrome phenotype with minimal or no extraocular manifestations. (Richards et al. 2000, Parma et al. 2002, Donoso et al. 2002, Donoso et al. 2003). PTC mutations are normally subjected to nonsense-mediated mRNA decay (NMD), which is a quality control-based surveillance mechanism that selectively degrades mRNAs that prematurely terminate translation because of a frame shift or nonsense mutation (see reviews Frischmeyer et al. 1999, Maquet 2004). However, PTC mutations have also been reported to cause nonsense-mediated altered splicing (NAS) leading to exon skipping (Dietz 1997, Valentine 1998, Cartegni et al. 2002). One reported mechanism for NAS involves disruption of positive cis regulatory elements within the mutated exon that are important for exon inclusion during the pre-mRNA splicing process (Cartegni et al. 2002). This predominantly ocular Stickler syndrome form is characterized by congenitally abnormal vitreous and an acquired RPRD, which can cause adult retinal detachments (Parma et al. 2002).

2.3.4.3 Stickler syndrome type III

Stickler syndrome type III (STL3) is an autosomal dominant non-ocular form of Stickler syndrome caused by mutations in the COL11A2 gene (Table 4). COL11A2 is encoded by the α2 chain of collagen XI. It is not expressed in the eye, where it is replaced by a similar polypeptide, the α2 chain of collagen V. Vikkula et al. reported non-ocular Stickler syndrome caused by a single-base mutation affecting splice sites at the 3’end of the triple-helical region of the COL11A2 gene (Vikkula et al. 1995). Sirko-Osada et al. also described a heterozygous 27-bp deletion in the triple-helical region associated with non-ocular Stickler syndrome (Sirko-Osada et al. 1998). A stop codon mutation in COL11A2 inducing exon skipping via NAS has also been reported in patients with non- ocular Stickler syndrome (Vuoristo et al. 2004). Patients with STL3 do not have the typical ocular findings of Stickler syndrome, and the phenotype manifests only in systemic features with a higher prevalence of sensorineural hearing loss than patients with classic Stickler (Mayne et al. 1993, Vikkula et al. 1995, Vuoristo et al. 2004). 33 2.3.5 Otospondylomegaepiphyseal dysplasia

Otospondylomegaepiphyseal dysplasia (OSMED, MIM #215150) is an autosomal recessively inherited disorder (Kääriäinen et al. 1993, Vikkula et al. 1995, van Steensel et al. 1997). Both homozygous and compound heterozygous mutations in the COL11A2 gene have been associated with the disease (Table 4). The phenotype overlaps with the autosomal dominant disorders, Stickler and Marshall syndromes, but can still be distinguished. The typical features for OSMED are short limbs, severe sensorineural hearing loss, enlarged epiphyses, abnormalities in the vertebral bodies, and a lack of ocular impairment. The typical facial findings are mid-facial hypoplasia, short upturned nose, depressed nasal bridge, cleft palate, and small mandibula. (Insley & Astley 1974, Kääriäinen et al. 1993, Vikkula et al. 1995, Pihlajamaa et al. 1998). Melkoniemi et al. reported ten mutations in patients suffering from OSMED. The patients had either homozygous or compound heterozygous COL11A2 mutations. Nine of ten mutations led to a premature termination codon and one was an in-frame deletion. These mutations are proposed to cause a complete loss-of-function of the α2(XI) chain (Melkoniemi et al. 2000). 34 Table 4. Type II and XI collagenopathies

Disorder Gene Inheritance Mutation Phenotype

Achondrogenesis COL2A1a AD Gly substitutions Short trunk, extremities and tubular type II bones, underossified axial skeleton, fetal disruption Hypochondrogenesis COL2A1a AD Gly substitutions Short stature, oval and flattened face, cleft palate, severe respiratory distress SED COL2A1a AD In-frame deletions, Abnormal epiphyses, flattened vertebral duplications, Gly bodies, myopia, retinal detachment substitutions Kniest dysplasia COL2A1a AD In-frame deletions Short trunk, kyphoscoliosis, enlarged (exons 12-24) joints, craniofacial findings, abnormal vertebra and epiphyses STL1 COL2A1a AD Null-allele, structural Typical extraoculard and oculare findings for STL, congenital membraneous vitreous, minor hearing impairment Predominantly COL2A1a AD PTC, structural Predominant oculare findings typical for ocular Stickler STL, absence of extraoculard findings

STL2 COL11A1b AD SSf, missense, in- Typical extraoculard findings for STL, frame deletions, Gly less severe ocular findings, moderate-to- substitutions severe hearing impairment Marshall COL11A1b AD SSf in 54 bp exons, Short stature, less severe eye findings particularly exon 50 than STL, early-onset hearing loss, pronounced maxillary hypoplasia STL3 COL11A2c AD SSf, PTC, in-frame Typical extraocular findings for STL, deletion higher prevalence of sensorineural OSMED COL11A2c AR Homozygous and Short limbs, severe sensorineural compound hearing loss, enlarged epiphyses, heterozygous abnormal vertebral bodies Gene chromosomal location a 12q13.11-q13.12, b 1p21 and c 6p21.2. d Typical extraocular findings for STL are join degeneration and hypermobility, cleft palate, flat face, small chin, and different ranges of hearing impairment. e Typical ocular findings for STL are RPRD, congenital synergetic vitreous, retinal detachment, presenile cataract, and glaucoma. f SS, splice site mutation.

2.4 Genetic factors in hearing loss

Hearing loss is the most common sensory impairment affecting one of every 500 newborn infants, and one of every 1000 children is likely to have severely or profoundly impaired hearing before adulthood. Genetic factors are involved in more than 50 % of the 35 cases with congenital hearing impairment and more than 120 independent genes have been identified. About 70 % of the hearing loss is nonsyndromic. Many of the syndromes responsible for syndromic hearing loss have already been described and the genes have been mapped. The genes involved in non-syndromic hearing loss have only recently begun to be identified. Some identified genes causing syndromic hearing loss are also responsible for isolated forms of hearing loss. (Mehl & Thomson 1998, Mehl & Thomson 1992, 1999, Chen et al. 2005, see reviews Piatto et al. 2005, Bayazit & Yoilmaz 2006). Cases with nonsyndromic deafness are classified by the mode of inheritance as DFNA, dominant, DFNB, recessive, or DFN, X-linked. The loci are numbered in the order of discovery. (See reviews Piatto et al. 2005, Bayazit & Yoilmaz 2006). The diversity of the genes and genetic loci associated with hearing loss shows the complexity of the genetic basis of the condition. (Keats & Berlin 2002, Aggarwal & Saed 2005). Many different genes have been shown to associate with nonsyndromic hearing impairment. The identification of the normal function of the gene product responsible for inherited hearing impairment will help us to understand the pathogenesis of the condition. (Mueller et al. 2002).

2.4.1 Nonsyndromic hearing loss

In most cases with hearing impairment, the signs of additional phenotypical symptoms are absent, supporting the diagnoses of nonsyndromic hearing loss. Nonsyndromic hearing loss is a genetically heterogeneous condition. So far 92 loci have been mapped and 41 genes have been associated with the dominant, recessive, or X-linked inherited forms of nonsyndromic hearing loss. (see review Petersen 2002, Hone & Smith 2003, Chen et al. 2005). Collagens are involved in auditory function and the auditory system due to their expression in bone and cartilage. Mutations in collagens are found in many syndromes that are associated with hearing loss including osteogenesis imperfecta (OI) (MIM #166200, #166220, #259420), Alport syndrome (MIM #301050, #203780, #104200), OSMED (MIM #215150), and Stickler syndrome (MIM #108300, #604841, #184840). Mutations in the COL11A2 gene have also been shown to cause autosomal dominant non- syndromic hearing loss at the DFNA13 locus (McGruit et al. 1999, Chen et al. 2005). The most predominant form of hereditary nonsyndromic hearing loss is autosomal recessive. It is sensorineural due to defects in the cochlea and differs from syndromic forms, where the hearing loss is mainly conductive or mixed-type. (see review Petersen 2002). McGruit et al. identified heterozygous missense mutations in American and Dutch families in the COL11A2 gene with autosomal dominant nonsyndromic hearing loss. (McGruit et al. 1999). Chen et al. described a missense mutation in the triple-helical region of COL11A2 near the N-terminal end in an Iranian family segregating with the autosomal recessive nonsyndromic hearing loss (ARNSHL). (Chen et al. 2005). 36 2.5 Eye

2.5.1 Anatomy and function

Probably the most crucial sensory system for the human being is the eye (Figure 1). It is an externalized portion of the brain. Light coming into the optic system is converted into electrical impulses, which are transported to the central nervous system via the optic nerve. The eye is composed of three different layers that form the eyeball, which fills the orbita, the bony cavity covering the eye. The layers from inside to outside of the eye are the retina, uvea, and the sclera. In addition, three different cavities exist inside the layers, that form the eyeball and also provide the basis for the physiology and function of the eye. The cavities are named for their anterior and posterior positions within in the eye and the vitreous cavity. Other important structures for the optic system of the eye are the iris, lens, ciliary body, and optic nerve. (Leeson et al. 1988). The retina forms the inner layer of the eye and is composed of two parts, the posterior light-sensitive portion and the anterior non-sensitive portion. These two different zones are separated from each other by the ora serrata, the border line. (Leeson et al. 1988). The middle layer between the retina and sclera is the uvea, being the only vascularized layer in the eye. The uvea consists of three different parts, namely the choroidal layer, the uveal portion of the ciliary body, and the anterior part of the iris. The uvea vascularizes the anterior part of the iris, whereas the choroid is the primary vascular layer covering two thirds of the posterior uvea. The ciliary body is located in the anterior sides of the uvea and is continued as choroid towards the posterior uvea. (Leeson et al. 1988, Tran et al. 2000). The outer layer of the eyeball is the sclera which is functionally and structurally divided into two parts, the sclera and cornea. The sclera covers the outermost layer of the eye, and it is composed of collagen and elastic fibers that are produced by fibroblasts. It determines both the shape and axial length of the eye and has a role in refraction function. The cornea forms a transparent, avascular layer as a part of the sclera and contains numerous free nerve endings (Leeson et al. 1988, Austin et al. 2002). The collagen architecture of the corneal stroma has an important function in the transparency of the cornea (Chakravarti et al. 2000). The anterior chamber of the eye is located between the cornea and anterior part of the iris, and the posterior chamber is located between the posterior part of the iris and lens. The third cavity of the eye, the vitreous body, fills up the space between the lens and retina. The human vitreous body is divided into anatomical regions defined as the central vitreous, the basal vitreous, vitreous cortex, the vitreoretinal interface, and the zonule. The vitreous is normally acellular except in the vitreous cortex and the basal vitreous, which both contain a low concentration of cells named hyalocytes. The vitreous gel is composed of highly hydrated ECM. The gel structure is maintained by a dilute network of thin collagen fibrils that are comprosed of collagens II, V/XI, and IX. The glycosaminoglycan (GAG) hyaluron is the major component filling the space between the collagen fibrils in mammalian vitreous. The vitreous gel provides the metabolic 37 requirements for the lens and excludes cells and other macromolecules from the vitreous cavity to maintain transparency. Some studies have also suggested that the vitreous has a role in coordinating eye growth. (Bishop 2000). In addition to the eyelids, the iris has the most important role in regulating the amount of light entering the eye. The iris contains two muscles, the sphincter pupillae and dilator pupillae. Regulation of the incoming light occurs through dilatation (mydriasis) and contraction (miosis) of the iris, depending on the intensity of the light. (Thompson 1987, Leeson et al. 1988). The crystalline lens is positioned behind the iris and refracts the light entering the eye through the pupil and finally focuses it on the retina. It is avascular, obtaining nutrition from the surrounding fluids. The lens is entirely surrounded by a capsule. Under the anterior capsule is a layer of epithelial cells. The lens maintains its own clarity and provides the refractive power of the eye by contributing to the optical system. It also functions in the accommodation and absorption of ultraviolet light. (Cotlier 1987). The ciliary body has a crucial role in maintaining the intraocular pressure (IOP) by secreting the aqueous structure for the vitreous cavity. It has been proposed that several vitreous connective tissue macromolecules and also some basement membrane proteins are mainly expressed in the ciliary body. In particular, the posterior half of the non- pigmented ciliary epithelium has been found to have secretory activity and has therefore been suggested to have a pivotal role in eye development. The non-pigmented epithelial layer is the innermost layer of the ciliary body, being apically joined with the pigmented epithelial layer (Bishop et al. 2002). The optic nerve connects the retina with the higher visual centers. It is rather a track of the central nervous system than a peripheral nerve. (Cohen 1987).

Fig. 1. Anatomy of the eye. 38 2.5.2 Proteins expressed in the eye

In recent years there has been a dramatic increase in the knowledge of the structural components of the eye. In this section, proteins expressed mainly in the structures of the optic system of the eye are reviewed. The SLRP proteins, opticin, lumican, fibromodulin, and PRELP, have a wide expression in different structural parts of the eye. PRELP, fibromodulin, and lumican are expressed in the sclera, while PRELP and lumican are also found in the cornea, where lumican has been suggested to have a crucial role in maintaining the normal architecture of the collagen fibrils. Opticin is the major glycoprotein found in the human vitreous, but has also been detected in the ciliary body, particularly from the non-pigmented epithelium, iris, and retina. (Heinegård et al. 1986, Grover et al. 1996, Chakravarti et al. 1998, Ezura et al. 2000, Bishop 2000, Hobby et al. 2000, Bengtsson et al. 2002). Ramesh et al. studied the immunolocalization of opticin in the human eye and revealed that its expression is mostly found in the vitreous humor, particularly in the basal and cortical vitreous gel, while the central vitreous showed less intensive staining for opticin. In that study, opticin was also found in the internal limiting membrane and posterior capsule of the lens (Ramesh et al. 2005) Previous studies have demonstrated opticin expression in the posterior non-pigmented ciliary body. These data have suggested that the ciliary body has an active role in secreting the aqueous humor of the vitreous cavity (Bishop et al. 2002). Proteins of the SLRP superfamily have important functions related to binding activity. Targets of these bindings in the eye are other extracellular matrix proteins such as collagens (Bishop et al. 2002) Members of the large collagen family are also found in the eye. Collagen II is the predominant collagen in the vitreous contributing to its gel-like structure, while also modulating the proliferative processes by binding growth factors. The IIA splice variant is predominant in the eye. (Ryan & Sandell 1990, Bishop 2000). Collagens V and XI are closely related and very similar in structure and function. They form mixtures of α chains derived from these collagen types. Collagen V is found in the cornea, but collagen V/XI is found in the heterotypic fibrils from vitreous. Also, collagens IX and VI are found in small amounts in human vitreous. Thin collagen fibrils composed of three different collagens (II, IX, and V/XI) maintain the gel-like structure of the vitreous humor by forming a dilute meshwork. (Bishop 2000). Other collagen types found in the eye are collagen XVIII localized at the internal limiting lamina (ILM), at the basement membrane of the iris, and in the vitreous and retina, and collagen IV in the inner limiting lamina (ILL) (Fukai et al. 2002, Passos- Bueno et al. 2005, Ramesh et al.2005). Collagen XVIII is proposed to interact with the opticin molecule in the adhesion of vitreous collagen fibrils (Ramesh et al.2005). Collagen I is the predominant collagen in the connective tissue of sclera, interacting with small amounts of other fibrillar and fibril-associated collagens (Marshall et al. 1993, Mayne 2002, Jobling et al. 2004). Together with collagen I, collagen V comprises almost 20 % of corneal fibrillar collagens (Gordon et al. 1994, Segev et al. 2006). 39 2.6 Myopia

2.6.1 Clinical diagnosis and classification of myopia

In nearsightedness, or in medical terms myopia, the light entering the eye is not focused correctly. Parallel rays of light coming into the eye are focused anterior to the retina, when they normally, in the emmetropic eye, are brought to focus on the retina. A consequence of this situation is a condition where near objects are seen clearly, but distant objects do not come into proper focus. Myopia is the most common eye condition resulting in a mismatch between the refractive power and axial length of the eye. (McBrien & Gentle 2003). The myopic eye shows features such as increased axial length of the eye, thinning of the sclera, retinal detachment, and liquefaction of the vitreous gel (McBrien et al. 2001, see review Jacobi 2005). These findings might occur due to different pathological pathways in the development of the eye or progression of eye-associated disorders, environmental factors, and aging (see review Jacobi 2005). Myopia can be classified into different categories based on inheritance, severity, age of onset, progression, and pathological consequences. It is also divided into three categories depending on the refractive state of the eye. Physiological or low myopia occurs when the refractive power of the eye is from -1.5 to -3 diopters (D), whereas intermediate myopia is classified as a refractive power of -3 to -5 D. When the degree of myopia is above -6 D, it is termed pathological or high myopia. (McBrien 2003, see review Jacobi 2005, Morgan 2005).

2.6.2 Prevalence of myopia

Being the most prevalent ocular condition, myopia has a wide variability in prevalence between different ethnic groups. The prevalence varies from 10-20 % in Africa and 30-40 % in Europe and the USA, to as high as 70-90 % in Asia (Chow et al. 1990, Saw et al. 1996, Zadnik et al. 1997, Wong et al. 2000). Pathological or high myopia, variably defined as a refractive error of at least -6 or -8 D, has been estimated to affect 1-3 % of the population (Fuchs 1960, Sperduto et al. 1983, Fredrick 2002).

2.6.3 Pathogenesis of myopia

Myopia is a result of the failure of the eye growth control processes. Manifestations of the disturbed growing processes are increased axial length of the eye, thinning of the sclera, liquefaction of the vitreous gel, and retinal detachment. As a consequence of these findings, the refractive power of the eye is not in balance with the size and structural components of the eye. (Morgan 2005). 40 Normal processing in the eye, when the axial length of the eye is actively matched to the optical power of the cornea and lens, is called emmetropisation. Animal models of myopia have suggested an important role for emmetropisation, but there is also evidence that the development of myopia is influenced by genetic factors. Several studies have proposed that environmental factors and the amount of near work are associated with myopia, but this connection is poorly defined. (see review Schaeffel et al. 2003). Studies in animal models have clearly demonstrated that the axial elongation of the eye happens due to the outer coat of the eye, the sclera. Particularly active remodeling of the sclera has been shown to occur rather than passive stretching (Jobling et al. 2004). The sclera is a connective tissue consisting of heterogenous collagen fibrils, with collagen I being most predominant. Studies of a mammalian model of myopia have shown that changes occur in the collagen architecture and spacing, such as degradation of collagen fibrils and a reduction in the diameters of the fibrils (Gentle et al. 2003, Jobling et al. 2004). Gentle et al. reported that changes in scleral collagen fibril synthesis are mainly caused by reduced collagen I production. Also, short time increases in the ratio of newly synthesized collagen III/I and collagen V/I influence the small diameter of scleral collagen fibrils (Gentle et al. 2003). Fibromodulin and lumican, the proteglycans expressed in human sclera, have been shown to have binding sites on collagens I and II (Hedbom & Heinegård 1993, Svensson et al. 2000). In addition, PRELP can bind these collagens through its leucine rich repeat domain. It has been proposed to function as an anchoring molecule between the basement membrane and the underlaying connective tissue (Bengtsson et al. 2002). In addition to collagen alterations, changes in scleral proteoglycans have also been reported to be associated with the scleral processing. These may happen due to reduced synthesis or sulfation of their glycosaminoglycans side chains (Norton & Rada 1995, McBrien et al. 2000, Jobling et al. 2004). These changes result in an abnormally thin sclera. In the high myopic eye, the liquefaction of the vitreous is suggested to happen due to disturbed spacing and organization of the collagen fibrils. This may possibly be a consequence of aging, when the fibrils become less sticky. Also, the vitreoretinal adhesion of the collagen fibrils may be weakened because of the age-related changes of collagen fibrils in the human vitreous. Mutations in the genes expressed in the structural components of the human vitreous have also been suggested to play a role in the development of high myopia. Together with the liquefaction and weakened adhesion of the vitreous, the abnormalities within in the vitreous collagen network may also predispose to posterior vitreous detachment. (Bishop 2000).

2.6.3.1 Genetic factors in myopia

Myopia is observed in a great amount of conditions including over 150 genetic syndromes defined with various levels of myopia. It has become clear that myopia has an etiologically heterogeneous nature and both genetic and environmental factors contribute to the development of myopia. (see review Jacobi et al. 2005). Children of myopic parents are more likely to have myopia than children with non- myopic parents (Goldschmidt 1981, see review Jacobi et al. 2005). Twin studies of 41 myopia also provide strong support for the role of inheritance in myopia (Hammond et al. 2001). The genetic heterogeneity is seen in nonsyndromic myopia, since ten chromosomal loci, MYP1-MYP10, have been detected so far (Table 5). To date, no mutations have been reported at these loci. (Young et al. 1998, Naiglin et al. 2002, Paluro et al. 2003, Stambolian et al. 2004, Hammond et al. 2004).

Table 5. Myopia loci.

MYP1 MYP2 MYP3 MYP4 MYP5 MYP6 MYP7 MYP8 MYP9 MYP10 Xq28 18p11.31 12q23-24 7q36 17q21-22 22q12 4q12 3q26 8p23 11p13

Only a few candidate genes for high myopia have been studied so far. TGF-β-induced factor (TGIF) was shown to be associated with high myopia (Lam et al. 2003), while Lipin 2 (LPIN2) was excluded as a candidate on the MYP2 locus (Zhou et al. 2005). Some homeobox genes have been suggested to be important for eye development, such as the PAX and CHX10 genes. Particularly the PAX6 gene was proposed to be a candidate gene for high myopia in twin studies, but an analysis of this gene has not been performed. (Hammond et al. 2004, see review Jacobi et al. 2005). Recently, a 5’-regulatory region SNP in the LUM gene was associated with high myopia (Wang et al. 2006). Since the ECM of the cornea and sclera and the ECM components of the vitreous have key roles in determining the refractive state by changing the axial length of the eye and the focusing power of the cornea and crystalline lens, mutations in the ECM genes are likely to be important particularly in high myopia (Bishop 2000, Gentle et al. 2003). Therefore, the predominant collagen in the vitreous, collagen II, and the ocular SLRPs have been proposed to have a role in the pathogenesis of myopia.

3 Outlines of the present research

There has been a long debate whether the two overlapping phenotypes, STL2 and Marshall syndromes, are separate entities or just variable phenotypes of one disease. Even though the clinical features and genetic findings overlap it seems that there are characteristic manifestations for both allelic syndromes that allow them to be distinguished from one another. Stickler syndrome type II is an autosomal dominant connective tissue disorder resulting from mutations in the COL11A1 gene. Marshall syndrome (Marshall et al. 1958) is also an arthro-opthalmopathy inherited in an autosomal dominant manner as a consequence of mutations in the COL11A1 gene (Annunen et al. 1999). The clinical phenotype overlaps with that of Stickler syndrome, and therefore it may be difficult to classify the patients as having Marshall or Stickler syndrome. It has been suggested that splicing mutations in the 54-bp exons in the 3’half of COL11A1 lead to Marshall syndrome, rather than Stickler syndrome. Therefore, we initiated a large study to analyze the genotype-phenotype correlation in type XI collagenopathies. STL1 is known as Stickler syndrome, with ocular and extraocular findings such as hearing loss, cleft palate, and early-onset osteoarthritis. The disease-causing gene, COL2A1, has two different splicing variants. Mutations in the alternatively spliced exon 2, expressed only in the eye, are likely to cause a predominantly ocular Stickler syndrome without any of the extraocular manifestations typical for Stickler syndrome. Hearing loss is a common feature of type II and XI collagenopathies, but approximately 80 % of the hearing loss is nonsyndromic. The COL11A1 gene is located in the same region as the nonsyndromic deafness locus (DNFB32) defined by linkage analysis. Therefore, COL11A1 was proposed as a candidate gene for nonsyndromic hearing loss. Myopia is the most common eye condition and has a marked risk for blindness due to the complications of high myopia. So far, ten chromosomal loci for high or pathological myopia have been identified, but no mutations have been implicated. Ocular SLRPs of the ECM have been suggested to have a role in the development of myopia based on their role in the development and growth of the eye and interactions with collagenous eye components of the ECM. 43 The aims of the study were: 1. to clarify the role of COL11A1 in Stickler and Marshall syndromes, 2. to study the mechanisms by which COL2A1 gene mutations lead to predominantly ocular Stickler syndrome, 3. to analyze COL11A1 as a candidate gene for nonsyndromic hearing loss at the DFNB32 locus, 4. to study the role of four SLRP proteins, opticin, lumican, fibromodulin, and PRELP, in the development of high myopia.

4 Materials and methods

Materials and methods used in this thesis are described in detail in the original articles I- IV.

4.1 Patients and controls (I-IV)

Altogether 44 subjects clinically diagnosed with Stickler syndrome type II, but having an overlapping phenotype with Marshall syndrome and lacking mutation in the COL2A1 gene, were included in the study (article I). Clinical, radiological, auditory, and ophthalmologic examinations were performed on all the study subjects. In the original article II, two patients with STL1 with no or minimal extraocular findings and one patient with high myopia were included in the study (article II). They were initially clinically examined by a specialist. Opthalmologic examination was performed and a blood sample taken for the DNA isolation. A large Tunisian family including twenty-two individuals with autosomal recessive nonsyndromic hearing loss was analyzed in the study (article III). Individuals were evaluated for hearing loss by a clinical examination. Also, audiological tests including pure-tone audiometry and evoked response audiometry were performed. Thresholds of air-conduction pure-tone average (ACPTA) were calculated from all the subjects for each ear to clarify the severity of the hearing impairment. The categories and limits for diagnoses were as follows: mild (25 dB ≤ ACPTA ≤ 39 dB), moderate (40 dB ≤ ACPTA ≤ 69 dB), severe (70 dB ≤ ACPTA ≤ 89 dB), and profound (ACPTA ≥ 90 dB). Subjects were clinically diagnosed to be otherwise healthy. Eighty-five English and 40 Finnish unrelated individuals with high myopia, defined as a refractive error of at least – 6 D, were included in the study (article IV). All the subjects had a positive family history of high myopia, having at least one affected member in their immediate family. Ophthalmologic examinations were performed by a specialist. DNA samples from family members were available for three of the Finnish patients and one of the English patients. The control groups in this study consisted of 208 English and 100 Finnish individuals with a refractive error within 2 D of emmetropia. 45 Genomic DNA was extracted from EDTA anti-coagulated blood samples from the study subjects using standard protocols. Signed informed consent was obtained from all the study subjects.

4.2 CSGE (I, III, IV)

Conformation-sensitive gel electrophoresis (CSGE) was used for mutation screening. (see review Ganguly et al. 1993, Körkkö et al. 1998, Annunen et al. 1999b). Primers for polymerase chain reaction (PCR) were designed to amplify the target sequences including exons and the corresponding exon-flanking sequences. Sequences from exons 14 to 68 of COL11A1 (Annunen et al. 1999b) and exon boundaries were amplified to create products ranging from 182 bp to 487 bp (article I). In the original article III, all 68 exons and exon boundaries of COL11A1 were amplified by PCR. In the myopia study (article IV), exons 1-8 of OPTC (Hobby et al. 2000), 1-2 of FMOD (see review Hocking et al. 1998), 2-3 of LUM (Grover et al. 1995), and 2-3 of PRELP (Grover et al. 1996) were amplified to obtain products with sizes from 232 bp to 497 bp. The quality and quantity of the PCR products were determined on a 1.5 % agarose gel. Approximately 20 ng of each PCR product was used for heteroduplex analyses by CSGE as previously described (Körkko et al. 1998), with the exception that the gels were stained with SYBR Gold nucleic acid gel stain (Probes, Eugene, Oregon, USA). Because CSGE reveals only the heterozygous variations, samples with suspected homozygous variations were mixed before the heteroduplexing phase with an equal amount of control PCR product (article III). Products containing heteroduplexes were sequenced.

4.3 Sequencing (I-IV)

PCR products containing heteroduplexes were sequenced using a DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech, Buckinghamshire, England) and ABI PRISMTM 377 (Applied Biosystems) (articles I-IV) or BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) and ABI PRISMTM 3100 (Applied Biosystems) (articles I, II and IV) to define the underlying sequence variations of the analyzed gene. PCR or nested primers were used for sequencing. RT-PCR products were sequenced using primers designed for the cDNA sequence of COL2A1 (article II). 46 4.4 Minigene analyses (II)

4.4.1 COL2A1 constructs (II)

Minigene analyses were performed to study the consequences of COL2A1 exon 2 mutations on alternative splicing. One minigene, named “COL2A1 E1-E54”, was synthesized by ligating two fragments amplified by PCR using commercially available genomic DNA (Boehringer Mannheim) as a template. The first fragment contained exon 1 and part of intron 1 (article II), and the second fragment spanned from intron 51 to the 3’-UTR, containing the intervening exons 52-54 and introns 52-53 (Table 3, article II). Amplified PCR products were ligated into the expression vector pcDNA3.1(+) (Invitrogen). A second COL2A1 minigene was utilized in the present study, named “COL2A1 E1- E3”. This 5.9 kb minigene consisting of exons 1, 2, and 3 with full-length intervening introns 1 and 2 has been described in a previous study (McAlinden et al. 2005). These three fragments of genomic DNA were subsequently ligated and cloned in the pcDNA3.1(+) vector (Invitrogen). Two mutant “COL2A1 E1-E54” minigenes were constructed by amplifying exon 2 of COL2A1 from the genomic DNA of an unaffected individual and ligated into the pGEMT easy-vector (Promega). The QuikChange™ XL site-directed mutagenesis kit (Stratagene) was used to create Cys57Tyr and Cys64Stop mutations within exon 2 according to the manufacturer’s instructions, and the presence of the mutation was confirmed by DNA sequencing. Either the wild-type or mutant exon 2 fragment was then inserted into the pcDNA3.1/COL2A1 E1-E54 construct using the EcoRV restriction enzyme site. To create a positive control, exon 27 of COL9A2 with the splicing mutation IVS26-2A>C (Noponen-Hietala et al. 2003) together with 102 bp and 66 bp of flanking introns 26 and 27, respectively, was amplified using primers with EcoRV restriction sites (Table 3, article II) and ligated into this minigene. Six different “COL2A1 E1-E3” minigenes were synthesized containing mutations in exon 2 associated with the predominantly ocular form of Stickler syndrome (Table 2, article II). Mutations were introduced into the exon 2-containing fragment of the minigene in the pSP73 vector (Promega) using the QuikChange™ site-directed mutagenesis kit. An additional mutant minigene was made by deleting a twelve nucleotide stretch in exon 2. This region was predicted to contain a splicing enhancer cis element when the COL2A1 exon 2 sequence was entered into a web-based program (http://exon.cshl.org/ESE) (Cartegni et al. 2003).

4.4.2 Cell culture and transfections (II)

CHO (Chinese-hamster ovary) cells were grown on 100 mm plastic dishes in DMEM (Dulbeccos’s modified Eagles’s medium; Biochrom KG) with 10 % (v/v) FBS (fetal bovine serum; GB Perbio HyClone), 0.1 % Penicillin (Sigma), 0.01 % Fungizone (Cambrex Bio Science), 0.1 % L-glutamine (Sigma), and 5 % Na-bicarbonate (Sigma). 47 Cells were transfected with different minigene constructs using FuGENE reagent according to product instructions (Roche). The final ratio of minigene DNA:FuGENE (µg/µl) was 2:3. Cells were incubated for 24, 48, and 72 hours and harvested with trypsin (Sigma). RNA was extracted using Trizol reagent (GibcoBRL) followed by cDNA synthesis with the SuperScript First-Strand Synthesis System (Invitrogen). The COL2A1 E1-E3 minigene was transfected into human embryonic kidney cells (HEK-293) or rat chondrosarcoma (RCS) cells using FuGENE 6 reagent (Roche Applied Science). The final ratio of minigene DNA:FuGENE (µg/µl) was 1:4. After transfection (48 h), RNA was harvested using the Qiagen RNeasy kit.

4.4.3 RT-PCR (II)

Total RNA for proband one and a control individual were extracted from Epstein-Barr virus-transformed lymphoblasts. The cDNA synthesis was carried out with the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen). The first PCR was performed with a pair of primers that corresponded to exons 1 and 8 of COL2A1 (Table 3, article II), and the second was performed with nested PCR primers that corresponded to exons 2 and 7 (Table 3, article II). To analyze the “E1-E54” minigene construct, the total RNA extracted from the cells was used in a reverse transcriptase reaction with the SuperScript First-Strand Synthesis System (Invitrogen). The cDNA was then used as a template for RT-PCR (Invitrogen), and followed by amplification with two sets of PCR primers. Two different primer sets were used to amplify the cDNA from exon 1 to exons 53 and 54 of COL2A1 (Table 3, article II). In addition, a forward primer from exon 2 (Table 3, article II) was used together with exon 53 and 54 reverse primers. To analyze the “E1-E3” minigene, approximately 1µg of RNA was reverse transcribed using random primers. To amplify cDNA derived only from the exogenously transfected COL2A1 mini-gene, primers pcDNA3-COL2A1-Exon 1 and sp6 (P1 and P2 in Figure 4A) were used in the presence of [α-32P] dCTP (10mCi/ml, 3000Ci/mmol; Amersham Biosciences). The linear range for these primers was established and PCR was carried out for 20 cycles: 95 °C for 30 s; 55 °C for 30 s; 72 °C for 30 s. PCR products were electrophoresed through 6 % polyacrylamide gels at 200 V. Gels were dried, exposed to a PhophsorImager screen, and scanned on a STORM™840 PhosphorImager (Amersham Biosciences).

4.5 Genotyping and linkage analyses (III)

Genome-wide screening was performed using 400 microsatellite markers at intervals of 10 CM (ABI PRISM 1 Linkage Mapping Set 2, Applied Biosystems). Multiplex PCR was performed with four markers, and PCR products were analyzed following electrophoresis on a 6 % polyacrylamide gel. To confirm the linkage and to fine-map the genetic interval, 19 additional polymorphic microsatellite markers were typed comparing 18 published (http://cedar.genetics.soton.ac.uk/pub/chrom1/map.html) and one new 48 marker (D1S21401) found by searching for possible (CA) repeats in the sequence data of BAC clones between markers D1S1166 and afmb014zb9. Linkage analyses were performed using the linkage 5.1 program. Two-point lodscores were calculated with the Mlink program.

4.6 PolyPhen, Ensembl, and statistical analyses (IV)

To analyze the consequences of the observed missense mutations in article IV, the PolyPhen (Polymorphism Phenotyping) automatic tool for predicting the impact of an amino acid substitution on protein structure and function was used. PolyPhen analyses are based on straightforward empirical rules which are applied to the sequence and phylogenetic and structural information, characterizing the substitution at the protein level (http://tux.emblheidelberg.de/ramensky/). The Ensembl system was used to analyze the conservativeness of the locations of the opticin, lumican, and fibromodulin variations within species (www.ensembl.org). Statistical analyses were performed using Fisher’s exact test with GraphPad software (www.graphpad.com).

4.7 Homology model for the opticin LRR domain (IV)

Examination of the sequence of human opticin and bovine decorin, for which a crystal structure at a resolution of 2.15 Å is available (Scott et al. 2004), suggested that a model of the LRR domain of opticin could be constructed using the software package Modeller 8v0 (see review Marti-Renom et al. 2000). Template reconstruction of opticin was done using XFIT (McCree 1999), with sequence alignments modeling the opticin LRR domain generated with Clustal W (Higgins et al. 1994).

5 Results

5.1 Analyses of Stickler and Marshall patients (I)

Forty-four patients with suspected Stickler syndrome were screened for mutations in the COL11A1 gene to detect the genotype-phenotype correlations in the type XI collagenopathies. All the patients had gone through the COL2A1 analyses in the Medical Genetics laboratory at the University of Ghent and no mutations were found. Sequences were compared to the wild-type sequences (GenBank accession number NM_001854) and the nucleotides were numbered starting from the first base of the initiation codon (ATG) of the reference sequence. (den Dunnen & Antonarakis 2001).

5.1.1 COL11A1 sequence variations (I)

Screening of 44 unrelated subjects for sequence variations in COL11A1 revealed altogether ten heterozygous disease-causing mutations. The mutations included seven splice site mutations, two deletions, and one glycine substitution in the coding region of COL11A1. All the splicing mutations were located in the 3’ half of COL11A1, spanning exons 47 to 55. Four of these mutations altered splicing consensus sequences of intron 50, with three of them being at position c.3816+1G>A, and one at c.3816+2insT. Four other splice site mutations were located in introns 47 (c.3655-2A>G), 53 (c.4033-1G>C), and 55 (c.4140+1G>A). In addition, one patient had a deletion of 29 nucleotides starting from the last nucleotide of exon 44 (c.3438delT) and including 28 nucleotides of intron 44 (c.3438+1_28del). This mutation is also likely to result in abnormal splicing. Another deletion observed was an in-frame deletion of 18 nucleotides located in exon 36 (c.2760_2777del). In addition, mutation analyses revealed a missense mutation in exon 59 of COL11A1, a glycine substitution by aspartate (c.4412G>A; p.Gly1471Asp). 50 5.1.2 Genotype-phenotype correlation (I)

All the patients included in this study had a phenotype very suggestive of either STL2 or Marshall syndrome. Four of ten patients with a mutation in COL11A1 could be classified as having Marshall syndrome rather than Stickler syndrome, while the six remaining patients showed an overlapping Marshall-Stickler phenotype. Based on the facial dysmorphism, patients GE-03-023 (Figure 1b, article I), GE-02- 021 (Figure 1c, article I), GE-02-130 (Figure 1d, article I), and GE-04-041 (Figure 1e, article I) were diagnosed with Marshall syndrome. The clinical phenotype of these patients included a severe midfacial hypoplasia with short nose, anteverted nares, and bulging of the eyes. Patients GE-03-023, GE-02-021, and GE-02-130 had sensorineural hearing loss (SNHL). The hearing status of patient GE-04-041 could not be determined because the patient was still an infant. Only one of the patients (GE-03-023) had short stature. All these patients had a splice site mutation in intron 50 of COL11A1. The six patients with an overlapping Marshall-Stickler phenotype had milder facial dysmorphism with less pronounced midfacial hypoplasia. Also, the nasal bridge was not significantly depressed compared to those of the patients with Marshall syndrome. Three of the six patients had SNHL. Five of the patients had high myopia, and one additional affected individual had experienced a spontaneous retinal detachment. The sixth patient’s ophthalmological evaluation revealed an empty vitreous with abnormal membranes, but no evidence of high myopia or retinal degeneration. Three of the six patients with an overlapping Marshall-Stickler phenotype had a splice site mutation in introns 47, 53 (Figure 1f, article I), and 55 (Figure 1g, article I). Two of the patients had deletions in exons 36 (Figure 1a, article I) and 44, and one had a glycine substitution in exon 59 (Figure 1h, article I).

5.2 Analyses of patients with predominant ocular findings typical for Stickler syndrome (II)

Two probands with predominant ocular findings typical for Stickler syndrome and one patient with only high myopia were analyzed for mutations in exon 2 of COL2A1. The consequence of the observed mutations was further studied with cell culture and RT-PCR techniques.

5.2.1 Analyses of COL2A1 sequence variations and RT-PCR (II)

Genetic analysis of exon 2 of COL2A1 showed that patients 1 and 2 had the same heterozygous mutation, c.192C>A, (Genebank accession # NM_001844). The mutation converted the codon TGC for cysteine at position 64 to the stop codon TGA (Cys64Stop; Genebank accession # NP_001835) (Figure 2). Patient 3 had a c.170G>A change in exon 51 2 that converted the codon TGT for cysteine to TAT for tyrosine (Cys57Tyr). Neither of the mutations was found in 248 control chromosomes. To study the consequence of the PTC mutation at the mRNA level, RT-PCR analysis was performed using the total lymphoblast RNA of patient 1. RNA samples from patients 2 and 3 were not available. Analysis of the RT-PCR products on agarose gel showed the presence of both the long and short forms of COL2A1. Sequence analysis of the long form revealed that only the normal allele was present in the cDNA, even though both alleles were present in the genomic DNA (Figure 2). These results indicated that the stop codon mutation resulted either in complete loss of the allele by NMD or the skipping of exon 2 via NAS.

Fig. 2. Sequencing of the exon 2 of COL2A1 from patient 1 with p.Cys64Stop mutation. A. Genomic DNA with the heterozygous C>A change that creates a stop codon. B. The sequence of the longer RT-PCR product showed the presence of only the normal allele.

5.2.2 Minigene analyses for COL2A1 (II)

To study the effect of Stickler-associated mutations in exon 2, two COL2A1 minigene constructs were created, one containing exons 1, 52-54, truncated intron 1 (300 bp), and full-length introns 52 and 53 (Figure 3B), and another containing full-length introns spanning the alternatively-spliced exon 2 (Figure 3C). 52

Fig. 3. COL2A1 alternatively spliced isoforms and minigene constructs. A. Two isoforms of procollagen II (COL2A1) exist by alternative splicing of pre-mRNA. Procollagen IIA contains sequence coded by exon 2 in the N propeptide while procollagen IIB lacks this sequence. B. “COL2A1 E1-E54” mini-gene (3.0 Kb) consisting of exon 1, part of intron 1, exon 2, part of introns 2 and 51, followed by exons 52-54 with the corresponding introns and 3’-UTR. Restriction enzyme sites used for subcloning this mini-gene and inserting the exon 2 sequence are shown. A COL9A2 fragment of exon 27 and flanking intronic sequences with IVS26-A>C splice site mutation was inserted to produce a positive control mini-gene. C. The 5.9 Kb “COL2A1 E1-E3” mini-gene consisting of exons 1, 2 and 3 and full-length introns 1 and 2 (32). 53 Cells were transfected with the minigene contructs containing exon 2 with the normal sequence and the p.Cys64Term and p.Cys57Tyr changes. CHO cells were used for the “E1-E54” minigene analysis. RT-PCR analysis of the non-mutated COL2A1 E1-E54 minigene with 1F1 and 54R primers resulted in a 810-bp product containing sequences for exons 1, 2, 52, 53, and part of exon 54 (Figure 4A, lane 3). Only a short product (603 bp) was obtained from constructs with the mutated exon 2 (p.Cys57Tyr and p.Cys64Stop) containing exons 1 and 52-54 (Figure 4A, lanes 1 and 4). Similar results were obtained using primers 1F2 and 53R, with the fragment sizes being 579 and 372 bp for the non- mutated and mutated constructs, respectively (Figure 4A). However, RT-PCR with primers from exon 2 to exon 53 amplified the long form from all constructs with exon 2, indicating that the long form was present in the cDNA.

Fig. 4. Alternative splicing of wild type and mutant COL2A1 E1-E54 mini-genes. Reverse transcriptase amplifications with two primer sets from exon 1 to exons 53 or 54 (left top and middle panels). Lane 1: _x174 DNA molecular standard, lane 2: Cys57Tyr mutation, lane 3: wild-type exon 2, lane 4: Cys64Stop mutation and lane 5: COL9A2 exon 27 splice site mutation. The left bottom panel shows the amplification product with primers from exon 2 to exon 53 frommini-genes containing either wild-type or mutant exon 2. The mechanisms of alternative splicingof the mini-gene to produce either the IIA or IIB isoform or the positive control with the COL9A2 exon 27 splice site mutation are presented in line diagrams on the right. Numbers represent the nucleotide sizes of the exon/intron regions. The primers used to amplify cDNA derived from the mini-gene are indicated with arrows. 54 A previously identified splice site mutation in COL9A2 (IVS26-2A>C) was introduced in the mini-gene to serve as a control for the splicing the mechanism. The control construct with COL9A2 exon 27 (IVS26-2A>C) resulted in two products. In the larger product (1004 bp), the mutation prevented the splicing of intron 1. The short product (603 bp) resulted from the skipping of exon 27 (Figure 4A, lane 5). A similar result was obtained with the use of primer set 1F2 and 53R, with the fragment sizes being 806 and 372 bp. The result showed that the intron preceding the mutation was not spliced correctly, congruent with result of the patient analysis (Noponen-Hietala et al. 2003). Figure 5B shows the IIA and IIB alternative splicing patterns of the wild-type and mutant minigenes in either HEK-293 or RCS cells. The wild-type splicing patterns differed between the cell types, where the ratio of IIA:IIB spliced isoforms was higher in HEK-293 cells compared to RCS cells. This was also shown in a previous study (McAlinden et al. 2005) and confirms the efficacy of this minigene system; mature chondrocytes (i.e. the RCS cells) would be expected to have a lower COL2A1 IIA:IIB ratio since during differentiation of the chondrocytes, the cells switch from producing the IIA form to the IIB form. There was a decrease in the ratio of IIA:IIB spliced isoforms in three mutant minigenes which correspond to mutations p.Trp47Stop, p.Cys57Tyr, and p.Cys64Stop (Figure 5B). This suggests that these three mutations have disrupted an enhancer splicing cis element that is required for the inclusion of exon 2. This is in agreement with the results in Figure 4 utilizing the COL2A1 E1-E54 minigene with the p.Cys57Tyr and p.Cys64Stop mutations. 55

Fig. 5. Alternatively splicing of wild type and exon 2 mutant COL2A1 E1-E3 minigenes. A. Mechanisms of alternative splicing of the mini-gene to produce either the IIA or IIB isoform. Numbers represent the nucleotide sizes of the exon / intron regions. Primers P1 and P2 were used to amplify cDNA derived from the mini-gene. Respective sizes of IIA and IIB cDNA isoforms are shown. B. Phosphor-images of IIA and IIB spliced isoforms derived from the wild-type or mutant mini-genes that were transfected into either human embryonic kidney (HEK-293) cells or rat chondrosracoma (RCS) cells. Lanes 1-7 show splicing of wild type (lane 1) compared to mutant COL2A1 mini-genes (lanes 2-7). The exon 2 mutations are listed in the right panel. These mutations have been reported in patients with the ocular phenotype of Stickler syndrome. The asterisks highlight the mutations that resulted in an apparent decrease in the ratio of IIA:IIB spliced products. 56 To determine if COL2A1 exon 2 contains cis elements that regulate alternative splicing, the 207-bp exon 2 sequence was entered into a web-based program that predicts the presence of potential cis elements (http://exon.cshl.edu/ESE) (Cartegni et al. 2003). The highest-scoring sites were located near or overlapped with the exon 2 Stickler mutations reported previously as well as in the current study (Table 1, article II). A deletion COL2A1 E1-E3 minigene was made devoid of 12 nucleotides that span a high- scoring cis element predicted to be a binding site for the splicing factor SRp55. In both HEK-293 and RCS cells, deletion of this region dramatically decreased the IIA:IIB ratio, proving that we had deleted an important splicing enhancer cis element. This evidence supports our hypothesis that some Stickler-related exon 2 mutations may result in the insertion of a premature stop codon that leads to nonsense-mediated decay, whereas others may disrupt splicing cis elements resulting in an altered splicing pattern and synthesis of an alternatively spliced isoform of procollagen II, namely type IIB (Figure 6).

Fig. 6. Differential effects of exon 2 mutations on alternative splicing of COL2A1. This model shows two potential outcomes of Stickler-related exon 2 mutations. One scenario is a mutation (X) in exon 2 resulting in a premature stop codon that does not overlap with any potential enhancer splicing factor protein binding sites in exon 2. Normal splicing of pre- mRNA would occur followed by degradation by nonsense-mediated decay (NMD) and haploinsufficiency. The second scenario is disruption of a splicing cis element by the Stickler mutation resulting in nonsense-mediated altered splicing (NAS) and production of an alternatively spliced protein (procollagen IIB in this case). 57 5.3 Analyses of patients with congenital profound autosomal recessive deafness (III)

A large Tunisian family consisting of seventeen unaffected and five affected individuals with congenital profound autosomal recessive deafness was studied to map the gene responsible for the defect. The linkage analyses revealed a novel deafness locus, DNFB32, on chromosome 1. The DFNB32 region contained the COL11A1 gene, which was analyzed as a candidate gene for autosomal recessive nonsyndromic deafness.

5.4 Linkage analyses (III)

Initially, linkage analyses were performed of the previously identified loci (http://www.uia.ac.be/dnalab/hhh, Table 6) to map the gene responsible for the deafness. No linkage was found, suggesting a novel locus for this Tunisian family. Genome-wide screening with 400 microsatellite markers at intervals of 10 cM indicated a novel locus on chromosome 1, defined as DFNB32. A maximum two-point lodscore (LOD) of 4.96 was obtained for the new polymorphic marker D1S21401 identified in this study. Fine mapping the genetic interval was performed with nineteen additional polymorphic microsatellite markers. Significant two-point lodscores were obtained with five microsatellite markers with a maximum LOD of 4.36 for D1S2896. Haplotype analyses definined the centromeric boundary of the disease interval between markers D1S1166 and afmb014zb9. 58 Table 6. Loci and gene for autosomal receissive, non-syndromic deafness (Modified from Petersen & Willems 2006).

Locus Chromosomal location Gene DFNB1 13q11–q12 GJB2 (Connexin 26) GJB6 DFNB2 11q13.5 MYO7A DFNB3 17p11.2 MYO15 DFNB4 7q31 SLC26A4 DFNB5 14q12 DFNB6 3p21 TMIE DFNB7 9q13–q21 TMC1 DFNB8 21q22.3 TMPRSS3 DFNB9 2p23.1 OTOF DFNB10 21q22.3 TMPRSS3 DFNB11 9q13–q21 DFNB12 10q21–q22 CDH23 DFNB13 7q34–q36 DFNB14 7q31 DFNB15 3q21.3–q25.2/19p13.3–p13.1 DFNB16 15q15 STRC DFNB17 7q31 DFNB18 11p15.1 USH1C DFNB20 11q25–qter DFNB21 11q23–q25 TECTA DFNB22 16p12.2 OTOA DFNB23 10q21.1 PCDH15 DFNB26a 4q31 DFNB27 2q23–q31 DFNB29 21q22.1 CLDN14 DFNB30 10p11.1 MYO3A DFNB31 9q32–q34 WHRN DFNB32 1p22.1–p13.3 DFNB33 9q34.3 DFNB35 14q24.1–q24.3 DFNB36 1p36.3 ESPN DFNB37 6q13 MYO6 DFNB38 6q26–q27 DFNB39 7q11.22–q21.12 DFNB40 22q11.21–q12.1 DFNB42 3q13.31–q22.3 DFNB44 7p14.1–q11.22 DFNB46 18p11.32–p11.31 DFNB48 15q23–q25.1 DFNB49 5q12.3–q14.1 DFNB53 6p21.3 COL11A2 DFNB55 4q12–q13.2 NLDb 1p35–p33 GJB3 NLDb 6q21–q23.2 GJA1 NLDb 7q22.1 SLC26A5 a Dominant modifier DFNM1 suppresses recessive deafness DFNB26. b No locus designation. 59 5.4.1 Analyses of the COL11A1 gene (III)

The COL11A1 gene is located in the DFNB32 region and was therefore analyzed as a candidate gene for the congenital profound autosomal recessive deafness. Mutations in this gene have been linked to two forms of syndromic deafness, Marshall and Stickler syndromes. All of the 68 coding exons of COL11A1 and their surrounding intronic sequences were first analyzed by CSGE, and about half of the exons were also analyzed by sequencing using an ABI 377 automated sequencer. Screening for sequence variations revealed altogether 21 variations in the coding region of COL11A1. All these variants have previously been detected in several unaffected individuals, suggesting that the variations are likely to be neutral.

5.5 Candidate gene analysis of patients with myopia (IV)

Eighty-five English and 40 Finnish unrelated individuals diagnosed with high myopia with a refractive error of at least – 6 D were included in the study. Altogether twelve DNA samples were available from family members of one English and three Finnish patients. Four SLRP genes, OPTC, LUM, FMOD, and PRELP, were selected as candidate genes and analyzed for sequence variations in the patients.

5.5.1 OPTC, LUM, FMOD and PRELP sequence variations

Screening of the OPTC gene revealed four unique variations found only in high myopic patients. A c.530C>G converted the codon ACA for Thr177 to AGA for Arg (Figure 7), and a c.989G>A variation changed the codon CGC for Arg330 to CAC for His in two Finnish patients. Among the English patients we found a c.686G>A change altering CGC for Arg229 to CAC for His in one patient (Figure 7). A c.985G>A variation altered the codon GGC for Gly329 to AGC for Ser and was found in two individuals. In addition, a c.973C>T variation changed the codon CGG for Arg325 to TGG for Trp in two high myopes, but was also identified in two control samples. Additional findings were a synonymous c.402C>T (p.L134L) change found in one high myopic patient and a non-synonymous c.803T>C change leading to an amino acid change, p.Leu268Pro (Figure 7). The latter was observed both in the patient and control sets with similar frequencies. Screening of the LUM gene revealed one unique variation changing an amino acid; a c.596T>C change converted the codon CTG for Leu199 to CCG for Pro in one English subject (Figure 7). In addition, one unique intronic variation was observed, c.893 -69 A>C. Both of these variations were not found in the controls. Screening of the FMOD gene revealed one unique sequence variation in a subject with high myopia. A c.971G>A variation converted the codon AGG for Arg 324 to AAG for Thr (Figure 7). The p.Arg324Thr variation was not found in the controls. 60 Only one unique intronic variation was found in the flanking sequence of exon 3 of the PRELP gene (c.1188+27T>C). Also, one synonymous variation at position c.408C>T coding for Asp149 was observed in a patient with high myopia (Figure 7). These variations were not observed in control individuals. (Table 2, article IV).

Fig. 7. Internal LRR organization of four SLRPs. Each box represents one structural element: N represents conserved N-terminal disufide bonded cap, boxes numbered 20–26, individual LRRs containing 20–26 amino acids, and E, extended ear repeat (Modified from McEwan et al. 2006). The asterisks highlight the mutations that located in the LRR residues.

5.5.2 Family studies

We studied the co-segregation of high myopia and the OPTC variations Thr177Arg, Arg325Trp, Arg330His and Arg329Ser in the families of four probands. In addition to the proband, the Thr177Arg variation was observed in one myopic family member, but was absent from two non-myopic relatives, the patient’s father and uncle. The Arg325Trp variation was also found in the patient’s mother, who had a refractive error - 5.25 D in both eyes, and a brother with moderate myopia with a refractive status of – 3.5/-3.75 D. Neither the emmentrophic father nor a sister with low myopia carried the variation. The Arg330His variation was found in the patient’s high myopic sister and emmentrophic father, whereas the mother with no myopia did not carry the variation. Both parents for the English patient with the Arg329Ser variation were analyzed. The variation was observed from the emmetrophic mother, but the father with low myopia was not a carrier. (Figure 1, article IV) 61 5.5.3 PolyPhen, Ensembl and statistical analyses

The PolyPhen analyses to predict the impact of each observed missense mutation on the protein structure and function indicated five probably or possible damaging changes: Thr177Arg, Arg325Trp and Gly329Ser in opticin, Leu199Pro in lumican, and Met157Val in PRELP (Table 2, article IV). The Ensembl system was used to analyze the conservativeness of the opticin, lumican, and fibromodulin variations within species and revealed that the variations Thr177Arg, Arg229His, Arg325Trp and Gly329Ser in opticin, Leu199Pro in lumican and Pro46Leu, Glu147Asp, and Arg324Thrn in fibromodulin were located in highly conserved positions (Figure 2, article IV). The total number of variations observed in these four SLRP genes showed no significant difference between the patients and controls in statistical analyses. However, slightly more variations were observed in the English patients in the OPTC gene with a p- value of 0.0422. The c.893-105G>A variation in the LUM gene was seen significantly more often in the English controls (16,8 %) than it was in the English patients (4,7 %), with a p-value of 0.0043. 6 Discussion

6.1 Role of the COL11A1 gene in patients with Stickler and Marshall syndromes

Stickler syndrome is an autosomal dominant connective tissue disorder resulting from mutations in at least three different genes: COL2A1, COL11A1, and COL11A2. The clinical phenotype of the patients is mainly characterized by ocular, orofacial, and articular abnormalities. Besides the genetic heterogeneity, the clinical findings also vary between the three subtypes. (Stickler et al. 1965, Brunner et al. 1994, Vikkula et al. 1995, Sirko-Osada et al. 1998, Stickler et al. 2001). Marshall syndrome is an arthro-opthalmopathy inherited in an autosomal dominant manner and caused by mutations in the COL11A1 gene (Marshall et al. 1958, Griffith et al. 1998, Annunen et al. 1999, Melkoniemi et al. 2003, Poulson et al. 2004). The clinical phenotype overlaps with STL2. This study was performed to clarify the role of COL11A1 in Stickler and Marshall syndromes and to perform a genotype-phenotype correlation. All patients included were initially analyzed for mutations in the COL2A1 and no mutations were found. Altogether 44 patients were included in the study and a total of ten heterozygous mutations were detected in the COL11A1 gene. Seven of the mutations were splice site mutations, all located in the C-terminal half of the gene spanning introns 47 to 55. Four of the splice site mutations were in intron 50, and three were located in introns 47, 53, and 55. One patient had a 29-nucleotide deletion consisting of the last nucleotide of exon 44 and the first 28 nucleotides of intron 44. This deletion included the donor splice site of the exon and is thus likely to cause exon skipping. In addition, one patient had an 18-bp in-frame deletion in exon 36, and one glycine to asparagine substitution was found in exon 59. The clinical phenotypes and genotypes of the patients were analyzed and compared to each other. To date, 25 different COL11A1 mutations have been reported. Eighteen of the mutations affect a splice site, and 17 of them are located in the C-terminal half of the gene. In addition, single or multi-exon deletions and missense mutations resulting in glycine substitutions have been reported. (Richards et al. 1996, Griffith et al. 1998, 63 Annunen et al. 1999, Martin et al. 1999, Melkoniemi et al. 2003, Poulson et al. 2004) (Table 7). Eleven of seventeen mutations affecting a splice site in the 3’ half of COL11A1 have been classified as Marshall syndrome rather than Stickler syndrome. Classification was based on early onset hearing-loss and pronounced facial dysmorphism with severe midfacial hypoplasia, short nose, and a low nasal bridge. (Richards et al. 1996, Griffith et al. 1998, Annunen et al. 1999, Martin et al. 1999, Melkoniemi et al. 2003, Poulson et al. 2004). Five of these eleven mutations were found in intron 50. These patients had the clinical features of Marshall syndrome including short stature, facial dysmorphism, and early onset SNHL. Ocular anomalies such as vitreoretinal degeneration and retinal detachment were not so common in these cases. A few cases showed a thick calvarium, intracranial calcification, and abnormal frontal sinuses (Griffith et al. 1998, Annunen et al. 1999). Four other splice site mutations, one deletion affecting splice site, and a glycine substitution were described to have an overlapping phenotype of Marshall and Stickler syndrome. These patients had milder facial dysmorphism including less pronounced midfacial hypoplasia and a less depressed nasal bridge. Patients more often had a severe eye phenotype, while the SNHL was less common. (Richards et al. 1996, Annunen et al. 1999, Martin et al. 1999, Poulson et al. 2004). The correlation between the vitreous phenotype and molecular defect in Stickler syndrome has been suggested to be one of the key components in the classification of the molecular subtypes (see review Snead & Yates 1999). However, the congenital membranous vitreous phenotype I associated with mutations in the COL2A1 gene was diagnosed in this study by an experienced ophthalmologist in one patient with a COL11A1 mutation. There are also other studies suggesting that the vitreous anomaly does not always allow the prediction of the defective gene in Stickler and Marshall syndrome. Parentin et al. found a linkage to the COL11A1 gene in a family with a membranous type I vitreous anomaly (Parentin et al. 2001). McLeod et al. reported a case with a COL11A1 mutation and a “change” in vitreous phenotype from beaded vitreous type II to membranous type I vitreous (McLeod et al. 2002). All this data suggest that the vitreous anomaly is not a certain feature for the clinical diagnosis but is still a useful finding that may be indicative for the molecular basis of the syndrome. In the present study, it was difficult to classify the patients as having Marshall or Stickler syndrome. Basically, based on the facial dysmorphism four patients, GE-03-023, GE-02-021, GE-02-130, and GE-04-041, were initially diagnosed as Marshall patients (Table 1, article I). They all shared a severe midfacial hypoplasia, short nose, anteverted nares, and bulging of the eyes. Three of patients also had SNHL. The hearing status for one patient (GE-04-041) could not be determined because the patient was still an infant. One of the patients had short stature. Ocular findings of these patients included high myopia, type I vitreous anomaly, and only one of the four cases had retinal detachment. Signs of ectodermal dysplasia were not observed in any of these patients as described in the original family with Marshall syndrome (Marshall et al. 1958). All four patients had a splice site mutation in intron 50 of the COL11A1 gene. Based on the clinical findings, the remaining six patients had an overlapping Marshall- Stickler phenotype (Table 1, article I). The facial dysmorphisms were milder than in Marshall syndrome, including less pronounced midfacial hypoplasia and a better developed nasal bridge. Early-onset hearing loss was present in three of six cases. All 64 except one patient had myopia, and one had a spontaneous retinal detachment. Molecular analyses revealed three splice site mutations all outside the hot spot region of intron 50. In addition, two different deletions, one causing an in-frame deletion and one affecting the exon 44 donor splice site likely to result in exon skipping, were observed together with one glycine substitution in the other three patients. Based on our study and data from previous studies, it can be concluded that Marshall and Stickler syndromes are distinct entities. In the diagnosis of patients with Marshall syndrome, attention should be paid to facial features, such as midfacial hypoplasia, bulging eyes, and a short nose with anteverted nares, as well as early-onset hearing loss. This initial diagnosis of Marshall syndrome should lead the molecular diagnosis towards the COL11A1 gene. (Annunen et al. 1999, see reviews Snead & Yates 1999, article I). This study also shows that intron 50 is a hot spot region for splice site mutations leading to Marshall syndrome. This finding is supported by previous data. (Annunen et al. 1999). COL11A1 mutations outside the hot spot region seem to lead to a less specific phenotype that is not easily distinguished from Stickler syndrome due to mutations in COL2A1.

Table 7. COL11A1 mutations

Mutation in COL11A1 Phenotype Reference IVS14-2DelA Stickler Annunen et al. 1999, Martin et al. 1999, Poulson et al. 2004 18-bp in-frame del in exon 36 Stickler/Marshall Annunen et al. 1999, Article I IVS38+2T>C Marshall Annunen et al. 1999 IVS38+1G>T Marshall Annunen et al. 1999 IVS43-2A>G Marshall Annunen et al. 1999 28-bp del exon/intron 44 Stickler/Marshall Article I IVS45+3G>A Robin sequence/Marshall Melkoniemi et al. 2003 IVS47-2A>G Marshall Annunen et al. 1999, Article I IVS50+3insT Marshall Annunen et al. 1999, Melkoniemi et al. 2003, Article I IVS50+3A>C Marshall Annunen et al. 1999 IVS50+1G>C Marshall Annunen et al. 1999 4-bp del exon/intron 50 Marshall Annunen et al. 1999 IVS50+1G>A Marshall Griffit et al. 1998, Article I IVS52+1G>A Stickler Poulson et al. 2004 9-bp del exon 52 Stickler Annunen et al. 1999 Del from -85IVS52 to +23IVS53 Marshall Annunen et al. 1999 IVS53-1G>C Stickler/Marshall Article I IVS54+1G>A Marshall Annunen et al. 1999 IVS55+1G>A Stickler/Marshall Article I IVS60-2A>G Stickler Poulson et al. 2004 40-Kb del multiple exons Stickler Martin et al. 1998 G148Ra Stickler Annunen et al. 1999 G998V Stickler Annunen et al. 1999 G1471D Stickler/Marshall Article I G97V Stickler Richards et al. 1996 a Amino acids numbered from the start of the triple helix 65 6.2 Structural and premature termination codon mutations in the alternatively spliced exon 2 of COL2A1 causing an ocular variant of Stickler syndrome

Mutations in COL2A1 encoding the α1 chain of collagen II are the most associated with Stickler syndrome. Procollagen II undergoes alternative splicing resulting in two different splicing variants, type IIA (long form) including exon 2 and IIB (short form) consisting of 53 exons without exon 2. (Ryan & Sandell 1990, Sandell et al. 1991). Stickler syndrome type I is a disorder caused by mutations in the COL2A1 gene. Disease- causing mutations are spread throughout the gene and several mutations leading to a premature termination codon (PTC) have been reported. Mutations involving exon 2 of COL2A1 seem to lead a predominantly ocular phenotype with minimal or no extraocular manifestations. The short form is predominantly expressed by differentiated chondrocytes in adult cartilage. The long form is expressed during early development in chondroprogenitor cells and in the vitreous of the eye. The vitreous is the only adult tissue containing procollagen IIA. (Ling et al. 1993). Thus, the tissue-specific expression of these two isoforms may explain the lack of extraocular manifestations in predominantly ocular Stickler syndrome. (Richards et al. 2000, Gupta et al. 2002, Parma et al. 2003, Donose et al. 2003, Yoshiba et al. 2006). Two patients with predominantly ocular Stickler syndrome and 125 high myopic patients were analyzed for mutations in exon 2 of COL2A1. A PTC mutation (Cys64Stop) and a novel structural mutation (Cys57Tyr) were detected in two patients. The same PTC mutation was also found in one patient with high myopia. The clinical phenotype of the patient with the Cys57Tyr mutation did not differ from that of the patients with PTC mutations presented in this and previous reports (Richards et al., 2000, Gupta et al., 2002, Donoso et al., 2002, Parma et al., 2002, Donoso et al., 2003). The consequences of these mutations were analyzed at the RNA level to clarify the underlying mechanism of these different mutations leading to a similar clinical phenotype. All previously reported mutations leading to an ocular variant of Stickler syndrome were PTC mutations. PTC mutations may result in nonsense-mediated mRNA decay (NMD). NMD is a quality control-based surveillance mechanism that selectively degrades mRNAs that prematurely terminate translation because of a frameshift or nonsense mutation (see reviews Frischmeyer & Dietz 1999, Maquet 2004). PTC mutations have also been reported to cause nonsense-associated altered splicing (NAS). (Dietz 1997, Valentine 1998, Cartegni et al. 2002). Other studies have shown evidence that some missense mutations located in exonic splicing enchancers (ESEs) can disturb splicing (Ars et al., 2000, Moseley et al., 2002, Fackenthal et al., 2002), and therefore cause splicing insufficiency. Glycine substitutions in the triple-helical region of fibrillar collagen, such as collagen II, result in a more severe phenotype than PTC mutations. They lead to reduction or delay in the formation of supramolecular assemblies or alter the molecules structure and function by dominant negative mechanism (see review Myllyharju & Kivirikko 2004). PTC mutations result in a decreased amount of procollagen, leading to a quantitative defect in procollagen biosynthesis. McAlinden et al. identified for the first time regulatory cis elements within the conserved double-strand 66 stem region that are functional only in the context of the natural weak 5’ splice site of exon 2. These cis elements modulate the cell-specific alternative splicing of exon 2 during cartilage development, and mutations in these regulative regions may result in abnormal splicing and an interrupted ratio between the two splice isoforms IIA and IIB (Shiga et al. 1997, Liu et al. 2001, McAlinden et al. 2005). Results from the expression studies in the present study indicated that Cys57Tyr and Cys64Stop mutations, and the previously described Trp47Stop mutation (Richards et al. 2000), are present in or near the functional cis regulatory elements in COL2A1 exon 2 and lead to an alteration of alternative splicing. High scoring cis elements that may serve as binding sites for positive-acting splicing factors were found using a web-based resource (Exon Splicing Enhancer (ESE) finder: exon.cshl.edu/ESE) (Cartegni et al. 2003). Some of these cis elements were close to or overlapped with the exon 2 Stickler- associated mutations. Deletion of one of the predicted cis elements was shown to dramatically affect the alternative splicing pattern, decreasing the IIA:IIB ratio. This supports the hypothesis that the mutations described in this study may result in altered splicing and promote procollagen IIB synthesis rather than production of misfolded protein. The results further emphasize the importance of exon 2 in the development and function of the eye. Patients displaying eye phenotypes such as high myopia and retinal detachment in the absence of extraocular effects should be analyzed first for mutations in exon 2 of COL2A1.

6.3 DFNB32 locus associated with recessively inherited nonsyndromic hearing loss and the role of the COL11A1 gene

Hearing impairment is the most frequently inherited sensory defect. One in 1000 newborn children have profound hearing loss (Parving 1999). Genetic causes are involved in over half of all cases (Steel 2000, Hone & Smith 2003). The inheritance pattern of the hearing impairment has been divided into four categories: autosomal dominant, autosomal recessive, X-linked, and mitochondrial inheritance. Isolated deafness is the most genetically heterogeneous trait. Hereditary hearing loss can be clinically considered as syndromic (SHL) or nonsyndromic hearing loss (NSHL). Research during the last decades has revealed over 70 genetic loci in NSHL (Smith et al. 1999, Bitner-Glindzics 2002, see review Petersen 2002). The Connexin 26 gene (Cx26, GJB2) was the first identified causative gene for DFNB (DFNB1) and is frequently observed in cases with recessive hearing loss (see review Petersen & Willems 2006). For families with autosomal recessive deafness in which Cx26 involvement has been excluded, further analyses of candidate loci or genes are needed. However, the families are often small and linkage analyses are impossible to perform. Mutation analyses may be possible, but the gene involved is known only for part of the loci. (Kelsell et al. 1997, Kremer & Hoefsloot 2002). Several DFNB genes have been shown to be responsible for isolated and syndromic forms of deafness (http://webhost.ua.ac.be/hhh/). One example is the MYO7A gene, which has been defined to cause both nonsyndromic and syndromic hearing impairment (Weil et al. 1995, Weil et al. 1997, Liu et al. 1997). 67 A large Tunisian family with congenital profound autosomal recessive nonsyndromic deafness was studied to identify the disease-causing mutation in this family. Linkage analyses revealed a novel deafness locus, DFNB32, on chromosome 1. It contains the COL11A1 gene, which is known to be responsible for two syndromes, Marshall and Stickler syndromes, characterized by hearing deficit. (Richards et al. 1996, Griffith et al. 1998). Also, the Col11a1 mouse model for chondrodysplasia (cho) shows a phenotype with strong hearing impairment (Szymko-Bennett et al. 2003). COL11A1 was analyzed as a candidate gene for the disease based on its chromosomal location and expression in the cochlea. The detection of mutations was done by CSGE, which has indicated to have sensitivity and specifity close to 100 % (Körkkö et al. 1998). The screening of the patients for mutations in COL11A1 revealed no mutations in association with the hearing impairment. However, the promotor region of COL11A1 was not analyzed in the study. It is possible, that another gene in the linked region, 1p13.3-22.1, is responsible for the phenotype as the region contains twenty-two different genes.

6.4 Role of the four SLRP genes, OPTC, LUM, FMOD, and PRELP, in the development of high myopia

The main ocular determinants of refraction are the length of the eye and the focusing power of the cornea and crystalline lens. Since in myopia either the cornea or lens curvature is too strong or the eyeball is too long (axial myopia), the image is focused in front of the retina, whereas a normal emmetropic eye focuses the light on the retina. Myopia is the most common eye condition, with a wide variability in prevalence between ethnic groups, from 10 % to as high as 90 % (Chow et al. 1990, Saw et al. 1996, Zadnik et al. 1998, Wong et al. 2000). Approximately 1-3 % of the population have been affected with high myopia defined as a refractive error of at least -6 or -8 dioptres (D) (Fuchs 1960, Sperduto et al. 1983, Fredrick 2002). The genetic component of intermediate and high myopia has been well established, but no candidate genes have been reported to date (Goss et al. 1988, Young et al. 1998). To study the role of SLRP proteins in the development of high myopia we screened patients, controls, and family members for sequence variations in the LUM, FMOD, OPTC, and PRELP genes. Twenty-three different sequence variations were found. Six non-synonymous changes and one synonymous change in addition to two intronic variations were found in the patients, but not in any of the 308 controls analyzed. Eight of the variations located at highly conserved positions. Two disease-related sequence variations have been reported earlier in these genes (Friedman et al. 2002, Wang et al. 2006). Friedman et al. observed the Arg325Trp change in patients with sporadic primary open-angle glaucoma or normal- tension glaucoma, but also found the variation in the control group. Also a 5’-regulatory region SNP in the LUM gene was associated with high myopia (Wang et al. 2006). However, there are no functional studies of this mutation. Fmod and Lum knockout mice suggest that these proteins have a role in the pathogenesis of myopia due to their expression in the optic system of the eye and the observed defects in the mutant mice, which show structural changes related to high myopia such as thin sclera, axial length elongation, and retinal detachment (Chakravarti et al. 2003). The Lum/Fmod double 68 knockout mice also show skeletal abnormalities such as short stature, joint laxity, and age-dependent osteoarthritis (Chakravarti 2003). Patients included in the present study were not analyzed for skeletal abnormalities. The observed variations, a Leu199Pro change in LUM, Glu147Asp and Arg324Thr in FMOD, and Thr177Arg and Arg229His in OPTC, are localized to the conserved LRR domains. These domains are believed to be functionally important structural modules for the SLRP molecules and used in molecular recognition processes such as cell adhesion, signal transduction, DNA repair, and RNA processing. They are also involved in the protein/protein interactions and packing of the SLRP molecules. LRRs are used to achieve strong protein/protein interactions that are believed to occur due to the non- globular shape of the SLRP molecules and exposed face of the parallel β-sheet. The collagen-regulating activity is mediated by the core protein, mainly via the central LRR4- 6. GAG chains, on the other hand, maintain the interfibrillar space by extending outward from the protein core. In the case of fibromodulin, binding sites in the C-terminal end of the protein are also required for the inhibition of fibrillogenesis. Of the variations found in this study, Arg324Thr is located in the C-terminal end of FMOD, whereas Arg229His is located in the central LRR4 domain of OPTC. (see reviews Kobe & Deisenhofer 1994, Hocking et al. 1998, Iozzo 1998, Iozzo 1999). The OPTC variations Thr177Arg, Arg325Thr, Gly329Ser, and Arg330His were found in all the high myopic relatives analyzed. Three of them, Arg325Thr, Gly329Ser, and Arg330His, occurred also in family members with low myopia or emmetropia. Arg325 and Gly329 are highly conserved between species and are located in a cluster at the COOH terminus after a conserved Cys residue, supporting the assumption of their role as a genetic risk factor in high myopia. The presence of these variations also in family members with low myopia or emmetropia may be explained by incomplete penetrance. Incomplete penetrance is not unexpected in complex diseases (Zlotogora 2003). High myopia is also proposed to be a polygenic disease with a possible environmental component. (Farbrother et al. 2004). The OPTC variations, Arg229His, Arg325Trp, and Thr177Arg, are likely to lead to misfolding of the LRR domains and cause interrupted secretion and /or function of opticin either directly or through aggregation driven by exposure of the hydrophobic core. The Arg229His variation causes a shorter side-chain and is less likely to be protonated at physiological pH, as Arg325Trp changes the positively charged amino acid to an uncharged hydrophobic residue. The Thr177Arg variation introduces an additional positively charged amino acid to the region of opticin already dominated by positively charged amino acids, leading to a highly destabilizing effect. This study suggests that a number of genes expressed in eye may be involved in different ways in the development of high myopia and that these four SLRP genes may contribute but are not unambiguously behind the disease. Polygenic inheritance of high myopia is supported also by the incomplete penetrance of the novel mutations found in this study and a number of different loci found earlier in linkage studies. This study reveals pilot findings that need to be repeated in a larger case/control set in the future. 7 Conclusion

Isolated high myopia and hearing loss are common clinical findings, and they are also found in several syndromic disorders, such as Stickler and Marshall syndromes. High myopia is a polygenic disease. Although several loci have been identified through linkage studies, no candidate genes have yet been identified. The findings in this thesis give support for the role of genetic factors in the development of high myopia. We showed that variations in four SLRP genes, OPTC, LUM, FMOD, and PRELP, associate with high myopia. Five missense variations (OPTC: T177R, R229H, R330H, LUM: L199P, FMOD: R324T) and two intronic variations (LUM: c.865-69A>C, PRELP: c.1150*27T>C) were found in patients with high myopia, but not in controls. A novel deafness locus, DFNB32, was identified on chromosome 1p13.3-22.1 in a Tunisian family with congenital profound autosomal recessive deafness. As the COL11A1 gene is located in this region, it was analyzed as a candidate gene. Mutation analysis, however, failed to show an association between this phenotype and the COL11A1 sequence variations. Stickler syndrome is a heterogeneous disorder caused by mutations in three collagen genes; STL1 is caused by mutations in COL2A1, STL2 by mutations in COL11A1, and STL3 by mutations in COL11A2. Marshall syndrome is caused by mutations in COL11A1 and it clinically overlaps with STL2. This thesis gives additional information about the correlation between the two overlapping phenotypes of STL2 and Marshall syndrome, revealing that intron 50 of COL11A1 is the hot spot for splice site mutations leading to the Marshall syndrome phenotype. Other mutations in COL11A1, such as glycine substitutions and splicing mutations outside the intron 50, lead to STL2 or an overlapping Stickler/ Marshall syndrome phenotype. Predominantly ocular Stickler syndrome is a variant of Stickler syndrome with only ocular findings and none of the extraocular manifestations typical of Stickler syndrome. We have shown here for the first time that mutations in exon 2 of COL2A1 cause predominantly ocular Stickler syndrome, most likely by alterering positive cis regulatory elements for splicing that results in a lower IIA:IIB isoform ratio. Patients with an ocular phenotype and minimal or no extraocular findings should first be analyzed for mutations in exon 2 of COL2A1. 70 The results of this thesis provide new information concerning genetic factors in high myopia. Further studies are needed, however, to confirm the specific role of the four SLRP genes in the pathogenesis of high myopia. The studies also provide new information on the genotype-phenotype correlation in Stickler and Marshall syndromes. This information provides better tools for clinical and molecular diagnostics of the disorders. Understanding the molecular mechanism by which COL2A1 exon 2 mutations lead to predominantly ocular Stickler syndrome will further clarify the importance of this exon for the development and normal function of the eye.

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Original articles

I Majava M, Hoornaert KP, Bartholdi D, Bouma MC, Bouman K, Carrera M, Devriendt K, Hurst J, Kitsos G, Niedrist D, Petersen MB, Shears D, Stolte-Dijkstra I, Van Hagen JM, Ala-Kokko L, Männikkö M & Mortier GR (2006) A Report on 10 New Patients with Heterozygous Mutations in the COL11A1 Gene and a Review of Genotype-Phenotype Correlations in Type XI Collagenopathies. Am J Med Genet, In press. II Majava M, McAlinden A, Bishop PN, Perveen R, Black G, Pierpoint MEM, Ala- Kokko L & Männikkö M (2006) Missense and nonsense mutations in the alternatively spliced exon 2 of COL2A1 cause the ocular variant of Stickler syndrome. (Manuscript) III Masmoudi S, Tlili A, Majava M, Ghorbel AM, Hardenoux S, Lemainque A, Zina ZB, Moala J, Männikkö M, Weil D, Lathrop M, Ala-Kokko L, Drira M, Petit C & Ayadi H (2003) Mapping of a new autosomal recessive nonsyndromic hearing loss locus (DFNB32) to chromosome 1p13.3-22.1. Eur J Hum Genet 11: 185-188. IV Majava M, Bishop PN, Hägg N, Scott PG, Rice A, Inglehearn C, Hammond CJ, Spector TD, Ala-Kokko L & Männikkö M (2006) Novel Mutations in the Small Leucine-Rich Repeat Protein/Proteoglycan (SLRP) Genes in High Myopia. Hum Mut, In press. Original publications are not included in the electronic version of the dissertation.

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896. Vuorialho, Arja (2006) Costs and effectiveness of hearing aid rehabilitation in the elderly 897. Ala-Kopsala, Minna (2006) Circulating N-terminal fragments of A- and B-type natriuretic peptides: molecular heterogeneity, measurement and clinical application 898. Haho, Annu (2006) Hoitamisen olemus. Hoitotyön historiasta, teoriasta ja tulkinnasta hoitamista kuvaviin teoreettisiin väittämiin 899. Kukkurainen, Marja Leena (2006) Fibromyalgiaa sairastavien koherenssintunne, sosiaalinen tuki ja elämänlaatu 900. Rautio, Katriina (2006) Effects of insulin-lowering drugs in PCOS: endocrine, metabolic and inflammatory aspects 901. Riekki, Riitta (2006) Late dermal effects of breast cancer radiotherapy 902. Leskelä, Hannu-Ville (2006) Human bone marrow stem cells—a novel aspect to bone remodelling and mesenchymal diseases 903. Hörkkö, Tuomo (2006) Growth and progression in colorectal cancer 904. Hinttala, Reetta (2006) Genetic causes of mitochondrial complex I deficiency in children 905. Niemi, Antti (2006) Röntgenhoitajien turvallisuuskulttuuri säteilyn lääketieteellisessä käytössä—Kulttuurinen näkökulma 906. Isokangas, Juha-Matti (2006) Endovascular treatment of 467 consecutive intracranial aneurysms in Oulu University Hospital. Angiographic and clinical results 907. Tähtinen, Tuula (2006) Insuliiniresistenssiin liittyvät kardiovaskulaariset riskitekijät suomalaisilla varusmiehillä. Tupakoinnin yhteys riskitekijöihin 908. Metsänen, Miia (2007) Thought disorder as a predictive sign of mental disorder. A study of high-risk and low-risk adoptees in the Finnish Adoptive Family Study of Schizophrenia 909. Herva, Anne (2007) Depression in association with birth weight, age at menarche, obesity and metabolic syndrome in young adults. The Northern Finland 1966 Birth Cohort Study 910. Lauronen, Erika (2007) Course of illness, outcome and their predictors in schizophrenia. The Northern Finland 1966 Birth Cohort study

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UNIVERSITY OF OULU P.O. Box 7500 FI-90014 UNIVERSITY OF OULU FINLAND ACTA UNIVERSITATIS OULUENSIS ACTA UNIVERSITATIS OULUENSIS ACTA D SERIES EDITORS Marja Majava MEDICA MarjaMajava ASCIENTIAE RERUM NATURALIUM Professor Mikko Siponen MOLECULAR GENETICS OF BHUMANIORA STICKLER AND MARSHALL Professor Harri Mantila SYNDROMES, AND THE ROLE CTECHNICA Professor Juha Kostamovaara OF COLLAGEN II AND OTHER DMEDICA Professor Olli Vuolteenaho CANDIDATE PROTEINS IN HIGH MYOPIA AND ESCIENTIAE RERUM SOCIALIUM Senior Assistant Timo Latomaa IMPAIRED HEARING FSCRIPTA ACADEMICA Communications Officer Elna Stjerna

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EDITOR IN CHIEF Professor Olli Vuolteenaho EDITORIAL SECRETARY Publications Editor Kirsti Nurkkala FACULTY OF MEDICINE, DEPARTMENT OF MEDICAL BIOCHEMISTRY AND MOLECULAR BIOLOGY, COLLAGEN RESEARCH UNIT, ISBN 978-951-42-8361-1 (Paperback) BIOCENTER OULU, ISBN 978-951-42-8362-8 (PDF) UNIVERSITY OF OULU ISSN 0355-3221 (Print) ISSN 1796-2234 (Online)