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FROM RARE SYNDROMES TO A
COMMON DISEASE
Mutations in minor cartilage collagen genes cause
Marshall and Stickler syndromes and intervertebral
disc disease
SUSANNA
ANNUNE N
Department of Medical Biochemistry
OULU 1999
SUSANNA ANNUNEN
FROM RARE SYNDROMES TO A
COMMON DISEASE
Mutations in minor cartilage collagen genes cause
Marshall and Stickler syndromes and intervertebral disc
disease
Academic Dissertation to be presented with the assent
of the Faculty of Medicine, University of Oulu, for public
discussion in the Auditorium of the Department of
Medical Biochemistry, on December 3rd, 1999, at 12
noon.
O U L U N Y L I O P I S TO , O U L U 1 9 9 9
Copyright © 1999
Oulu University Library, 1999
Manuscript received 01 October 1999
Accepted 13 October 1999
Communicated by
Docent Jaakko Ignatius
Professor Markku Savolainen
ISBN 951-42-5413-9
(URL: http://herkules.oulu.fi/isbn9514254139/)
ALSO AVAILABLE IN PRINTED FORMAT
ISBN 951-425412-0
ISSN 0355-3221
(URL: http://herkules.oulu.fi/issn03553221/)
OULU UNIVERSITY LIBRARY
OULU 1999
Annunen, Susanna, From rare syndromes to a common disease. Mutations in minor
cartilage collagen genes cause Marshall and Stickler syndromes and intervertebral
disc disease.
Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry,
University of Oulu, FIN-90220 Oulu, Finland
1999
Oulu, Finland
(Received 1 October 1999)
Abstract
Collagens IX and XI are quantitatively minor components of the collagen fibrils in cartilage. The
spectrum of the phenotypes caused by mutations in the COL2A1 gene coding for collagen II, the
main cartilage collagen, is relatively well defined, but there is little data on the phenotypes caused
by collagen IX and XI mutations.
The structure of the human COL11A1 gene coding for the α1 chain of collagen XI was
characterized here. It was found to consist of 68 exons and span 160 kb, excluding introns 1 and 4.
Over 50 kb of new intronic sequences were defined. The exon-intron organization coding for the
major triple helical domain was found to be identical to that of the human COL11A2 gene, which
codes for the α2 chain of collagen XI.
The sensitivity of conformation sensitive gel electrophoresis (CSGE) for mutation detection was
improved and tested with a large number of sequence variations in collagen genes. The sensitivity
with the revised conditions was found to be close to 100%. In addition, CSGE was found to be a
simple and practical method for analyzing large numbers of samples.
Fifteen mutations in the COL11A1 gene and eight in the COL2A1 gene were found by CSGE in
patients with Marshall or Stickler syndrome. The genotypic-phenotypic comparison indicated that
mutations leading to a premature translation termination codon in the COL2A1 gene resulted in
Stickler syndrome and splicing mutations of 54 bp exons in the C terminal half of the COL11A1
gene resulted in Marshall syndrome. The other COL11A1 mutations caused phenotypes
overlapping both syndromes.
In an analysis of the COL9A2 gene in 157 patients with intervertebral disc disease, six were
found to have a tryptophan for glutamine substitution in the central collagenous domain of the
collagen IX molecule. None of 174 control individuals had this substitution. The substitution
cosegregated with the phenotype in the families studied, and linkage and linkage disequilibrium
analyses supported the association of the locus and the disease with a joint lod score of over 11.
Keywords: mutation detection, sciatica
Acknowledgements
The research presented in this thesis was performed at the Department of Medical
Biochemistry, University of Oulu, and Biocenter Oulu, Finland, and at the Center for
Gene Therapy, the late Allegheny University of the Health Sciences, Philadelphia, USA,
during the last years of the second millenium Anno Domini.
I wish to express my sincere thanks to my supervisor, Docent Leena Ala-Kokko, for
always having time to advise me, and sometimes even to help in the practical lab work,
for her everlasting optimism, for arranging an opportunity for me to work at the Center
for Gene Therapy in Philadelphia for two summers, and most importantly, for guiding me
into the field of science. I would also like to thank Professors Kari I. Kivirikko and Taina
Pihlajaniemi, the leaders of the Collagen Research Unit, for providing excellent research
facilities and a high-standard scientific environment. Moreover, I wish to acknowledge
Professor Ilmo Hassinen, whose enormous knowledge and enthusiastic attitude towards
science inspired me as a first-year medical student to want to learn and create a piece of
the fascinating world of science. I also wish to express my gratitude to Professor Darwin
J. Prockop, Director of the Center for Gene Therapy, for giving me the honour of
working in his laboratory.
I am grateful to Professor Markku Savolainen and Docent Jaakko Ignatius for their
valuable comments on the manuscript of this thesis. I also wish to thank Malcolm Hicks,
M.A., for reviewing the language of the manuscripts of this thesis and the original articles
II and III.
I am indebted to all my collaborators. The clinicians who examined the patients and
took samples made an invaluable contribution to this research. I also learned a lot about
medicine from them. Harald H. H. Göring did much invaluable work in calculating the
lod scores and trying to make me understand even something of the statistical
dimensions.
My friends and colleagues at the Department of Medical Biochemistry deserve my
warmest thanks. The fruitful and relaxing scientific and non-scientific conversations and
the time spent outside the lab with Jarmo Körkkö, Petteri Paassilta, Tero Pihlajamaa,
Jussi and Mirka Vuoristo, Miia Melkoniemi, Merja Välkkilä, Merja Perälä, Jaana
Lohiniva, Veli-Antero Kaakinen and Joni Mäki have been of great value for me. I also
wish to thank Tero for teaching me the basics of lab work in the very first steps of this
research and Miia for helping me to keep pace with my medical studies. Special thanks
are due to Jarmo and Jaana Körkkö for their invaluable help in numerous things and all
the moments of leisure spent with them during my stays in Philadelphia. My sister and
colleague Pia Marttila deserves the greatest thanks for much wise advice concerning
practical work and the more tortuous sides of scientific life.
I wish to thank Aira Harju, Helena Lindqvist, Jaana Körkkö and Robert Hnatuk for
their skilful technical assistance, Pertti Vuokila for ordering all the reagents and Seppo
Lähdesmäki for helping with technical problems. I am grateful to Marja-Leena Kivelä
and Auli Kinnunen for their always friendly secretarial help. I also wish to acknowledge
Ari-Pekka Kvist and Juha Näpänkangas for maintaining the computer systems at the
department and helping with the problems concerning computers.
I wish to express my gratitude to my parents for their loving care and support and for
giving me an excellent basis for my life. I am also grateful to my sisters, brother, their
families and my friends for always being there for me even if I seemed to be constantly in
the lab. Above all, I owe my deepest gratitude to my fiancé Markku for all his love,
support and understanding and for brightening the dark moments and making the bright
moments even brighter.
This work was supported financially by the Emil Aaltonen Foundation and the Finnish
Medical Foundation.
Oulu, September 1999
Abbreviations
4Hyp
ACGII-HCG
BAP
bp
C
cDNA
COL
COLyAx
Colyax
COMP
CSGE
CT
DGGE
ECM
FACIT
GAG
kb
kDa
MACIT
MED
MRI
mRNA
MULTIPLEXIN
N
NC
OA
OMIM
OSMED
PSACH
RT-PCR
SED
SSCP
4-hydroxyproline
achondrogenesis type II-hypochondrogenesis
1,4-bis(acryloyl)piperazine
basepair
carboxy
complementary DNA
collagenous domain
human gene for the α(x) chain of collagen y
murine gene for the α(x) chain of collagen y
cartilage oligomeric matrix protein
conformation sensitive gel electrophoresis
computerized tomography
denaturing gradient gel electrophoresis
extracellular matrix
fibril-associated collagen with interrupted triple helices
glycosaminoglycan
kilobase
kilodalton
membrane-associated collagen with interrupted triple helices
multiple epiphyseal dysplasia
magnetic resonance imaging
messenger RNA
collagen with multiple triple helix domains and interruptions
amino
non-collagenous domain
osteoarthritis or osteoarthrosis
Online Mendelian Inheritance in Man
otospondylomegaepipyseal dysplasia
pseudoachondroplasia
reverse transcriptase PCR
spondyloepiphyseal dysplasia
single-strand conformation polymorphism
WZS
X
Y
Weissenbacher-Zweymüller syndrome
any amino acid
any amino acid
List of original articles
This thesis is based on the following articles, which are referred to in the text by their
Roman numerals:
I
Körkkö J, Annunen S, Pihlajamaa T, Prockop DJ & Ala-Kokko L (1998)
Conformation sensitive gel electrophoresis for simple and accurate detection of
mutations: comparison with denaturing gradient gel electrophoresis and
nucleotide sequencing. Proc Natl Acad Sci USA 95: 1681-1685.
II
Annunen S, Körkkö J, Czarny M, Warman ML, Brunner HG, Kääriäinen H,
Mulliken JB, Tranebjærg L, Brooks DG, Cox GF, Cruysberg JR, Curtis MA,
Davenport SLH, Friedrich CA, Kaitila I, Krawczynski MR, Latos-Bielenska A,
Mukai S, Olsen BR, Shinno N, Somer M, Vikkula M, Zlotogora J, Prockop DJ
& Ala-Kokko L (1999) Splicing mutations of 54 bp exons in the COL11A1 gene
cause Marshall syndrome but other mutations cause overlapping
Marshall/Stickler phenotypes. Am J Hum Genet 65: 974-983.
III
Annunen S, Paassilta P, Lohiniva J, Perälä M, Pihlajamaa T, Karppinen J,
Tervonen O, Kröger H, Lähde S, Vanharanta H, Ryhänen L, Göring HHH, Ott J,
Prockop DJ & Ala-Kokko L (1999) An allele of COL9A2 associated with
intervertebral disease. Science 285: 409-412.
In addition, some unpublished data are presented.
Contents
Abstract
Acknowledgements
Abbreviations
List of original articles
Contents
1. Introduction.................................................................................................................. 13
2. Review of the literature................................................................................................ 15
2.1. Collagens............................................................................................................... 15
2.1.1. Fibrillar collagens........................................................................................ 15
2.1.2. Non-fibrillar collagens ................................................................................ 16
2.1.3. Collagen biosynthesis.................................................................................. 18
2.2. Cartilage................................................................................................................ 18
2.2.1. Collagenous components of cartilage.......................................................... 19
2.2.2. Non-collagenous components of cartilage .................................................. 22
2.3. Cartilage collagens in non-cartilaginous tissues ................................................... 23
2.3.1. Inner ear ...................................................................................................... 23
2.3.2. Ocular vitreous body ................................................................................... 23
2.3.3. Intervertebral disc........................................................................................ 23
2.4. Disorders affecting cartilage collagens ................................................................. 24
2.4.1. Achondrogenesis type II-hypochondrogenesis (ACGII-HCG) ................... 25
2.4.2. Spondyloepiphyseal dysplasia (SED).......................................................... 25
2.4.3. Kniest dysplasia........................................................................................... 26
2.4.4. Stickler and Marshall syndromes ................................................................ 26
2.4.5. Otospondylomegaepiphyseal dysplasia (OSMED), WeissenbacherZweymüller syndrome (WZS) and non-ocular Stickler-like syndrome....... 28
2.4.6. Multiple epiphyseal dysplasia (MED)......................................................... 29
2.4.7. Osteoarthritis (OA)...................................................................................... 30
2.5. Animal models for disorders of the cartilage collagens ........................................ 30
2.5.1. Collagen II................................................................................................... 31
2.5.2. Collagen IX ................................................................................................. 31
2.5.3. Collagen XI ................................................................................................. 32
2.6. Intervertebral disc disease..................................................................................... 32
2.6.1. Cmd mice .................................................................................................... 33
2.7. Mutation detection methods.................................................................................. 33
2.7.1. Commonly used methods based on electrophoresis .................................... 33
2.7.2. Commonly used methods based on cleavage of a mismatch....................... 34
2.7.3. Other methods ............................................................................................. 35
3. Outlines of the present research ................................................................................... 37
4. Materials and methods ................................................................................................. 38
4.1. Subjects and clinical examination methods (I-III) ................................................ 38
4.2. Characterization of the COL11A1 gene (II).......................................................... 39
4.3. CSGE (I-III) .......................................................................................................... 39
4.4. Screening of the COL11A1 and COL2A1 genes of patients with Marshall and
Stickler syndromes for mutations by CSGE (II) ................................................... 40
4.5. RT-PCR (II) .......................................................................................................... 41
4.6. Screening of patients with sciatica for mutations in the COL9A2 gene (III) ........ 41
4.7. Statistical analysis (III) ......................................................................................... 41
5. Results.......................................................................................................................... 43
5.1. Optimization of CSGE (I) ..................................................................................... 43
5.1.1. Improved conditions for mutation detection by CSGE (I) .......................... 43
5.1.2. Testing the sensitivity and specificity of CSGE.......................................... 44
5.2. Characterization of the human COL11A1 gene (II).............................................. 44
5.2.1. Characterization of clones for the COL11A1 gene ..................................... 45
5.2.2. Genomic structure of the human COL11A1 gene ....................................... 45
5.2.3. Resequencing of the coding region ............................................................. 46
5.3. Identification of mutations in the COL11A1 and COL2A1 genes in patients
with Marshall or Stickler syndrome (II)................................................................ 46
5.3.1. Identification of mutations in the COL11A1 and COL2A1 genes by
CSGE........................................................................................................... 46
5.3.2. Genotypic-phenotypic comparison.............................................................. 47
5.4. Identification of an allele of the COL9A2 gene associated with intervertebral
disc disease (III) .................................................................................................... 50
5.4.1. Identification of a tryptophan for glutamine/arginine substitution in the
COL9A2 gene.............................................................................................. 50
5.4.2. Co-inheritance of the Trp allele and intervertebral disc disease.................. 50
5.4.3. Statistical analysis ....................................................................................... 50
5.4.4. Scanning the entire COL9A2 gene for mutations in patients with the Trp
allele ............................................................................................................ 52
6. Discussion.................................................................................................................... 53
6.1. Detection of mutations by CSGE .......................................................................... 53
6.2. Genomic structure of the human COL11A1 gene................................................. 54
6.3. COL11A1 and COL2A1 gene mutations in patients with Marshall or Stickler
syndrome ............................................................................................................... 55
6.4. The association of a COL9A2 allele with intervertebral disc disease ................... 57
7. References.................................................................................................................... 59
1. Introduction
Cartilage is an important form of supportive tissue. It is rich in extracellular matrix and
contains only a sparse population of chondrocytes. The most abundant type of cartilage in
the body is hyaline cartilage, which forms articular cartilage, growth plates and the
cartilaginous anlage for endochondral ossification. The extracellular matrix of hyaline
cartilage is highly hydrated and comprises cartilage-specific collagen fibrils and
proteoglycans.
Collagen II, the main component of the collagen fibrils in cartilage, is a homotrimer of
three identical α chains, α1(II)3, which are encoded by the COL2A1 gene. While collagen
II accounts for the majority of the collagens in cartilage, other significant types are
collagens IX and XI. Collagen IX is a heterotrimer of three dissimilar α chains, α1(IX),
α2(IX) and α3(IX), which are encoded by the COL9A1, COL9A2 and COL9A3 genes,
respectively. Collagen IX is known to be located on the surface of the cartilage collagen
fibrils, but its precise function is not currently known, although it has been suggested that
it may mediate the interaction between collagenous and non-collagenous cartilage
components and maintain the homeostasis of cartilage. Collagen XI is likewise a
heterotrimer of three distinct α chains, the α1(XI), α2(XI) and α3(XI) chains being
encoded by the COL11A1, COL11A2 and COL2A1 genes, respectively. Like collagen II,
collagen XI forms fibrils, and they are thought to be buried in collagen II fibrils. It has
been suggested that the function of collagen XI may be to regulate collagen fibril
diameter.
Collagens II, IX and XI are also expressed in the intervertebral disc, which consists of
an outer ring-like structure called the annulus fibrosus and an inner gel-like matrix called
the nucleus pulposus. Collagen II is the major collagenous component of the nucleus
pulposus, while collagen IX is found in minor amounts throughout the disc and collagen
XI accounts for a few percent of the collagens in the nucleus pulposus. In addition to
hyaline cartilage and the intervertebral disc, collagens II, IX and XI are also expressed in
the ocular vitreous body and the tectorial membrane of the inner ear.
Collagen II is the most intensively studied collagen type in cartilage, and a large
number of mutations in the COL2A1 gene have already been characterized. Depending
on the type of mutation, they cause a spectrum of phenotypes ranging from lethal
achondrogenesis type II-hypochondrogenesis to early onset osteoarthritis with mild
chondrodysplasia. Mutations characterized in the genes coding for collagen IX lead to
14
multiple epiphyseal dysplasia, and mutations in the genes coding for collagen XI lead to
Stickler and Marshall syndromes and heterozygous and homozygous otospondylomegaepiphyseal dysplasia. The number of mutations in these genes known at present is
small, however, and the spectrum of phenotypes caused by gene mutations in collagens
IX and XI is largely unknown.
Characterization of the genetic defects lying behind such phenotypes is important for
obtaining an understanding of the function of the particular molecule and of the whole
tissue. Furthermore, knowing the molecular defect that causes the disorder in question
helps in the development of a treatment for the disease and the prevention of symptoms,
and on the other hand, enables defining the exact genetic defect involved and genetic
prenatal diagnosis.
The goal of this thesis is to increase our knowledge of the role of collagens IX and XI
in disorders affecting cartilaginous tissues. It involves characterization of the genomic
structure of the COL11A1 gene, the description of 23 mutations in the COL11A1 and
COL2A1 genes causing Marshall and Stickler syndromes, definitions of the genotypic
and phenotypic relations of these syndromes, and a report of an association between an
allele of the COL9A2 gene bearing a putative mutation and intervertebral disc disease. In
addition, an improvement to the conformation sensitive gel electrophoresis (CSGE)
method for mutation detection is described.
2. Review of the literature
2.1. Collagens
Collagens are a significant component of the extracellular matrix (ECM). They are a
group of related proteins that share common structural characteristics. Collagens are
trimeric molecules consisting of three similar or dissimilar α chains, and they form
supramolecular structures in the ECM. All collagens have at least one triple helical region
in the molecule. The formation of triple helix requires the presence of glycine as every
third amino acid, because any bulkier amino acid would cause steric hindrance to helix
formation. So far, 19 collagen types encoded by over 30 different genes are known.
These can be divided into several groups based on their structural and other properties
(For reviews, see Vuorio & de Crombrugghe 1990, Kielty et al. 1993, Mayne & Brewton
1993, Pihlajaniemi & Rehn 1995, Prockop & Kivirikko 1995.)
2.1.1. Fibrillar collagens
Collagens I, II, III, V and XI aggregate into rod-like fibrils in a quarter-staggered array
that gives a characteristic banding pattern. They contain a large collagenous domain of
about 1000 amino acids. (See Kielty et al. 1993, Pihlajaniemi & Rehn 1995, Prockop &
Kivirikko 1995.) Collagens I, II and III are known as the major fibrillar collagens, since
they are quantitatively more significant than collagens V and XI, which are often called
minor fibrillar collagens.
Collagen I, II and III fibrils create the structural frameworks for many tissues, such as
bone, skin, cartilage, tendon and blood vessels. Collagen I is a heterotrimer of two α1(I)
chains and one α2(I) chain that are encoded by the COL1A1 and COL1A2 genes,
respectively. Homotrimers of α1(I) have also been observed (Uitto 1979). Collagen I is
expressed in various tissues, but it is the main structural component of bone, skin, tendon,
ligament, teeth and fasciae. Collagen II is a homotrimer of α1(II) chains that are encoded
by the COL2A1 gene. In adult tissues it is expressed in cartilage, vitreous humour, the
intervertebral disc and the inner ear (for more details on collagen II, see section 2.2.1.1.).
16
Collagen III is a homotrimer as well, consisting of three α1(III) chains that are encoded
by the COL3A1 gene and being expressed in various tissues, although it is most abundant
in elastic tissues such as the skin, blood vessels, gut and lung. The major fibrillar
collagens share highly similar characteristics. They are synthesized as large precursors
with C and N propeptides, which are later cleaved by specific C and N proteinases. Also,
all the genes coding for the α chains of major fibrillar collagens are similar in their
genomic structure. The numbers and the sizes of the exons are the same, with only a few
exceptions. (See Vuorio & Crombrugghe 1990, Chu & Prockop 1993, Prockop &
Kivirikko 1995.)
Collagens V and XI share many properties with the major fibrillar collagens, although
they also have some features that are different. The exon sizes and numbers of exons in
the genes for the minor fibrillar collagens characterized so far, the COL5A1 and
COL11A2 genes, are different from those of the genes that code for the major fibrillar
collagens (Takahara et al. 1995, Vuoristo et al. 1995). While the entire propeptides are
cleaved during post-translational modification in the major fibrillar collagens, the N
propeptides of collagens V and XI seem to be partially retained in the mature molecule.
Collagens V and XI form heterotypic fibrils with collagens I and II, respectively, and
they are thought to be located inside collagen I/II fibrils (Birk et al. 1988, Mendler et al.
1989). It has been suggested that they regulate fibril diameter and thus have a significant
role in fibril formation. Collagen V is codistributed with collagen I, and is composed of
three different α chains, α1(V), α2(V) and α3(V), encoded by the COL5A1, COL5A2
and COL5A3 genes. The most common composition for the collagen V molecule is
[α1(V)]2α2(V), but others also exist. Collagen XI usually consists of α1(XI), α2(XI) and
α3(XI) chains, coded by the COL11A1, COL11A2 and COL2A1 genes (for more details
on collagen XI, see section 2.2.1.3.). Collagen V and XI α chains can also appear in the
same heterotypic molecule, e.g. the α2(V) chain substitutes for α2(XI) in the vitreous
body of the eye. It has therefore been suggested that collagens V and XI could be
considered a single type, collagen V/XI. (See Fichard et al. 1994.)
2.1.2. Non-fibrillar collagens
Of the currently known collagen types, collagens IV, VI-X and XII-XIX are the ones that
do not form fibrils. According to their structural properties, they can be further classified
as network-forming collagens, beaded filament-forming collagen, collagen of anchoring
fibrils, fibril-associated collagens with interrupted triple helices (FACITs), membraneassociated collagens with interrupted triple helices (MACITs) and collagens with
multiple triple helix domains and interruptions (MULTIPLEXINs) (see Pihlajaniemi &
Rehn 1995, Prockop & Kivirikko 1995).
Collagens IV, VIII and X form network-like structures. Collagen IV has a large triple
helical domain that is interrupted by numerous short non-collagenous sequences, which
confer flexibility on the individual molecule. This allows aggregation of the monomers to
form the network-like structures that are found in basement membranes. The structures of
collagens VIII and X are very similar to each other but differ from that of collagen IV.
The triple helical domains of collagens VIII and X are only about 450 amino acids in
17
length and these types assemble into hexagonal lattices. The genes that code for collagens
VIII and X have an atypical structure, since they consist of only three exons, the third of
which codes for almost the entire protein. Collagen VIII is expressed primarily in
Descemet’s membrane in the eye, and collagen X is a highly specialized collagen of
hypertrophic chondrocytes in the deep-calcifying zone of cartilage. (See Kielty et al.
1993, Pihlajaniemi & Rehn 1995, Prockop & Kivirikko 1995.)
Collagen VI molecules form a unique structure of beaded filaments, which are found
in most connective tissues. It is located in the close vicinity of cells, large collagen fibrils
and some basement membranes, and may thus function as an anchorage between these
structures and the surrounding ECM. (See Chu et al. 1990.)
Collagen VII is the major component of the anchoring fibrils that attach the basement
membrane to the stroma of the tissues, subject to external frictional forces such as skin,
oral mucosa and cervix (see Burgeson et al. 1990, Kielty et al. 1993).
The FACITs (IX, XII, XIV, XVI, XIX) do not form fibrils themselves, but are found
to be associated with fibrillar collagens. They are not secreted as procollagens like the
fibrillar collagens are, and thus do not undergo proteolytic processing after secretion.
(See Shaw & Olsen 1991, Mayne & Brewton 1993, Prockop & Kivirikko 1995.)
Collagen IX, the best studied member of the FACIT group, is expressed in the same
tissues as collagen II and is covalently crosslinked to the surface of collagen II fibrils
(Eyre et al. 1987, Brewton & Mayne 1994, Diab et al. 1996; for more details on collagen
IX, see chapter 2.2.1.2.). Collagens XII and XIV bear structural similarities to collagen
IX, but their functions are unclear, although it has been suggested that they are associated
with collagen I fibrils. There is also evidence of collagen XII and XIV expression in
tissues from which collagen I is absent, however. Collagens XVI and XIX have also been
classified as FACITs in view of their homology with the other FACITs. (See Mayne &
Brewton 1993.) Even though the exact function of the FACITs is unclear, it has been
suggested that they may serve as molecular bridges and thus have a role in ECM
organization and integrity (see Shaw & Olsen 1991).
Collagens XIII and XVII are not homologous in structure but have a transmembrane
domain, and are therefore called MACITs. The mRNA for collagen XIII can be found in
a variety of tissues, but the function of the protein has not yet been defined. Even though
alternative splicing is relatively common in collagens, that found in collagen XIII is of an
unusual complexity, affecting the sequences for ten exons. Collagen XVII is expressed in
hemidesmosomes of the skin. Autoantibodies against it have been identified in cases of
bullous pemphigoid, an autoimmune disease that causes blistering, suggesting an
anchoring function for this collagen. (See Pihlajaniemi & Rehn 1995.)
Collagens XV and XVIII consist of multiple triple helical domains and interruptions
and are thus called MULTIPLEXINs. They are homologous proteins that are expressed in
a variety of tissues, with overlapping but not similar distributions (Kivirikko et al. 1995,
Saarela et al. 1998). A fragment of the C terminus of collagen XVIII is proteolytically
cleaved to form endostatin, which is known to prevent angiogenesis (O’Reilly et al.
1997).
18
2.1.3. Collagen biosynthesis
After the transcription and translation of collagen mRNA, the newly synthesized
polypeptide undergoes post-translational modifications both intracellularly, in the
endoplasmic reticulum, and extracellularly. The biosynthesis of collagen is a complex
multistage process that requires numerous enzymes. Following cleavage of the signal
peptide, prolyl residues in X-Pro-Gly sequences are hydroxylated to hydroxyprolines,
lysyl residues in X-Lys-Gly sequences to hydroxylysines, and prolyl residues in Pro4Hyp-Gly sequences to hydroxyprolines by the enzymes prolyl 4-hydroxylase, lysyl
hydroxylase and prolyl 3-hydroxylase, respectively. Prolyl 4-hydroxylase plays a central
role in collagen biosynthesis, since 4-hydroxyproline is essential for triple helix stability
at body temperature. Some of the hydroxylysyl residues are further glycosylated with
galactose or galactose and glucose by hydroxylysyl galactosyl transferase or galactosyl
hydroxylysyl glucosyl transferase, respectively. The formation of the triple helix
proceeds from the C terminus to the N terminus. The initial interaction between the noncollagenous domains of the α chains leads to nucleation of the C termini of the
collagenous domains and then to the propagation of triple helix formation in a zipper-like
manner. Correct chain selection is directed by molecular recognition sequences in the C
terminal non-collagenous domains (Lees et al. 1997). In addition, interchain disulphide
bonds in the C terminal non-collagenous domains, catalyzed by protein disulphide
isomerase (PDI), stabilize the chain assembly, but they are not essential for triple helix
formation (Bulleid et al. 1996, see also Bulleid 1996). The correctly folded procollagen
molecules are secreted through the Golgi complex. (See Kielty et al. 1993, Kivirikko
1995, Prockop & Kivirikko 1995.)
The C and N propeptides are cleaved from the procollagen molecules by specific C
and N proteinases in the extracellular space, removal of the propeptides being a
requirement for normal fibril formation. The cleavage of the propeptides converts the
soluble procollagen molecules to insoluble collagen molecules that are able to selfassemble and form fibrils spontaneously. The fibril-forming collagens undergo this
cleavage through the action of C and N proteinases, but many of the non-fibrillar
collagens retain the C and N terminal non-collagenous domains in the mature molecule.
After aggregation of the collagen molecules into fibrils, covalent cross-links are formed
to provide the supramolecular structures with tensile strength and mechanical stability.
The cross-links are formed either by disulphide bonds between cysteine residues or by
lysyl and hydroxylysyl aldehydes that are formed in the reaction catalyzed by lysyl
oxidase. (See Kielty et al. 1993, Kivirikko 1995.)
2.2. Cartilage
Cartilage is hyperhydrated supportive tissue that does not contain blood vessels, nerves or
lymphatics. It consists of about 80% water by weight, and the remaining 20% is mainly
collagen II and proteoglycans. Cartilage is rich in ECM, which is produced by
chondrocytes of mesenchymal origin. There are three types of cartilage. Hyaline cartilage
is the prototypic manifestation that forms articular cartilage, the cartilaginous anlage of
19
developing bones, the epiphyseal plates, the costochondral cartilages and the cartilages of
the respiratory tract. Fibrocartilage resembles hyaline cartilage but contains collagen I
fibres for tensile and structural strength. It is found in the annulus fibrosus of the
intervertebral disc, tendinous and ligamentous insertions, menisci, the symphysis pubis
and the insertions of joint capsules. Elastic cartilage constitutes the pinna of the ears, the
epiglottis and the arytenoid cartilages of the larynx. (See Schiller 1994, Muir 1995.)
2.2.1. Collagenous components of cartilage
Collagens provide the structural framework for hyaline cartilage. Collagen II, which
constitutes 80-85% of the total collagen content of cartilage, forms fibrils that contain
small amounts of collagens IX and XI. The quantities of these minor cartilage collagens
range from 1% to 10%, depending on the cartilage source and age. Small amounts of
collagens VI, XII and XIV have also been identified in cartilage. Expression of collagen
X is restricted to hypertrophic chondrocytes during endochondral ossification. (See
Mayne 1989, Eyre 1991, Brewton & Mayne 1994, Eyre & Wu 1995, Cremer et al. 1998.)
2.2.1.1. Collagen II
The COL2A1 gene that codes for the α chains of homotrimeric collagen II consists of 54
exons and is about 31 kb in size (Ala-Kokko & Prockop 1990, Ala-Kokko et al. 1995).
Its chromosomal location is 12q13.11-q13.12 (Takahashi et al. 1990). Besides its
expression in cartilage, the ocular vitreous body, intervertebral discs and the inner ear,
collagen II can be found in many non-chondrogenic tissues during development (see
Brewton & Mayne 1994). The sequences coding for the N terminal propeptide contain an
alternatively spliced exon that codes for a 69-amino acid cysteine-rich domain (Ryan &
Sandell 1990). Collagen II molecules including (IIA) or excluding this domain (IIB) have
distinct distributions in the stages of chondrogenesis, type IIA predominating in the prechondrogenic mesenchyme and differentiating chondrocytes, and type IIB in
differentiated chondrocytes (Nah & Upholt 1991, Sandell et al. 1991).
The collagen II molecules in the fibril overlap with each other by a distance of about a
quarter of their length, thus forming a banded fibril. The molecules are covalently crosslinked between the triple helical domain and the N or C terminal telopeptides of adjacent
collagen II molecules, i.e. the short non-collagenous regions that are retained in
propeptide cleavage. The particular molecular properties that serve to adapt the collagen
for use in cartilage are unclear, but it has been suggested that the high hydroxylysine
content of the molecule results in a highly hydrated fibril suitable for cartilage. (See Eyre
1991, Kielty et al. 1993, Eyre & Wu 1995.)
20
2.2.1.2. Collagen IX
Collagen IX is a heterotrimeric molecule composed of three separate gene products,
α1(IX), α2(IX) and α3(IX) chains. Like collagen II, it is expressed in cartilage, the
ocular vitreous body, intervertebral discs and the inner ear (Ayad et al. 1982, Slepecky et
al. 1992b, see also Brewton & Mayne 1994). Collagen IX belongs to the group of
FACITs and consists of three collagenous (COL1-COL3) and four non-collagenous
(NC1-NC4) domains numbered from the C terminus of the molecule. The length of the
molecule is about two-thirds of that of the collagen II molecule. (See Kielty et al. 1993.)
The molecule is covalently crosslinked to the surface of collagen II fibrils in an
antiparallel manner; the N terminal regions of the COL2 domains of all three chains
being crosslinked to the N telopeptide of α1(II) and the central region of the COL2
domain of α3(IX) chain to the C telopeptide of α1(II) (Eyre et al. 1987, Vaughan et al.
1988, Wu et al. 1992, Diab et al. 1996). In addition, collagen IX molecules have been
shown to be crosslinked to one another between the N terminal COL2 domain of the
α1(IX) or α3(IX) chain and the C terminal NC1 domain of the α3(IX) chain (Wu et al.
1992, Diab et al. 1996). The length of the NC3 domain is different in all three α chains,
thus forming a kink in the molecule (McCormick et al. 1987). When the COL2 domain is
attached covalently to the surface of collagen II fibrils, the kink allows the COL3 and
NC4 domains to project away from the fibril surface (Bruckner et al. 1988, Vaughan et
al. 1988).
Collagen IX can also be classified as proteoglycan, because the molecule contains a
glycosaminoglycan (GAG) side chain (Bruckner et al. 1985, Bruckner et al. 1988). The
attachment site for the chondroitin sulphate side chain is located in the NC3 domain of
the α2(IX) chain (Huber et al. 1986, McCormick et al. 1987). The proportion of
molecules with glycosaminoglycan side chain substitution varies depending on the tissue
and its source, and the function of the glycosaminoglycan side chain is not known (Yada
et al. 1990, Diab et al. 1996, see also Brewton & Mayne 1994).
The α chains of collagen IX are encoded by the COL9A1, COL9A2 and COL9A3
genes. The COL9A2 and COL9A3 genes consist of 32 exons, while the COL9A1 gene
consists of 38 exons. The six additional exons of the COL9A1 gene code for the 243amino-acid globular NC4 domain that is formed only from the α1(XI) chain. There is an
alternative promoter and second transcription start site in the COL9A1 gene, giving rise
to a short form of the α1(IX) chain and the whole collagen IX molecule. In the short form
the alternative exon 1 in intron 6 is used and exon 7 is skipped, thus splicing the
alternative exon 1 straight to exon 8 of the COL9A1 gene (Nishimura et al. 1989). The
short form exists primarily in the eye. (See Brewton & Mayne 1994.) The exon-intron
structures of the corresponding regions of these genes are highly similar, but the genes
are very different in size. The COL9A1 gene spans about 90 kb, while the COL9A2 and
COL9A3 genes are only about 15 and 23 kb, respectively. (Pihlajamaa et al. 1998a,
Paassilta et al. 1999b.) The chromosomal locations of the genes are 6q12-q13 (COL9A1),
1p33-p32.2 (COL9A2) and 20q13.3 (COL9A3) (Warman et al. 1993b, Warman et al.
1994, Tiller et al. 1998).
The function of collagen IX is not currently known. The location of collagen IX
molecules on the surface of collagen II and the projection of the COL3 and NC4 domains
out from the surface of the fibril suggest a role in the intermolecular organization of
21
cartilage. The pI of the NC4 domain is about 10, thus making the domain a candidate for
interaction with the highly anionic glycosaminoglycans of cartilage (Vasios et al. 1988).
There is also evidence of a role of collagen IX in the maintenance of cartilage integrity
(Fässler et al. 1994, Hagg et al. 1998).
2.2.1.3. Collagen XI
Collagen XI, originally called 1α2α3α collagen, is similarly a heterotrimer, and is
quantitatively a minor member of the group of fibrillar collagens (Morris & Bächinger
1987). Like collagens II and IX, collagen XI is expressed in cartilage, the ocular vitreous
body and the nucleus pulposus (see Eyre & Wu 1987, Brewton & Mayne 1994), but the
question of its occurrence in the inner ear is controversial (Slepecky et al. 1992b,
Thalmann 1993). Furthermore, the mRNAs for all the α(XI) chains can be found in equal
or unequal amounts in a variety of tissues (Lui et al. 1995). The molecule consists of a
major triple helical region of about 1000 amino acids (COL1) that is flanked by a noncollagenous C terminal region (NC1) and an N terminal region that consists of a minor
triple helical region (COL2) flanked by non-collagenous domains (NC2 and NC3).
Collagens V and XI seem to resemble each other closely in their structural and biological
properties. In addition, their α chains can substitute for each other, so that a fraction of
α1(XI) in cartilage has been replaced by α1(V) (see Eyre & Wu 1987) and a fraction
α1(V) in bone has been replaced by α1(XI) (Niyibizi & Eyre 1989). Furthermore, the
ocular vitreous body contains the α1(XI) and α2(V) chains but no α2(XI) chain (Mayne
et al. 1993). These findings have led to the suggestion that collagens V and XI should be
classified as a single collagen type, collagen V/XI. (See Fichard et al. 1994.)
The α1(XI) and α2(XI) chains are products of the COL11A1 and COL11A2 genes,
respectively, while the α3(XI) chain is an overglycosylated form of the IIB splicing
variant product of the COL2A1 gene that also codes for the α chains of collagen II (Eyre
& Wu 1987, Wu & Eyre 1995). The COL11A1 gene is located in the p21 region of
chromosome 1 (Henry et al. 1988). The cDNA for it has been cloned, but only a minor
fraction of the exon-intron organization has been elucidated (Bernard et al. 1988,
Zhidkova et al. 1995). On the other hand, the genomic organization of the COL11A2
gene has been thoroughly defined. It spans 28 kb and consists of 66 exons. Even though it
shares some properties of the major fibrillar collagens, there are marked differences in the
number of exons, exon sizes and codon usage, indicating that the gene has not evolved
with those for the major fibrillar collagens. (Vuoristo et al. 1995.) The COL11A2 gene is
located at 6p21.2 (Kimura et al. 1989) or 6p21.3 (Hanson et al. 1989).
Both the α1(XI) and α2(XI) chains undergo alternative splicing in the N terminal
propeptide. The transcripts for the α1(XI) chain contain either exon IIA, coding for 39
amino acids, IIB coding for 51 amino acids, or neither of them, but not both. In addition,
exon IV, coding for 85 amino acids, may or may not be present in the mRNA. It is
notable that exons IIA, III and IV are highly acidic, while exon IIB is highly basic. The
alternative splicing of the α2(XI) chain involves exons 6, coding for 26 amino acids, and
7, coding for 21 amino acids. Furthermore, Lui et al. (1996) suggest additional splicing of
sequences in intron 6 of the transcript to the mRNA, yielding a truncated α2(XI) chain
22
because of the translation termination codons in the sequences. (Zhidkova et al. 1995.)
The N terminal domain of the α2(XI) chain has also been shown to give rise to
proline/arginine-rich protein (PARP) in cartilage (Neame et al. 1990, Zhidkova et al.
1993).
Collagen XI molecules are covalently cross-linked primarily to each other in head to
tail organization. The cross-links occur between the N telopeptides and the C terminal
ends of the helical regions. In addition, the N terminal end of the α1(XI) chain is attached
to the C telopeptide sequence of the α3(XI) or α1(II) chain, presumably to α1(II). These
data indicate that collagen XI may form primarily homopolymers, which are also capable
of cofibrillar assembly with collagen II. (Wu & Eyre 1995.) The coexistence of collagens
II, IX and XI in the cartilage collagen fibrils and the finding that collagen XI is
immunologically masked has led to the suggestion that collagen XI is buried inside the
fibril (Mendler et al. 1989). Its hypothesized function is regulation of fibril diameter. This
is supported by the finding that collagen XI is restricted to thin fibrils and that mouse and
human defective collagen XI occurs in abnormal thick, disorganized fibrils (Seegmiller et
al. 1971, Keene et al. 1995, Li Y et al. 1995, van Steensel et al. 1997). Partial retention
of the N propeptide has been proposed as the molecular basis for this function. (See
Fichard et al. 1994.)
2.2.2. Non-collagenous components of cartilage
While collagens form the structural network for cartilage, proteoglycans (also called
mucopolysaccharides) provide hydration and swelling pressure, enabling the cartilage to
withstand compressional forces. Proteoglycans are proteins that have one or more GAG
side chain, which may be chondroitin sulphate, dermatan sulphate, keratan sulphate,
heparan sulphate or heparin. (See Hardingham & Fosang 1992, Roughley & Lee 1994.)
The largest and most abundant proteoglycan in cartilage is aggrecan, which consists of a
multidomain core protein of over 200 kDa bearing over a hundred chondroitin and
keratan sulphate side chains in a bottle brush manner. The core protein of an aggrecan
molecule attaches to hyaluronan by non-covalent binding, which is stabilized by link
protein, thus forming huge multimolecular aggregates. (See Neame & Barry 1993,
Hardingham & Fosang 1995.) Many other proteoglycans, such as decorin, biglycan,
fibromodulin, lumican and PRELP (proline/arginine-rich end leucine-rich repeat protein),
have also been characterized in cartilage. These have relatively small core proteins and
only a few GAGs. While aggrecan is mainly responsible for the hydration of cartilage,
other proteoglycans in cartilage have distinct biological functions apart from their
hydrodynamic functions, e.g. regulation of collagen fibrillogenesis or cell adhesion and
migration. (Grover et al. 1995, Bengtsson et al. 1995, Kinsella et al. 1997, see also
Stanescu 1990, Roughley & Lee 1994.)
In addition to link protein, there are other non-collagenous proteins in cartilage that do
not belong to the proteoglycans, e.g. cartilage oligomeric matrix protein (COMP),
anchorin and chondrocalcin in cartilage (see Heinegård & Oldberg 1989).
23
2.3. Cartilage collagens in non-cartilaginous tissues
Besides cartilage, cartilage collagens are significant structural components of the tectorial
membrane of the inner ear, the ocular vitreous body and intervertebral discs.
2.3.1. Inner ear
Collagen II, which is the most studied cartilage collagen in the inner ear, has been found
in various tissues there, such as the tectorial, basilar and vestibular membranes and the
cartilaginous rest of the endochondral layer (Thalmann 1986, Slepecky et al. 1992b,
Thalmann 1993). Thalmann et al. (1987) have shown that collagen accounts for
approximately 40% of the total protein in the tectorial membrane of the guinea pig. This
membrane lies over the organ of Corti, and the stereocilia of the outer hair cells are
embedded in its gel-like matrix (see Moore 1992a). The co-localization of collagens II
and IX in the thick, unbranched radial fibres of the tectorial membrane is well
demonstrated, but there is some inconsistency in reports with regard to the expression of
collagens XI and V in the tectorial membrane and in the inner ear generally (Slepecky et
al. 1992a, Slepecky et al. 1992b, Thalmann 1993).
2.3.2. Ocular vitreous body
The ocular vitreous body is a gel-like structure consisting primarily of hyaluronan and
collagen fibrils (Ren et al. 1991, Brewton & Mayne 1992). The collagenous component
comprises collagen II, collagen IX and collagen resembling types V and XI (Swann &
Sotman 1980, Ayad et al. 1982, Ayad & Weiss 1984, Seery & Davison 1991). Further
studies by Mayne et al. (1993) demonstrated that the chains of the collagen V/XI fraction
of bovine vitreous body are α1(XI) and α2(V). Since neither of these is known to exist in
homotrimers (see Fichard et al. 1994), it is highly probable that they form heterotypic
trimers either with or without the α1(II) chain. Collagen IX in the vitreous lacks the NC4
domain, suggesting utilization of the alternative downstream promoter of the COL9A1
gene (Ren et al. 1991, Warman et al. 1993a). (See Brewton & Mayne 1994, Bishop
1996.)
2.3.3. Intervertebral disc
An intervertebral disc stabilizes the spine by anchoring adjacent vertebral bodies to each
other and simultaneously absorbs the compressive forces directed towards the spine.
While stabilization of the spine requires firmness, resilience is essential for shock
absorption. To fulfil both of these tasks, the intervertebral disc comprises concentrically
arranged compartments. The outermost structure is a ring-like outer annulus fibrosus,
24
which consists of highly oriented, densely packed collagen fibril lamellae, inside which is
the fibrocartilaginous inner annulus fibrosus. There is a transition zone separating the
central nucleus pulposus, a gel-like structure, from the layers of the annulus fibrosus, and
there are no clear boundaries between the structures of the intervertebral disc. The
cartilaginous endplates between the vertebral bodies and the intervertebral disc are often
considered part of the intervertebral disc. (See Humzah & Soames 1988, Moore 1992b,
Buckwalter 1995.)
Collagens I and II are the predominant collagens in the intervertebral disc. Collagen II
comprises almost all of the total collagen in the nucleus pulposus, the amount of which
decreases towards the outer structures so that it is absent or almost absent in the outer
annulus fibrosus, while collagen I comprises almost all of the total collagen composition
of the outer annulus fibrosus and decreases to absent or almost absent in the nucleus
pulposus (Eyre & Muir 1977, Buckwalter 1995, Nerlich et al. 1998). Many reports
indicate the presence of collagen IX in the intervertebral disc, even though Roberts et al.
(1991) failed to immunolocalize it in the normal human disc (Ayad et al. 1982, Newall &
Ayad 1995, Nerlich et al. 1998). Collagen IX has been found in all the compartments of
the intervertebral disc, although it comprises about 2% of the total collagen or even less.
Both short and long forms of collagen IX can be found, the short form predominating in
the nucleus pulposus (Newall & Ayad 1995). By contrast, collagen XI has been found
only in the nucleus pulposus, where it comprises about 3% of total collagen.
Proteoglycans, primarily aggrecan, are a significant component of intervertebral discs
accounting for a few percent of the dry weight of the annulus fibrosus, but about 50
percent of the nucleus pulposus. (See Buckwalter 1995.)
2.4. Disorders affecting cartilage collagens
Numerous conditions that affect the components of cartilage have been characterized.
The development and maintenance of cartilage can be affected at several stages in the
life-span: the defects can already occur during embryogenesis, in the early steps of
mesenchymal differentiation, or only after middle age. The mechanisms of the faulty
processes are various. Transcription factors, growth factors, growth factor receptors,
structural proteins or enzymes involved in their modification can be defective, or else the
process can be due to inflammatory autoimmune disease, trauma or simply wear and tear.
(See Horton & Hecht 1993, Mundlos & Olsen 1997a, Mundlos & Olsen 1997b,
Kuivaniemi et al. 1997, Cremer et al. 1998.) Only disorders affecting cartilage collagens
are discussed in the following.
In the case of a cartilage collagen defect, the symptoms may appear in all or some of
the tissues in which the collagens are expressed. Probably the most common symptom is
destruction of the hyaline cartilage of joints. The development of bony structures such as
the spine, long bones and facial bones is often defective due to the disruption of
endochondral ossification. Interestingly, the eye and ear can be severely affected: high
myopia, retinal detachments and hearing deficits are usual manifestations of cartilage
collagen defects. (See Spranger et al. 1994, Mundlos & Olsen 1997b.)
25
2.4.1. Achondrogenesis type II-hypochondrogenesis (ACGII-HCG)
Initially, achondrogenesis type II (Langer-Saldino type) and hypochondrogenesis were
considered two separate disorders (Langer et al. 1969, Saldino 1971, Maroteaux et al.
1983), but after individuals with an intermediate phenotype had been characterized they
often came to be regarded as different degrees of severity of the same disorder
(Borochowitz et al. 1986). Infants with ACGII-HCG (OMIM #200610) die perinatally or
within the first weeks of life. The disorder is characterized by a short, barrel-shaped
trunk, very short extremities, a large head, a soft cranium, a flat face and hydropic
appearance. Radiographically, the affected infants show varying degrees of
underossification of the axial skeleton. Histological and electron microscopic
examinations reveal hypercellular epiphyseal cartilage with poorly organized or absent
growth plate and diminished extracellular matrix that contains thick, irregular collagen
fibrils together with large chondrocytes with a dilated rough endoplasmic reticulum.
Biochemical studies of hyaline cartilage from ACGII-HCG infants have indicated
abnormal and diminished collagen II, even its absence, and the presence of collagen I.
(Eyre et al. 1986, Godfrey et al. 1988, Godfrey & Hollister 1988, see also Horton &
Hecht 1993, Spranger et al. 1994.)
As suggested by earlier biochemical data, the genetic defect causing ACGII-HCG was
found in the COL2A1 gene (Vissing et al. 1989). Over ten mutations causing ACGIIHCG have been reported, and all of them involve the replacement of glycine by a bulkier
amino acid in the triple helical region of the α1(II) chain. The disorder is caused by
heterozygous mutations, and naturally, because of the severity of the symptoms, the cases
are usually sporadic. (See Horton & Hecht 1993, Kuivaniemi et al. 1997.)
2.4.2. Spondyloepiphyseal dysplasia (SED)
Spondyloepiphyseal dysplasia (SED, OMIM #183900) is characterized by a short trunk,
short extremities, a barrel-shaped chest, kyphosis and lordosis, severe myopia, and often
retinal detachments, cleft palate and clubfoot. Affected infants usually survive.
Radiographs show defects in ossification of the spine and primarily proximal extremities.
(See Horton & Hecht 1993.) Histological studies show defects resembling those in
ACGII-HCG, and biochemical examination indicates abnormalities in collagen II (Yang
et al. 1980, Murray et al. 1989). SED refers to a heterogeneous group of disorders that
affect the spine and epiphyses, and as the symptoms resemble those of ACGII-HCG,
SED can be considered to represent the mild end of the spectrum comprising ACGIIHCG and SED phenotypes (see Spranger et al. 1994).
Most cases of SED show autosomal dominant inheritance, but an autosomal recessive
form of inheritance has also been suggested in some cases (Harrod et al. 1984, see also
Horton & Hecht 1993). Lee et al. (1989) identified a deletion of an entire exon in the
COL2A1 gene, leading to a lack of 36 amino acids in the triple helical region of the
α1(II) chain, and subsequently numerous mutations, including insertions, mutations
causing aberrant RNA splicing, glycine mutations and a cystein for arginine substitution,
have been characterized (see Vikkula et al. 1994, Kuivaniemi et al. 1997). However,
26
some investigations have excluded the COL2A1 gene as a locus for SED in certain
families, indicating that there must also be some other locus or loci for the disorder
(Wordsworth et al. 1988, Anderson et al. 1990).
2.4.3. Kniest dysplasia
Patients with Kniest dysplasia (OMIM #156550) have a round face, midfacial hypoplasia,
prominent eyes and high myopia. Cleft palate, hearing loss and retinal detachment are
also frequent manifestations of the disorder. The trunk and extremities are shortened and
the joints are enlarged and exhibit restricted mobility. Numerous abnormal characteristics
are seen in radiographic examinations. There is generally flattening of the vertebral
bodies, the long bones are shortened and of a dumbbell shape due to expanded
metaphyses, while the epiphyses are irregular and fragmented, the joint spaces are
narrow, and there is generalized osteopenia in their ends. Kniest dysplasia is usually more
severe than SED and is lethal in some cases. (Chen et al. 1980, see also Horton & Hecht
1993, Spranger et al. 1994.) Histologically, the cartilage is soft and vacuolar, and electron
microscopy shows abnormally thin and irregular collagen fibrils in the matrix and dilated
rough endoplasmic reticulum (Horton & Rimoin 1979, Poole et al. 1988, Gilbert-Barnes
et al. 1996.)
Kniest dysplasia is an autosomal dominant disorder. More than ten heterozygous
mutations in the COL2A1 gene have been reported to date, including that in the patient
originally described by Dr. Kniest in 1952. All except two of them lead to whole or
partial exon deletions, and all but one are clustered in the N terminal half of the molecule,
thus indicating that the cause of Kniest dysplasia lies in short in-frame deletions in this
particular region of the collagen II molecule. (Spranger et al. 1997, Fernandes et al. 1998,
see also Kuivaniemi et al. 1997.) Recent studies have shown that a significant fraction of
the defective collagen II molecules are secreted and incorporated in cartilage collagen
fibrils. Furthermore, it has been hypothesized that the normal α1(II) chains would form a
loop and thus allow in-register triple helix formation in both directions from the deletion
site. Moreover, due to the susceptibility of the loop to protease cleavage, this could also
account for the degenerate vacuolar appearance of the cartilage matrix in patients with
Kniest dysplasia. (Weis et al. 1998, Fernandes et al. 1998.)
2.4.4. Stickler and Marshall syndromes
Stickler syndrome (OMIM #108300), originally called hereditary progressive arthroophthalmopathy, is an autosomal dominantly inherited disorder. Patients have high
myopia, vitreoretinal degeneration and frequently retinal detachments and cataracts.
Midfacial hypoplasia and micrognathia are often accompanied by cleft palate or a lesser
degree of clefting, and sensorineural hearing defect is common. The skeletal
manifestations include juvenile progressive arthropathy, irregularity of the vertebral
bodies and hypermobile joints. Skeletal growth is usually normal. Mild epiphyseal
27
dysplasia and overtubulation of long bones are seen in radiological examination. (Stickler
et al. 1965, Stickler & Pugh 1967, see also Herrmann et al. 1975, Temple 1989, Snead &
Yates 1999.) In addition, an increased prevalence of mitral valve prolapse has been
reported (Liberfarb & Goldblatt 1986), but this was not supported in the
echocardiographic studies of over 100 patients (see Snead & Yates 1999).
Marshall syndrome (OMIM #154780) is also an autosomal dominantly inherited
disorder, and closely resembles Stickler syndrome. The ocular, facial and auditory
features are much alike, but patients with Marshall syndrome are usually short in stature
and have intracranial ossifications, abnormal frontal sinuses and thickened calvaria. In
addition, Dr. Marshall originally reported some kind of hypohidrotic ectodermal
dysplasia associated with the other symptoms, but it is thought nowadays that ectodermal
dysplasia is not involved in the syndrome. (Marshall 1958, O’Donnell et al. 1976, Aymé
& Preus 1984, Stratton et al. 1991, Shanske et al. 1997, Warman et al. 1998.)
Numerous new cases of Stickler or Marshall syndrome have been reported since the
original kindreds, and these have broadened the range of characteristics, so that it is now
clear that there is clinical heterogeneity within the disease phenotypes, especially in
Stickler syndrome (Herrmann et al. 1975, Zlotogora et al. 1992). The similar features of
Stickler and Marshall syndromes have led to a clinical debate as to whether the
syndromes are two distinct entities or just different manifestations of a single entity
(Baraitser 1982, Winter et al. 1983, Aymé & Preus 1984, Stratton et al. 1991, Shanske et
al. 1997). Furthermore, the diagnosis of Marshall syndrome has aroused some debate
(Shanske et al. 1998, Warman et al. 1998).
Another autosomal dominantly inherited syndrome with ocular manifestations is
Wagner syndrome, or hyaloideoretinal degeneration of Wagner (OMIM #143200). This
was originally characterized by myopia, cataract, vitreoretinal degeneration and often
retinal detachment, but no manifestations in non-ocular tissues. There are some reports of
Stickler-like non-ocular symptoms in Wagner syndrome, however, leading to a
suggestion that the two could represent the same disorder. (See Liberfarb et al. 1979,
Horton & Hecht 1993.)
Linkage studies have shown that the COL2A1 gene is a locus for Stickler syndrome
(Francomano et al. 1987, Knowlton et al. 1989). Altogether nine mutations have been
reported to date, all of them leading to premature termination of translation (Ahmad et al.
1991, Brown et al. 1992, Ahmad et al. 1993, Ritvaniemi et al. 1993, Brown et al. 1995a,
Ahmad et al. 1995, Williams et al. 1996). The same gene was also found to be a locus for
Wagner syndrome in one family (Körkkö et al. 1993), even though Wagner syndrome,
together with erosive vitreoretinopathy, is normally linked to 5q13-q14 (Brown et al.
1995b). The COL2A1 gene has also been excluded in several families with Stickler
syndrome (Knowlton et al. 1989, Vintiner et al. 1991, Bonaventure et al. 1992), and
Snead et al. (1994) have suggested that Stickler families with congenital vitreous
anomaly (Stickler syndrome type 1) have linkage to the COL2A1 gene, while in those
with congenitally defective vitreous gel architecture but no congenital vitreous anomaly
(Stickler syndrome type 2) linkage is excluded. Subsequently, Richards et al. (1996)
demonstrated linkage to the COL11A1 gene in a family with Stickler syndrome type 2,
and traced it to a missense mutation converting glycine to valine in the N terminal part of
the triple helix of the α1(XI) chain. In addition, Wilkin et al. (1998) have suggested still
another locus for Stickler syndrome apart from the genes coding for collagens II and XI.
28
It was also hypothesized that Marshall syndrome was caused by mutations in the
COL11A1 gene (van Steensel et al. 1997). A splicing mutation that led to a deletion of 18
amino acids in the C terminal half of the α1(XI) triple helical region was found to be
associated with the phenotype (Griffith et al. 1998).
2.4.5. Otospondylomegaepiphyseal dysplasia (OSMED), WeissenbacherZweymüller syndrome (WZS) and non-ocular Stickler-like syndrome
Otospondylomegaepiphyseal dysplasia (OSMED, OMIM #215150) is an autosomal
recessive chondrodysplasia characterized by sensorineural deafness, spondyloepiphyseal
dysplasia with large epiphyses, disproportionate shortness, midfacial hypoplasia and
often micrognathia and cleft palate, but no ocular manifestations (Giedion et al. 1982,
Kääriäinen et al. 1993, van Steensel et al. 1997). Electron microscopic examination of
cartilage shows disorganized matrix with extremely thick collagen fibres that consist of
aggregated thinner fibrils (van Steensel et al. 1997). Weissenbacher-Zweymüller
syndrome (WZS, OMIM #277610)) is highly similar to OSMED and is also thought to be
an autosomal recessive disorder. One characteristic of WZS is its favourable progression
during childhood. (Cortina et al. 1977, Chemke et al. 1992.)
Vikkula et al. (1995) demonstrated linkage to the COL11A2 gene in a family with
OSMED, and also identified a homozygous mutation in the gene. The mutation led to a
substitution of arginine for glycine in the N terminal half of the triple helical region of the
collagen XI molecule. The original patient with WZS described by Drs. Weissenbacher
and Zweymüller in 1964 was also found to have a mutation in the COL11A2 gene. The
mutation converted a glycine to glutamate in the C terminal end of the triple helix, and,
surprisingly, was heterozygous. (Pihlajamaa et al. 1998b.) Furthermore, a kindred with an
autosomal dominant disorder resembling Stickler syndrome but without eye involvement
was linked to chromosome 6 near to the COL11A2 gene (Brunner et al. 1994), and
subsequently Vikkula et al. (1995) reported of a heterozygous mutation in the kindred
causing exon skipping in the COL11A2 mRNA and thus deletion of 18 amino acids near
the C terminal end of the triple helical region in the α2(XI) chain. A mutation causing
deletion of 9 amino acids in the middle of the triple helical region was described in
another family with a Stickler-like syndrome without eye involvement (Sirko-Osadsa et
al. 1998).
The nomenclature for the chondrodysplasias caused by mutations in the COL11A2
gene was established before the common molecular background for the disorders had
been discovered, and thus the terminology is confusing, with various names applying to
the same disorders. Recent findings have led to a suggestion for a new nomenclature for
these disorders. The term heterozygous OSMED was introduced to include the former
WZS and non-ocular Stickler-like syndrome, while homozygous OSMED is
recommended for recessive OSMED. (Pihlajamaa et al. 1998b, Spranger 1998.)
29
2.4.6. Multiple epiphyseal dysplasia (MED)
The typical finding in patients with multiple epiphyseal dysplasia (MED; OMIM
#132400), an autosomal dominantly inherited disorder, is prominent and frequently
painful joints with restricted mobility. This usually appears in early childhood. The
disorder affects multiple joints rather than being generalized, and it leads to precocious
osteoarthritis. The patients have a waddling gait and may have short and stubby hands.
Stature is normal or moderately short with normal proportions. Radiographic examination
shows typically small, irregular epiphyses. (Barrie et al. 1958, Hoefnagel et al. 1967,
Spranger et al. 1974, van Mourik et al. 1998a.) The severity of symptoms varies
considerably, and some patients with radiological evidence of MED do not have any
complaints (van Mourik et al. 1998a). MED is often divided into two subgroups, the mild
Ribbing type and the severe Fairbank type (see International Working Group on
Constitutional Diseases of Bone 1998). Because of the wide phenotypic spectrum of
MED, not all patients with the disorder can be classified into either the Ribbing or the
Fairbank type, and it has been suggested that these terms should be abandoned (van
Mourik et al. 1998a, Haga et al. 1998). The Ribbing type has also been reported to be
recessive, but molecular genetic studies on such families suggest germline mosaicism as
an explanation (Deere et al. 1995, Loughlin et al. 1998).
Pseudoachondroplasia (PSACH, OMIM #177170) is an autosomal dominant disorder
that resembles MED but is more severe, causing disproportionate dwarfism and
ligamentous laxity. Radiographs show abnormalities in the epiphyses, metaphyses and
spine. (See Horton & Hecht 1993.)
Genetic linkage of both MED and PSACH to the pericentromeric region of
chromosome 19 has been demonstrated (Hecht et al. 1993, Briggs et al. 1993, Oehlmann
et al. 1994). This is where the gene for cartilage oligomeric matrix protein (COMP), a
pentameric calcium binding glycoprotein of the extracellular matrix, is located (Newton
et al. 1994). A large number of mutations causing either MED or PSACH have been
reported in the COMP gene (Briggs et al. 1995, Hecht et al. 1995, Ikegawa et al. 1998),
but it has also been excluded in a MED family, where linkage was demonstrated to a
region of chromosome 1 containing the COL9A2 gene (Briggs et al. 1994). Muragaki et
al. (1996) identified a splicing mutation of exon 3 in the COL9A2 gene, leading to a lack
of 12 amino acids in the COL3 domain of the α2(IX) chain. Recently, another mutation
in collagen IX was reported in a family with MED, causing a splicing defect of exon 3
with a similar consequence to that reported earlier but applying to the COL9A3 gene.
(Paassilta et al. 1999a.)
Similar dilatations of the rough endoplasmic reticulum in chondrocytes can be seen in
MED and PSACH (Stanescu et al. 1982, Stanescu et al. 1993). These were studied
further in a PSACH patient who was shown to have a mutation in the COMP gene. The
dilatations contained aggregates consisting of at least COMP and collagen IX, suggesting
a common pathogenic mechanism for MED and PSACH. (Maddox et al. 1997b.) No
dilatation of the rough endoplasmic reticulum was seen in patients with MED caused by
the COL9A2 mutation, however (van Mourik et al. 1998b).
30
2.4.7. Osteoarthritis (OA)
Osteoarthritis, or osteoarthrosis (OA), is the most common articular disorder and is
characterized by joint pain and tenderness, stiffness, crepitus and limitation of motion.
Radiographs typically show joint-space narrowing, osteophytes, subchondral bone
sclerosis and subchondral cyst formation. In severe cases deformity of the bone ends is
also seen. OA is classified into primary (idiopathic) or secondary subsets; in a primary
case there is no known predisposing factor, while in a secondary case such a factor is
known, e.g. trauma, infection, a mechanical factor, some other joint disorder or a
metabolic disease. Further classification divides OA into localized, in which only certain
joints are affected, and generalized subsets. Even though it is often considered only an
inevitable consequence of ageing, female sex, obesity, occupational load and heavy
sports exertion are risk factors for it. (See Moskowitz 1989, Altman 1995, Creamer &
Hochberg 1997.)
The suggestion of genetic factors underlying OA in certain families was made several
decades ago (Stecher 1941, Stecher et al. 1953, Kellgren et al. 1963). In 1989, Palotie et
al. demonstrated a linkage to the COL2A1 gene in a family with primary OA. Knowlton
et al. (1990) made a similar finding in a family with precocious OA and associated mild
chondrodysplasia, and Ala-Kokko et al. (1990) subsequently demonstrated a mutation in
this family. The mutation converted arginine to cysteine, an amino acid not normally
found in the triple helical domains of collagens, in the middle of the triple helix of the
collagen II molecule. Altogether five families with this same mutation have been reported
to date, and three of them have been shown to have a common ancestor (see Bleasel et al.
1995, Kuivaniemi et al. 1997, Bleasel et al. 1998). In addition, a mutation converting
arginine to cysteine at position 75 near the N terminus of the triple helix has been
reported in three families with OA associated with mild SED (see Bleasel et al. 1995,
Bleasel et al. 1996).
2.5. Animal models for disorders of the cartilage collagens
In order to fully understand the mechanisms of a cartilage disorder, histological and
molecular studies are essential. Human cartilage specimens for such studies are difficult
to obtain, because that would require a surgical operation, and perhaps more importantly,
spare cartilage is rarely available, at least in large amounts. Furthermore, it is well known
that chondrocytes dedifferentiate in cell culture (Grundmann et al. 1980). Animal models,
often mice, are thus used for cartilage disorders. The phenotype is either a natural variant
of the species or is generated by the scientist by introducing mutagenic agents into the
animal, or more elegantly, by manipulating the genome by modern techniques that allow
targeted mutation, inactivation or specific expression of virtually any gene. Large
amounts of cartilage specimens can be obtained from animal models, and disorders can
also be studied in vivo.
31
2.5.1. Collagen II
Several approaches have been used for studying collagen II defects in mice. Two
transgenic mouse lines with deletions in the triple helical domain have been generated,
and two with glycine substitutions. The phenotypes caused by these mutations are usually
perinatally lethal chondrodysplasias characterized by dwarfism, short snout, cleft palate,
disorganized growth plates and reduced ossification. The collagen fibrils are disorganized
and reduced in amount. (Vandenberg et al. 1991, Garofalo et al. 1991, Metsäranta et al.
1992, Maddox et al. 1997a.) A phenotype resembling this has also been produced in a
mouse line with disproportionate micromelia (Dmm) generated by irradiation (Brown et
al. 1981). These mice have a deletion in the region encoding the C propeptide of
procollagen II, which leads to a substitution of one amino acid for two (Pace et al. 1997).
Garofalo et al. (1993) overexpressed collagen II. The mice died at birth, but they did
not exhibit cleft palate, abnormal craniofacial features, or other skeletal deformities,
except for a slight reduction in the length of the long bones. Electron microscopy of the
cartilage showed abnormally thick, extensively branched fibrils, which led to the
suggestion that the imbalance in cartilage components caused by the overexpression of
collagen II disrupted the mechanism that controls the diameter of the fibrils. On the other
hand, total inactivation of the Col2a1 gene generated by S.-W. Li et al. (1995) in mice led
to severe perinatally lethal chondrodysplasia. This mouse model shed some light on the
role of cartilage in skeletal development by showing that collagen II is essential for the
development of endochondral bone and epiphyseal cartilage but not that of many other
skeletal structures such as membranous and periosteal bone.
2.5.2. Collagen IX
Three mouse lines with defective collagen IX have been generated. Mice expressing a
truncated α1(IX) chain, which lack part of the COL2 domain, the entire NC3 domain and
part of the COL3 domain, were shown to develop osteoarthritis, which was mostly
obvious in the knee joints. Ones with a higher level of transgene expression also
developed mild chondrodysplasia with mild proportionate dwarfism and a slight
ossification defect in the spine. Abnormally thin collagen fibrils were seen in electron
microscopy. (Nakata et al. 1993.) Furthermore, long-term follow-up of the mice revealed
accelerated intervertebral disc degeneration, which occasionally led to herniation and
osteophyte formation (Kimura et al. 1996).
Fässler et al. (1994) generated a mouse line lacking the α1(IX) chain. The mice
showed no detectable abnormalities at birth, but later developed a severe progressive
degenerative joint disease resembling human OA, which was most prominent in the knee
joints. The fibril morphology of the cartilage was nevertheless visually identical in the
homozygous and control mice. Absence of the α1(IX) chain was shown to lead to
absence of the α2(IX) and α3(IX) chains as well, even though they were transcribed.
Thus this mouse line exhibited functional inactivation of the entire collagen IX. These
findings lead to a suggestion that collagen IX is essential for the integrity of the cartilage
matrix, but not for skeletal development. (Hagg et al. 1997.)
32
Transgenic mice expressing multiple copies of a minigene coding for the NC4 domain
of collagen IX have also been created. This overexpression of the NC4 domain induced
osteoarthritis analogous to human OA. (Haimes et al. 1995.)
2.5.3. Collagen XI
One mouse model for collagen XI disorders has been reported. Mice with autosomal
recessive chondrodysplasia (cho) have disproportionately short limbs with wide, flared
metaphyses and abnormal facial features consisting of a short snout, short mandible,
protruding tongue and cleft palate. They have soft tracheal cartilage rings, which is
probably the cause of their perinatal death. Microscopy showed disorganized growth
plates and abnormally thick collagen fibrils. (Seegmiller et al. 1971.) Linkage studies
mapped the cho locus to the region containing the Col11a1 gene, and a frame shift
mutation causing premature translation termination was identified. The mutation thus
leads to absence of the α1(XI) chain. The finding indicates the importance of this chain
for skeletal morphogenesis and, more specifically, for the formation of proper collagen
fibrils in cartilage. (Li Y et al. 1995.)
2.6. Intervertebral disc disease
Degenerative changes in the intervertebral discs begin early in childhood. The
degeneration occurs in all parts of the discs, but the nucleus pulposus undergoes the most
extensive changes. The annulus fibrosus generally expands at the expense of the nucleus
pulposus, the number of viable cells in the disc decreases and the water concentration
also decreases. The amounts of proteoglycans decrease, and fragmentation of the
aggregating proteoglycans can be detected. The pathomechanism of these changes is not
known, but one potential cause could be a decline in nutrition due to the increasing
volume of the disc during growth and a simultaneous age-related decrease in the arteries
supplying nutrients and removing waste. Even if disc degeneration is an inevitable
consequence of ageing, it can vary greatly in rate and extent within the same person and
between individuals. (See Buckwalter 1995.)
The degeneration impairs the structural integrity of the disc tissue and increases the
probability of functional deficiency and disc herniation. In intervertebral disc herniation
part of the nucleus pulposus extrudes through the annulus fibrosus. The extruded material
may cause mechanical compression or chemical irritation to the adjacent nerve root and
lead to sciatica, i.e. radiating pain in the dermatome of the root. (See Hasue 1993,
Alaranta & Poussa 1994.) Herniation of the lumbar discs and sciatica, also called lumbar
disc syndrome, is a common disorder affecting the Finnish population with a prevalence
of about 5% (Heliövaara et al. 1987). Many environmental risk factors for intervertebral
disc disease have been proposed, including hard physical work, driving motor vehicles
and smoking (see Heliövaara 1989), but genetic predisposition to advanced disc
degeneration and intervertebral disc disease has also been suggested, and it has even been
33
claimed that this may be the primary factor (Varlotta et al. 1991, Scapinelli 1993, Battié
et al. 1995, Sambrook et al. 1999). In addition, polymorphisms in the vitamin D receptor
gene have been associated with intervertebral disc degeneration (Videman et al. 1998).
2.6.1. Cmd mice
Mice that are homozygous for cartilage matrix deficiency (cmd) have short limbs, trunk,
tail and snout, cleft palate and hearing impairment, and they die at birth, probably of
respiratory failure (Rittenhouse et al. 1978, Yoo et al. 1991). The mice were found to
lack aggrecan (Kimata et al. 1981), and a deletion causing premature translation
termination in the gene coding for aggrecan has been identified (Watanabe et al. 1994).
Despite the original understanding that the heterozygous mice are normal, Watanabe et
al. (1997) demonstrated that they show dwarfism and spinal misalignment at the age of
one year. They exhibited deformation of the vertebral bodies, advanced disc degeneration
and disc herniations, but no changes in other cartilaginous tissues. This finding supports
results obtained with mice expressing a truncated α1(IX) chain, indicating that mutations
in the genes coding for cartilage matrix protein can cause intervertebral disc disease.
Mice with hereditary kyphoscoliosis (ky) also develop advanced intervertebral disc
degeneration, but these changes are secondary to a neuromuscular disorder that leads to
atrophy of the muscles supporting the spine (Mason & Palfrey 1984, Bridges et al. 1992).
2.7. Mutation detection methods
Knowing the genetic defect behind a disease provides a basis for understanding the
disease and treating it. The detection of new, previously unknown mutations in sequences
of a candidate gene requires an appropriate method. Sequencing is a thorough method,
but if the sequences to be searched span thousands or tens of thousands of base pairs, the
procedure turns out to be expensive and excessively laborious. A large number of
methods for mutation detection, most of them based on PCR, have been introduced to
make it more efficient with regard to costs, time and work. The methods usually take
advantage either of the change in electrophoretic mobility produced by the mutation or of
recognition and cleavage of the mismatched site in a heteroduplex comprising a mutant
and a wild strand. (See Cotton 1989, Cotton 1993, Eng & Vijg 1997, Nollau & Wagener
1997.)
2.7.1. Commonly used methods based on electrophoresis
Single-strand conformation polymorphism (SSCP) is based on the fact that singlestranded DNA has a defined secondary structure under certain conditions. This secondary
structure changes if the sequence is altered, and this is detected in the form of mobility
34
shifts on a polyacrylamide gel. (Orita et al. 1989a, Orita et al. 1989b.) Band visualization
has to be performed with radiolabelling or silver staining, as ethidium bromide, which is
widely used to stain double-stranded DNA, does not stain single-stranded DNA
efficiently. The advantage of SSCP is the simplicity of the procedure, but its sensitivity,
which is best with fragments less than 200 bp, is only 60-95%. (See Cotton 1993, Eng &
Vijg 1997.)
The melting properties of double-stranded DNA depend on the sequence of the DNA
fragment. This phenomenon is utilized in denaturing gradient gel electrophoresis
(DGGE) by electrophoresing DNA fragments on a polyacrylamide gel under increasing
denaturing conditions. Thus sequence variations alter the melting and mobility of the
mutant fragment as compared with a control fragment. (Fischer & Lerman 1983, see also
Myers et al. 1987.) The original method was able to detect sequence variations only in
the low melting domains of the fragment, i.e. sequences with a low melting temperature,
giving a sensitivity of about 50%, but the addition of an artificial high melting GC-rich
sequence (GC-clamp) to one end of the fragment using PCR primers converts the whole
fragment to a low melting region and greatly improves the sensitivity (Sheffield et al.
1989). Fragments of up to 1 kb can be analyzed, but ones of 150-500 bp are usually most
appropriate. The advantage of DGGE is its high sensitivity of almost 100%, but
optimization of the melting properties of the fragment and electrophoresis conditions is
laborious and time-consuming. (See Cotton 1989, Cotton 1993, Ganguly & Prockop
1995.)
Heteroduplex analysis (HA or HET) is based on the different mobilities of
homoduplexes and heteroduplexes on a non-denaturing polyacrylamide gel.
Heteroduplexes, i.e. double-stranded DNA which contains one strand of the mutant type
and one of the wild type, are formed by heat denaturation and subsequent reannealing of
the two forms of DNA. (See Glavac & Dean 1995.) HA is a simple method that does not
require optimizing, but its sensitivity is estimated to be about 80% (Cotton 1993). Many
modifications of HA have been used to improve the sensitivity (see Glavac & Dean
1995).
In conformation sensitive gel electrophoresis (CSGE), mildly denaturing agents are
used to enhance the conformational changes in the heteroduplexes. Sensitivity is also
improved by using 1,4-bis(acryloyl)piperazine (BAP) instead of the commonly used Bis
as a crosslinker in the polyacrylamide gels. Even though CSGE is a simple method, it has
been reported to detect most mutations in fragments of 200-800 bp. (Ganguly et al. 1993,
Ganguly & Prockop 1995.)
2.7.2. Commonly used methods based on cleavage of a mismatch
A mismatch in a heteroduplex creates a short region of single-stranded DNA, which can
be detected by an enzyme or a chemical that cleaves single-stranded DNA. In enzyme
mismatch cleavage (EMC), bacteriophage T4 endonuclease VII and/or T7 endonuclease I
is used to cleave the site of the mismatch. EMC can be used for fragments of up to 1.5 kb.
The method is relatively sensitive, but some unspecific background can also be observed.
(Youil et al. 1995, Mashal et al.1995.) Chemical mismatch cleavage (CMC) is a two-
35
phase procedure in which the DNA is first treated with hydroxylamine and osmium
tetroxide, which react with mismatched cytosines and thymines, respectively, and
piperidine is then added to cleave the modified mismatched base. Fragments up to 2 kb
can be screened with a sensitivity of 100%, but the toxic reagents are a major drawback.
(Cotton et al. 1988, see also Cotton 1993, Nollau & Wagener 1997.)
2.7.3. Other methods
Carbodiimide (CDI) is a bulky reagent that reacts with thymine and guanine residues.
The CDI modification procedure for mutation detection is based on the observation that
CDI reacts more rapidly with unpaired bases than with paired ones. Electrophoresis,
nuclease cleavage, immuno electron microscopy and primer extension have been used to
detect the modified bases (Novack et al 1986, Thomas et al. 1988, Ganguly et al. 1989,
Ganguly & Prockop 1990). In the primer extension method, which is probably the most
practical of these, CDI blocks primer extension and shorter products can be identified by
electrophoresis. DNA fragments of up to 1 kb can be used, and the location of the
mutation can be estimated to an accuracy of about 15 bp. The advantage of the immuno
electron microscopy method is that a mutation can be detected in a heteroduplex of
several kilobases. (See Cotton 1993.)
Cleavase fragment length polymorphism (CFLP) is based on the secondary structure
changes that a sequence variation generates. Cleavase I endonuclease cleaves the singlestranded DNA at the bifurcated end of a double-stranded region, and thus generates DNA
fragments of different lengths depending on the sequences. (Lyamichev et al 1993, see
also Nollau & Wagener 1997.)
A mismatch binding protein MutS, which is a component of the Eschericia coli DNA
repair system, can also be used to detect mutations in heteroduplexes. Various methods,
including mobility shift assays, biotinylated MutS, solid-phase assay with immobilized
MutS and an optical biosensor have been used to detect MutS binding (Lishanski et al.
1994, Wagner et al. 1995, Geschwind et al. 1996, Gotoh et al 1997). Advantage has also
been taken of the protective feature of the MutS protein against endonucleases for
detection purposes (Ellis et al. 1994).
The protein truncation test (PTT) is specific to mutations causing premature
translation termination. A T7 promoter and a eucaryotic translation initiation sequence
are added to the fragment of interest by PCR, after which the fragment is transcribed and
translated. A premature translation termination codon results in shortening of the
translation product, which can be detected in electrophoresis. (Roest et al. 1993, see also
Nollau & Wagener 1997.)
The most recent methods for mutation detection require special equipment and
computerized analysis. Denaturing high-performance liquid chromatography (DHPLC) is
based on the high resolution separation of heteroduplexes from homoduplexes under
controlled partially denaturing conditions. The method is highly sensitive and the
commercially available procedure (Transgenomic WAVETM System) is fully automated.
(Hayward-Lester et al. 1995, Liu et al. 1997/98, O’Donovan et al. 1998.) Oligonucleotide
microarray (DNA chip) technology will be a promising method for mutation detection in
36
the future. It is based on hybridization of the sample to a large number of immobilized
oligonucleotide microarrays fabricated by advanced manufacturing processes. The
method has an enormous capacity, and many applications have been demonstrated for it.
(See Marshall & Hodgson 1998, Ramsay 1998, Hacia 1999.)
3. Outlines of the present research
Collagen II is the main collagen in cartilage, and mutations in the COL2A1 gene coding
for the collagen II homotrimer are known to cause a spectrum of chondrodysplasias
ranging from lethal ACGII-HCG to early-onset OA associated with mild
chondrodysplasia. In general, a certain phenotype predicts a certain type of mutation in
the COL2A1 gene. Kniest dysplasia, for example, is usually caused by a mutation leading
to a short in-frame deletion in the N terminal half of the triple helix, while a glycine
substitution at the C terminal end of the triple helix often leads to ACGII-HCG.
Collagens IX and XI are minor cartilage collagens which are also expressed in the
intervertebral disc, ocular vitreous body and tectorial membrane of the inner ear. At the
time when this research project was initiated, only one mutation had been reported in
collagen IX. This was in the COL9A2 gene and led to MED. Four mutations in collagen
XI had been reported, two of which were in the COL11A1 gene, one leading to Stickler
syndrome and the other to Marshall syndrome, while two of them were in the COL11A2
gene, one leading to OSMED and the other to Stickler-like syndrome without ocular
involvement.
Based on the collagen II studies it was hypothesized that mutations in collagens IX
and XI would also cause a spectrum of phenotypes. The object of this work was therefore
to increase our knowledge of the role of collagens IX and XI in disorders affecting
cartilaginous tissues. The specific aims were:
1. to improve the sensitivity of conformation sensitive gel electrophoresis (CSGE),
2. to characterize the genomic structure of the human COL11A1 gene and to set up the
CSGE method for the gene,
3. to clarify the relation between the Marshall and Stickler syndromes by analyzing the
COL11A1 and COL2A1 genes in patients with Marshall and Stickler syndromes for
mutations by CSGE and comparing the two syndromes on the basis of the genetic and
clinical data, and
4. to analyze the COL9A2 gene in patients with intervertebral disc disease for mutations
by CSGE.
4. Materials and methods
The materials and methods are described in detail in the original papers I-III.
4.1. Subjects and clinical examination methods (I-III)
Altogether 30 patients with a suspected diagnosis of Marshall or Stickler syndrome were
referred to us from several departments of genetics, ophthalmology and surgery in
Finland, Israel, the Netherlands, Norway, Poland and the USA. The clinical diagnosis
was established by a clinician familiar with this morphology and experienced with these
disorders. Informed consent was obtained from the patients.
157 unrelated Finnish patients with sciatica, defined as unilateral pain of duration over
one month radiating from the back to below the knee, and the family members of four of
the patients were referred to us from Department of Physical Medicine and Rehabilitation
and the Department of Internal Medicine at Oulu University Hospital. The patients were
evaluated clinically and radiologically by experienced clinicians. Altogether 174 Finnish
controls consisting of 101 asymptomatic subjects, 54 patients with OA and 19 patients
with various chondrodysplasias but no history of sciatica were included in the study. The
OA patients were referred to us from the Department of Surgery at Kuopio University
Hospital.
The radiological examinations of the patients with sciatica were performed by
magnetic resonance imaging (MRI) in 156 cases and by computed tomography (CT) in
one case. The MRI scans, obtained with a 1.5-T imaging system (Signa, General Electric,
Milwaukee, Wisconsin), consisted of sagittal images with a TR/TE of 4000/95 ms and
axial images with a TR/TE of 640/14 ms. The CT images (Hi Speed Advantage, GE
Medical Systems, Milwaukee, Wisconsin) consisted of scans through the L2-L3
interspace to the L5-S1 interspace. The radiographs were read by two neuroradiologists
blinded to the results of the clinical and genetic examination and the clinical histories of
the patients. The following radiological findings were considered indications of
intervertebral disc disease: (1) disc extrusion, (2) any herniation at two or more levels, (3)
endplate degeneration at one or more levels in patients under 30 years of age, at two or
more levels in patients aged 30-50 years or at four or more levels in patients over 50
39
years, or (4) bulging and/or protrusion at four or more levels (Jensen et al. 1994,
Weishaupt et al. 1998).
Genomic DNA was extracted from the venous blood or cultured fibroblasts by
standard protocols and used for the analyses.
4.2. Characterization of the COL11A1 gene (II)
To characterize the genomic structure of the COL11A1 gene and obtain intronic
sequences of the gene required for the CSGE analysis, screenings for genomic clones
were ordered from Genome Systems, Inc. PCR probes were designed based on the
published cDNA sequences (Bernard et al. 1988) and the sequences obtained here by
sequencing intronic PCR products amplified with exon specific primers. PCR probes for
the subsequent screenings were designed based on sequences defined from clones
obtained in previous screenings. The probes were from 5’UTR, exon/intron 2, intron 4,
intron4/intron5, exons 27/28, intron 28/intron 29, intron 52/intron 53 and 3’UTR.
Altogether five clones, one P1, two PACs, and two BACs were found, each containing a
part of the gene. The P1 clone was transferred from the Escherichia coli NS3529 strain to
the NS3516 strain, which gives a higher yield, via transduction according to the
instructions provided by Genome Systems. The plasmid DNA preparations were
performed using a standard plasmid isolation protocol recommended by the
manufacturer.
The genomic organization and intronic sequences were defined, either directly from
the clones or from PCR products amplified from the clones, using an automated
sequencer (ABI PRISM 377 Sequencer and dRhodamine or BigDye Termination Cycle
Sequencing Kit with AmpliTaq DNA Polymerase, FS, Perkin-Elmer; Expand Long
Template PCR System, Boehringer Mannheim). For regions that were not included in the
clones, the PCR amplifications for sequencing were performed from genomic DNA. The
sequencing primers were designed based on the cDNA sequences or sequences defined in
this work.
The sizes of the introns up to about 1.5 kb were defined by sequencing, and the sizes
of the other introns were defined by PCR amplification (Expand Long Template PCR
System, Boehringer Mannheim). Unspecific amplification was ruled out either by
sequencing the ends of the PCR products or by using two different sets of PCR primers
for each intron.
4.3. CSGE (I-III)
The PCR products for CSGE analysis were amplified from genomic DNA. The primers
were designed to amplify the target sequence and about 50 bp of both the 5’ and 3’
flanking sequences. The primers were designed initially to generate PCR products of
200-800 bp, as suggested in the previous reports (Ganguly et al. 1993, Ganguly &
Prockop 1995), but during experiments designed to improve the method the size
40
requirement of the PCR product was changed to 200-450 bp. The PCR amplifications
were typically carried out in a reaction volume of 40 µl containing 50-100 ng of genomic
DNA, 200µM of each dNTP, 0.25 µM of each primer and 1 unit of Taq polymerase
(AmpliTaq Gold, Perkin-Elmer). The cycling conditions consisted of initial denaturation
for 10 min at 95°C followed by 30-35 cycles of 95°C for 20-40 sec, 54-60°C for 30-40
sec and 72°C for 20-40 sec, followed by a final extension at 72°C for 4-10 min.
Heteroduplexes were generated at the end of the PCR cycling by denaturing the PCR
products at 95-98°C for 3-5 min and reannealing at 68°C for 30 min. An aliquot of 50100 ng was mixed with loading buffer (10 x stock solution of 30% glycerol - 0.25%
bromphenol blue - 0.25% xylene cyanol FF) and loaded onto a CSGE gel.
CSGE analysis was performed with a 1 mm gel in a standard DNA sequencing
apparatus with 33 x 40 or 37.5 x 45 cm glass plates. The initial gel composition was 10%
of a 1:99 ratio of 1,4-bis(acrolyol)piperazine (BAP; Fluka) to acrylamide (Intermountain
Scientific), 10% ethylene glycol (Sigma), 15% formamide (Gibco), 0.1% ammonium
persulphate (APS; U.S. Biochemicals) and 0.07% N,N,N’,N’-tetramethylethylenediamine
(TEMED; Sigma) in 0.5 xTTE buffer (44mM Tris - 14.5 mM taurine - 0.1 mM EDTA,
pH 9.0), according to the previously published protocol, and the gel was electrophoresed
at 400 V for 16 hours (Ganguly et al. 1993, Ganguly & Prockop 1995). In the course of
experimentation to improve the method, the gel composition was changed to contain 1215% BAP-acrylamide, and the electrophoresis was performed at 40 W for 6 to 8.5 hours
depending on the size of the glass plates. After the electrophoresis, the gel was stained
with ethidiumbromide (1 µg/ml) on a glass plate and a relevant piece was cut out,
transferred onto a UV transilluminator and photographed with a high quality chargecoupled device (CCD) camera (Fotodyne or UVP).
4.4. Screening of the COL11A1 and COL2A1 genes of patients with
Marshall and Stickler syndromes for mutations by CSGE (II)
PCR primers suitable for CSGE analysis of the COL11A1 gene were designed based on
the intronic sequences defined in this work. The entire coding region of the gene,
excluding exons 2 and 4, was amplified in 67 fragments. The CSGE analysis for the
COL2A1 gene was performed by the procedure set up by Körkkö et al. (submitted),
according to the protocol described in section 4.3. After amplification, the PCR products
were analyzed on an agarose gel to check the quantity and quality of the products and to
reveal any large deletions or insertions. The CSGE analysis was then performed using the
improved conditions described in paper I. The samples showing heteroduplexes in CSGE
were subjected to automated sequencing (ABI PRISM 377 Sequencer and dRhodamine
or BigDye Termination Cycle Sequencing Kit with AmpliTaq DNA Polymerase, FS,
Perkin-Elmer) using one of the PCR primers as the sequencing primer. The PCR products
had been treated prior to this with exonuclease I and shrimp alkaline phosphatase to
remove residual PCR primers and nucleotides (Werle et al. 1994, Hanke & Wink 1994).
The physicians of the patients found to have a mutation were asked to provide
complete clinical data to substantiate the diagnosis of either Marshall or Stickler
41
syndrome and to perform a genotypic-phenotypic comparison. All available family
members of such patients were also analyzed for the presence of the mutation.
4.5. RT-PCR (II)
RT-PCR analysis was performed on a patient, who had a sequence variation at the +3
position in intron 50 of the COL11A1 gene to study the effect of the variation in splicing.
Total RNA was extracted from cultured skin fibroblasts (RNeasy Midi Kit, Qiagen) and
used as a template for first strand synthesis (Superscript Preamplification System, Gibco
BRL), which was performed with the oligo(T) primer under the conditions suggested by
the manufacturer for transcripts with a high GC content. The subsequent PCR
amplifications of the illegitimate transcripts were performed using α1(XI) cDNA specific
primers, and the products obtained were sequenced.
4.6. Screening of patients with sciatica for mutations in the COL9A2
gene (III)
The patients with sciatica were screened for mutations in the COL9A2 gene using PCR
primers suitable for CSGE analysis designed on the basis of previously defined sequences
(Pihlajamaa et al. 1998a). Initially, a subset of patients with sciatica were screened for
mutations in the COL9A2 gene by the method described in section 4.3. After a unique
sequence variation in exon 19 had been found in one of these patients, exon 19 was
amplified in the samples from all the other patients and controls using the PCR primers
5’-TGG ATC TCA GTT TCC CTA CCT G (-92 to -71 in intron 18) and 5’-CAA GAG
GTG GTG ATT GAG CAA GAG C (+99 to +75 in intron 19). The PCR products were
analyzed for sequence variation by CSGE and sequencing (ABI PRISM 377 Sequencer
and dRhodamine or BigDye Termination Cycle Sequencing Kit with AmpliTaq DNA
Polymerase, FS, Perkin-Elmer), and some of the PCR products were cloned prior to
sequencing (T7 Sequencing Kit, Pharmacia).
The family members of four patients who were found to have the sequence variation
in exon 19 were evaluated clinically and radiologically, and genetic analysis for the
presence of the mutation was performed to study the co-segregation of the genotype with
the phenotype.
4.7. Statistical analysis (III)
The statistical analysis to evaluate the significance of the sequence variation was
performed by Harald H. H. Göring at the Department of Genetics and Development of
Columbia University, New York, USA, using the ILINK program of the FASTLINK
computer package.
42
According to the clinical and radiological examinations, pedigree members of the four
families who tested positive for the disease in either the clinical or the radiological test
were coded as affected, and those testing negative in both tests were coded as unaffected.
Individuals who were not examined or examined only clinically or radiologically and
found to be normal were coded as being of unknown phenotypic status. In addition,
artificial pedigrees were created from the case-control data to enable linkage and linkage
disequilibrium analyses to be performed jointly on the pedigrees and singletons by coding
two case or control individuals as the parents of a hypothetical offspring of unknown
phenotype (Kainulainen et al. 1999).
5. Results
5.1. Optimization of CSGE (I)
To test and improve the sensitivity of CSGE for mutation detection, genes coding for
collagen α chains were selected for examination. Collagen genes are challenging targets
for mutation detection because they are complex, consisting of large numbers of exons,
and the sequences are repetitive and GC-rich due to the numerous glycine and proline
residues. Previously known sequence variations in the COL1A1, COL1A2, COL2A1,
COL3A1, COL9A1 and COL9A2 genes were used as references in the optimization. In
order to improve the sensitivity of CSGE, the lengths of the PCR products used for the
analyses, the electrophoresis conditions and the acrylamide concentrations in the gel were
altered to test the sensitivity under different conditions.
5.1.1. Improved conditions for mutation detection by CSGE (I)
The original report on CSGE suggested that the procedure could be applied to PCR
products of size 200-800 bp (Ganguly et al. 1993). However, sequence variations were
detected less frequently in PCR products of 500-800 bp than in shorter products, and a
common polymorphism in exon 25 of the COL1A2 gene (Constantinou et al. 1990) was
not detected in a PCR product of 755 bp whereas it was clearly detected when the region
containing the polymorphism was amplified to generate a PCR product of less than 300
bp (Fig. 1 in paper I). Some other PCR products of over 500 bp were also found to
contain polymorphisms using shorter PCR products (data not shown). This finding led to
a suggestion that only PCR products of up to 450 bp should be used to ensure the
sensitivity of the method.
The electrophoretic conditions were altered to test whether the detection of poorly
detectable sequence variations could be improved and to speed up the procedure. The
electrophoresis was performed with 40 W for 6 hours instead of a overnight run with 400
V. The separation of heteroduplexes on the gel was improved under the altered
electrophoretic conditions in several cases (see Fig. 2A and 2B in paper I), and a specific
44
test with 48 heteroduplexes detected previously under the original conditions did not
indicate any disappearance of these (data not shown). Furthermore, a few previously
undetected sequence variations became detectable with the new electrophoretic
conditions (data not shown).
In addition, the BAP-acrylamide concentration in the gel was increased from 10% to
15% to see if it would improve the separation of the heteroduplex bands in CSGE. With
the higher concentration of the polymer the running time was increased to 8.5 hours. This
improved the detection of a single base polymorphism in intron 11 of the COL1A1 gene,
which was previously poorly detected (Fig. 2C and 2D in paper I). To test these
alterations further, over 200 single base pair changes that had previously been detected
using 10% polyacrylamide gels and electrophoretic conditions of 400 V overnight or 40
W for 6 hours were analyzed using 15% polyacrylamide gels with running conditions of
40 W for 8.5 hours. The separation was found to be better under the new conditions.
5.1.2. Testing the sensitivity and specificity of CSGE
Altogether 76 previously identified single base changes detected by nucleotide
sequencing of the COL1A1 gene (Körkkö et al. 1998), DGGE analysis of the COL1A1
and COL2A1 genes (Ritvaniemi et al. 1993, Ritvaniemi et al. 1995, Körkkö et al. 1997),
or sequencing of the cDNAs for α chains of collagens I and III (see Ganguly et al. 1993)
were used to test the sensitivity of CSGE. All the changes except one, which was not
consistently revealed, were detected using the improved CSGE procedure (Table 1 in
paper I).
The specificity of the method was tested by sequencing 223 PCR products that
generated heteroduplexes in CSGE. In addition, over 200 PCR products that did not
generate heteroduplexes in CSGE were sequenced as controls. All the products that
generated a heteroduplex yielded at least one sequence variation, while no sequence
variations were found in the controls (Table 2 in paper I). These results indicate that
CSGE does not generate false-positive or false-negative results to any significant extent.
A sequence variation in the PCR product resulted in a unique heteroduplex band that
was usually readily distinguishable from other possible sequence variations or their
combinations in the product (Fig. 6 in paper I). Only rarely were heteroduplex bands
observed that looked similar but were generated by different sequence variations in the
PCR product (data not shown).
5.2. Characterization of the human COL11A1 gene (II)
All three genes coding for collagen IX α chains have been characterized (Pihlajamaa et
al. 1998a, Paassilta et al. 1999b) as have the COL11A2 and COL2A1 genes, which code
for the α2(XI) and α3(XI) chains of collagen IX (Ala-Kokko et al. 1995, Vuoristo et al.
1995). For the COL11A1 gene, which codes for the α1(XI) chain, only the cDNA
sequence and a few exon-intron boundaries had previously been known (Bernard et al.
45
1988, Zhidkova et al. 1995). To complete the information on the genomic structures of
the genes coding for collagen XI and to set up a mutation screening method for this gene,
the human COL11A1 gene was characterized here.
5.2.1. Characterization of clones for the COL11A1 gene
Altogether five clones for the human COL11A1 gene, one P1, two PAC and two BAC
clones, were obtained from the screenings ordered from Genome Systems, Inc. The
locations of the introns and the intronic sequences flanking the exons were defined by
sequencing with exon-specific primers and primers designed on the basis of the intronic
sequences defined here. The exons were numbered in a manner corresponding to the
numbering of exons in the COL11A2 gene (Vuoristo et al. 1995). The P1 clone (address
DMPC-HFF#1-140(B10)) was found to contain the sequences from intron 4 to intron 29,
both of the PAC clones (addresses PAC-84:8A and PAC-154-1M) contained the
sequences from intron 4 to intron 54, one of the BAC clones (address 170O21) contained
the sequences from intron 4 to intron 63, and the other BAC clone (address BACH-86N5)
contained the sequences from an undefined distance from the 5’-end of the gene to intron
1 (Fig. 1 in paper II).
5.2.2. Genomic structure of the human COL11A1 gene
Although PCR probes for screening of the P1, PAC and BAC libraries were used from
exon/intron 2, intron 4, and 3’-UTR, no clones containing the sequences from intron 1 to
intron 4 or sequences in the 3’-direction of intron 63 were found. Most of the structure
and sequences of these regions were defined by sequencing PCR products amplified from
genomic DNA, but several attempts to amplify introns 1 and 4 failed and thus their size
remained undefined.
The gene was found to consist of 68 exons (Fig. 1 in paper II, Table 1 in paper II),
which were numbered from 1 to 67, using the numbers 6A and 6B for the sixth and
seventh exons (previously called IIA and IIB), because they are alternatively spliced and
do not exist in the same mRNA (Zhidkova et al. 1995). The exon numbers 9-15
previously used by Bernard et al. (1988) correspond to exons 16-22 in this numbering.
The locations of the exon-intron boundaries in the major triple helical region of the
COL11A1 gene were exactly the same as those in the COL11A2 gene (Table 1 in paper
II, Vuoristo et al 1995).
The sizes of the introns were defined by sequencing or PCR amplification. The size of
the gene, excluding introns 1 and 4, was about 160 kb (Table 1 in paper II). The intronic
sequences flanking the exons were sequenced and over 50 kb of new sequences were
defined for the gene. The sequences were submitted to GenBank (accession numbers
AF101079-AF101112).
46
5.2.3. Resequencing of the coding region
Resequencing of the coding region indicated some differences relative to the published
cDNA sequence (Bernard et al. 1988). No cysteinyl residue was found in the triple
helical region, and the amino acid sequence at positions 413-416, calculated from the first
glycine of the main triple helical domain, was Lys-Asp-Gly-Leu instead of the previously
reported Arg-Met-Gly-Cys. In addition, the amino acid at position 690 was methionine
instead of tryptophan, an amino acid rarely found in collagen triple helices. These
sequences were defined from 150 alleles.
5.3. Identification of mutations in the COL11A1 and COL2A1 genes
in patients with Marshall or Stickler syndrome (II)
The CSGE method was set up for the COL11A1 gene based on the sequences defined
here and a corresponding analysis for the COL2A1 gene was performed by the procedure
set up by Körkkö et al. (submitted). A group of 30 patients who had been referred to us
with a suspected diagnosis of Marshall or Stickler syndrome were screened for mutations
in these two genes. Altogether 23 mutations were identified, fifteen in the COL11A1
gene and eight in the COL2A1 gene.
5.3.1. Identification of mutations in the COL11A1 and COL2A1 genes
by CSGE
All exons and exon boundaries except those of exons 2 and 4 of the COL11A1 gene were
amplified by PCR and heteroduplexes were generated. The PCR products were analyzed
on an agarose gel to check their quantity and quality and reveal any large insertions or
deletions. The agarose gel analysis of the products for exon 53 suggested a heterozygous
deletion of about 150 bp in one patient. The sequencing indicated a 162 bp deletion that
contained 85 bp of intron 52, exon 53, and 23 bp of intron 53. CSGE analysis was
performed on the rest of the patients, and several unique heteroduplexes were observed.
Sequencing identified ten mutations that altered the splicing consensus sequences, two
small in-frame deletions in the coding sequences, and two glycine substitutions (Table 2
in paper II).
A similar procedure was performed to analyze the COL2A1 gene in the patients for
whom no mutations were found in the COL11A1 gene, and altogether eight novel
mutations were found by CSGE analysis and sequencing. Six of these caused a frameshift leading to premature translation termination codons, and two of them altered the
splicing consensus sequences (Table 2 in paper II). In the familial cases the mutation was
found to cosegregate with the phenotype (data not shown).
All except two of the mutations altering the splicing consensus sequences affected the
conventional AG-dinucleotide at the acceptor splice site or the GT-dinucleotide at the
donor splice site. One of the remaining two was an A to C change and the other was an
47
insertion of T at position +3 in intron 50 of the COL11A1 gene. To study the effect of the
A to C change on splicing, RT-PCR analysis was performed for illegitimate mRNA
transcripts extracted from the patient’s cultured skin fibroblasts. Two PCR products were
obtained, one corresponding to the wild type cDNA sequence and one lacking the
sequences for exon 50. No RNA was available from the patient with the insertion, but
DNA was obtained from the parents, who were unaffected, and analyzed for the presence
of the insertion. Neither parent had the insertion (data not shown).
In the familial cases those family members who were available were examined for the
presence of the mutation and for clinical symptoms. The mutation was found to
cosegregate with the phenotype (data not shown).
5.3.2. Genotypic-phenotypic comparison
All the COL11A1 mutations were found in the region coding for the major triple-helical
domain. Ten out of fifteen mutations altered the splicing consensus sequences for 54 bp
exons, and one genomic deletion led to the loss of a 54 bp exon. Eight of these mutations
affected exons 48-54, four of them exon 50. The analysis also identified two in-frame
deletions of 18 bp and 9 bp in exons 36 and 52, respectively. In addition, two glycine
substitution mutations were found (Fig. 1, table 2 in paper II).
Fig. 1. Schematic drawing of the COL11A1 transcript and the locations of the mutations. The
major triple helix is left blank and all the other regions are shaded. The areas corresponding
to the exons are separated by the horizontal lanes and are drawn to scale. The locations of all
the mutations found in the COL11A1 gene are marked with the identification number of the
corresponding patient (Table 2 in paper II). The mutations marked above the drawing are
splicing mutations in the C terminal half of the molecule (1 to 8 and 10) and a genomic
deletion including a 54 bp exon (9). Those marked below the drawing are glycine substitution
mutations (12 and 15), small in-frame deletions (13 and 14), and a splicing mutation in the N
terminal end of the triple helix (11).
48
The patients who were found to have a mutation, were evaluated clinically, with
particular emphasis on the major findings characteristic of the Stickler and Marshall
syndromes, in order to evaluate the phenotypic consequences of the COL11A1 and
COL2A1 mutations. Particular attention was paid to the characteristics reported to differ
between these syndromes. It has been suggested that the syndromes differ in that patients
with Marshall syndrome are more often of short stature, deaf and have abnormalities in
cranial ossification and more pronounced dysmorphic features, including a retracted
midface with flat nasal bridge, short nose, anteverted nostrils and a long philtrum.
Furthermore, it has been suggested that retinal detachment occurs less frequently in
patients with Marshall syndrome. (O’Donnell et al. 1976, Aymé & Preuss 1984, Stratton
et al. 1997.)
In general, the phenotypes in the COL11A1 and COL2A1 genes resembled each other
(Table 1, table 3 in paper II). The clinical findings in the patients who had a mutation in
either of the genes typically consisted of high myopia, midfacial hypoplasia, palatal
defects and retro/micrognathia.
There were some major differences, however. The vast majority of patients with
COL11A1 mutations had moderate to severe hearing impairment that was congenital or
detected in early childhood, whereas the patients with COL2A1 mutations had normal
hearing or minor hearing loss that usually developed later in life. The ocular findings
were generally more severe in the patients with COL2A1 mutations than in those with
COL11A1 mutations, in that almost all of the former had vitreoretinal degeneration and
retinal detachment. Furthermore, cataracts were more common in patients with COL2A1
mutations than in the patients with COL11A1 mutations. Slight differences were also
seen in stature and facial characteristics (Table 3 in paper II). Midfacial hypoplasia, a
short nose and a flat nasal bridge were more clearly pronounced in Marshall syndrome,
and did not disappear when the child grew older (Fig. 2 in paper II). Radiographic
skeletal evaluations could be performed only on a few patients, as radiographs were
available only for those with skeletal complaints. Osteoarthritic changes seemed to be
more common in the cases with COL2A1 mutations, but many of the patients were too
young to be evaluated for such changes. Cranial radiographs were available only for six
cases with COL11A1 mutations and two with COL2A1 mutations. None of the latter had
cranial abnormalities, but four of the former group had abnormalities such as abnormal
frontal sinuses, intracranial ossifications and/or a thickened calvarium.
The patients with COL11A1 and COL2A1 mutations could be divided into three
groups based on the phenotype and mutational type (Table 1). The first group had a
splicing mutation of a 54 bp exon or a genomic deletion of a 54 bp exon in the COL11A1
gene region coding for the C terminal half of the α1(XI) molecule, and their
characteristics resembled those reported in Marshall syndrome (Aymé & Preuss 1984,
Shanske et al. 1997, Griffith et al. 1998). The second group had other mutations in the
COL11A1 gene and phenotypes overlapping both the Marshall and Stickler syndromes,
while the third group had a mutation in the COL2A1 gene and a phenotype that closely
resembled the classical Stickler syndrome (Stickler et al. 1965, Stickler & Pugh 1967,
Herrmann et al. 1975).
49
Table 1. Summary of the clinical data.
Findings
COL11A1 mutations
patients 1-10
a
patients 11-15
COL2A1 mutations
b
patients 16-23
moderate to severe hearing loss
10/10
4/5
0c/7
ocular findings
high myopia
retinal detachment
vitreoretinal degeneration
cataract
10/10
1/10
3/9
4/10
4/5
2/5
3/5
3/5
8/8
6/8
5/7
5/7
facial features
short nose
anteverted nares
midface hypoplasia
flat nasal bridge
palate defect
micro/retrognathia
long philtrum
epicanthus
hypertelorism
lowered auricles
10/10
10/10
10/10
10/10
8/10
8/10
7/9
7/9
6/10
4/8
4/5
4/5
4/5
4/5
4/5
3/5
3/5
2/5
0/4
0/3
5/7
3/6
7/7
6/7
8/8
4/6
4/6
3/5
2/7
1/4
stature
short
tall
6/10
0/10
3/5
1/5
2/7
2/7
cranial findings
abnormal frontal sinuses
intracranial ossifications
thick calvarium
2/3
1/3
2/4
1/1
1/2
1/2
0/2
0/2
0/2
dental abnormalities
1/9
0/2
1/3
a
b
c
splicing mutation of a 54 bp exon or genomic deletion causing a loss of 54 bp in exons
coding for the C terminal half of the α1(XI) molecule
other mutations in the COL11A1 gene
3 of the cases had a minor hearing deficit
50
5.4. Identification of an allele of the COL9A2 gene associated with
intervertebral disc disease (III)
5.4.1. Identification of a tryptophan for glutamine/arginine substitution
in the COL9A2 gene
The CSGE method for the COL9A2 gene was set up based on the published sequences
(Pihlajamaa et al. 1998a), and ten patients with intervertebral disc disease were initially
analyzed for mutations in the gene. A unique CSGE pattern was found in the PCR
product for exon 19 in one of the patients (Fig. 1 in paper III), and sequencing identified
a heterozygous substitution of Trp (TGG) for Gln (CAG) at amino acid position 326.
Analysis of exon 19 in all 157 patients with intervertebral disc disease indicated that six
of them had the Trp substitution. The analysis was also performed on the 174 controls,
who consisted of 101 asymptomatic individuals, 54 with OA and 19 with various
chondrodysplasias but no history of sciatica. None of the controls had the Trp
substitution. Sequencing of exon 19 also indicated that Arg (CGG) was found relatively
often at position 326, suggesting that the Trp allele had arisen from the Arg allele by a
single base substitution (Fig. 2 in paper III). The frequency of the Arg substitution was
about the same in the patient group and all of the control groups, indicating that it was a
neutral polymorphism (Table 1 in paper III).
5.4.2. Co-inheritance of the Trp allele and intervertebral disc disease
To study whether the Trp allele is co-inherited with intervertebral disc disease, the
families of four patients with this allele were included in the study. The two other
families were not available for examination. The families were evaluated clinically and
by CT (family A) or MRI (families B, C and D), and analyzed for the presence of the Trp
allele. As stated in the Materials and methods section, the individuals were considered
affected if they had either clinical or radiological evidence of the disease, or both. All the
family members who had inherited the allele had intervertebral disc disease (Fig. 2).
5.4.3. Statistical analysis
Linkage analysis and linkage disequilibrium analysis were performed to evaluate the
statistical evidence for a connection between the Trp allele in the COL9A2 gene and
intervertebral disc disease. Since the aetiology of the disease is multifactorial (Heliövaara
1989), a high phenocopy rate was specified. In the selected model the disease locus
accounts for 10% of cases, in other words, 90% of patients with intervertebral disc
disease are affected for reasons unrelated to this particular disease locus. A fully
penetrant dominant disease model was chosen for the analysis and the disease prevalence
51
Fig. 2. The pedigrees of the four families studied. The right half of each symbol indicates the
clinical findings, and the left half the radiological findings. The symbol is black when the
finding is positive, and a dot indicates that the family member was examined and found to be
normal. A blank symbol indicates that the individual was not examined. The proband is
marked with an arrow. The family members with the Trp allele are indicated by (+), and the
members who did not have the allele are indicated by (-).
52
was set at 4.8%, which is the estimated frequency of sciatica in Finland (Heliövaara et al.
1987).
Artificial pedigrees were created from the case-control data to enable a linkage and
linkage disequilibrium analysis to be carried out jointly on the pedigrees and singletons
(Kainulainen et al. 1999). Linkage disequilibrium analysis gave a lod score of 4.5 at a
recombination fraction of 0.12. Subsequent linkage disequilibrium given the presence of
linkage was analyzed, and an additional lod score of 7.1 was obtained. The joint lod score
was therefore 11.6 (Table 2 in paper III). A similar analysis was performed with the
recombination fraction fixed at 0.0 (Table 3 in paper III). The lod scores were lower, but
that does not necessarily contradict the hypothesis that the Trp allele itself caused the
disease. The explanation is rather the inaccuracy of the model used. For common diseases
such as intervertebral disc disease it is practically impossible to specify a model with
100% accuracy, and thus reduced lod scores and overestimated recombination fractions
are obtained as a result of model inaccuracies. (Clerget-Darpoux et al. 1986, Risch &
Giuffra 1992, see also Terwilliger & Ott 1994.)
To examine the extent to which the results were dependent on the assumed disease
model, individual parameters of the model were varied keeping the predicted population
prevalence unchanged at 4.8%. The linkage and linkage disequilibrium lod scores
remained high for a dominant model when the proportion of cases explained by the
disease locus was varied and when high but incomplete penetrance was used (Table 2 in
paper III).
5.4.4. Scanning the entire COL9A2 gene for mutations in patients with
the Trp allele
To determine whether there were other sequence variations in the COL9A2 gene among
the patients whose disease was linked to this gene, all the exons and exon boundaries
were analyzed by CSGE. The analysis did not identify any other possible disease causing
mutation of coding sequences or RNA splice sites, but it did identify two additional
neutral polymorphisms, an A to G change at the third nucleotide in the codon for proline
at nucleotide 9 in exon 21 and a G to A change at nucleotide +17 in intron 30. These were
analyzed in 90 and 286 individuals, respectively, but were not found in any individuals
other than those with the Trp allele.
6. Discussion
6.1. Detection of mutations by CSGE
The detection of unknown mutations in one or more candidate genes is a challenging
task, especially with large, complex genes. Sequencing would be a thorough method, but
it is too laborious and expensive. Numerous PCR-based screening methods, each having
typical advantages and disadvantages, are available for mutation detection (see Cotton
1989, Cotton 1993, Eng & Vijg 1997, Nollau & Wagener 1997), but typical drawbacks
are the radioactive or toxic chemicals required for the procedure and problems of
sensitivity or specificity. Screening a large number of samples in complex genes requires
an easy, powerful method that maintains sensitivity in all sequence contexts but does not
require separate optimization for different PCR products. In addition to being large in
size and containing dozens of exons, collagen genes are problematic for mutation
detection due to their repetitive sequences and high content of G and C residues. This is a
consequence of the obligatory sequence for collagens, which consists of a glycine residue
as every third amino acid, which is coded by a codon that always starts with two G
residues, and has a high content of proline residues, which are coded by codons that
always start with two C residues.
CSGE is a relatively recent method that has many advantages, including nonradioactivity, a standard procedure for all fragments, large capacity and the use of
standard equipment. Furthermore, it has been reported to have relatively high sensitivity,
detecting 60 out of 63 single base pair changes in PCR fragments of 200-800 bp.
(Ganguly et al. 1993, Ganguly & Prockop 1995.) No reports on its sensitivity have been
published, however, since the initial one by Ganguly et al. (1993).
To study CSGE further, modifications of the procedure were developed and tested
with large number of known sequence variations, including the three changes that were
not detected in the initial report. The procedure was improved by using shorter PCR
products, a higher polymer concentration in the gel and more powerful electrophoretic
conditions. To test the sensitivity of the improved method, 76 previously identified
sequence variations were tested and all the changes except one, which was not
consistently identified, were detected. Furthermore, 223 samples with unique CSGE
patterns, along with over 200 PCR products with only homoduplexes on the gel, were
54
sequenced to test the specificity of the procedure. All of the samples with heteroduplexes
on CSGE and none of the samples with only homoduplexes had sequence variations. This
indicates that the sensitivity and specificity of CSGE under the improved conditions are
close to 100%.
In addition to the high sensitivity and specificity, CSGE proved to be a simple and
practical method. No special optimization is required, which allows different fragments
to be analyzed simultaneously. The generation of heteroduplexes, which can be added to
the end of the PCR program, is the only special treatment required for the samples.
Standard sequencing apparatus is used for the electrophoresis, and the loading of multiple
samples of different sizes into the same lane provides for a high capacity. Unique CSGE
patterns are usually seen for the different sequence variations on the gel. This makes it
easy to avoid sequencing the same neutral polymorphisms in all the samples carrying it,
and thus reduces the cost of the analysis and the work required.
Although CSGE proved to be a highly sensitive and specific method, it has the same
drawbacks as the other PCR-based procedures that make use of the formation of
heteroduplexes. The alleles with a deletion or some other sequence variation at the PCR
primer binding site of the fragment to be analyzed may not be amplified and thus will not
be detected by CSGE, while insertions between the PCR primer sites may lengthen the
fragment to such an extent that the allele is not amplified. Furthermore, equal
amplification of both a normal and mutant allele cannot be ensured, and thus unequal
amplification due to a low amount of the mutant allele in the PCR reaction, or its
absence, can cause false negatives in the analysis. In addition, as the formation of
heteroduplexes is based on the presence of two alleles with different sequences at the site
of the mutation, the mutation will remain undetected if it is present in both alleles, as in
the case of a homozygous mutation in a recessive disease. This can be overcome by
mixing the patient sample with a control sample.
6.2. Genomic structure of the human COL11A1 gene
Characterization of the human COL11A1 gene in P1, PAC and BAC clones and in
genomic DNA indicated that the gene consists of 68 exons and spans at least 160 kb. The
sizes for introns 1 and 4 remained undefined because no clones containing these
sequences were found and attempts to amplify them by PCR failed. The exon-intron
organization of the major triple helical region of the COL11A1 gene was identical to that
of the COL11A2 gene, indicating a common evolution for these genes differing from that
of the genes for the major fibrillar collagens, which have different numbers and sizes of
exons (Vuorio & de Crombrugghe 1990, Vuoristo et al. 1995). The COL11A1 and
COL11A2 genes are completely different in size, however, the former being over 160 kb
and the latter only 28 kb. The COL11A1 gene resembles COL5A1 in size, the latter being
about 150 kb excluding the first intron, the size of which has not been defined (Takahara
et al. 1995). These two genes are also identical in the exon-intron organization of the
major triple helical region, except for one area in which there are two exons of 54 bp in
the COL11A1 gene corresponding to one exon of 108 bp in the COL5A1 gene.
Furthermore, the exon-intron organization of the C propeptides of these genes is
55
identical. These findings support the previous assumption of a close relation between the
α chains of collagens V and XI. The size of intron 1 of the COL5A1 gene has been
thought to be at least 600 kb, and that of intron 4 is 26 kb (Takahara et al. 1995). This
suggests that the failure to amplify the sequences of introns 1 and 4 of the COL11A1
gene by PCR was caused by their large size.
6.3. COL11A1 and COL2A1 gene mutations in patients with Marshall
or Stickler syndrome
Two mutations in the COL11A1 gene have been identified, one in Marshall syndrome
and the other in Stickler syndrome (Richards et al. 1996, Griffith et al. 1998). The
COL11A1 gene is the only locus for Marshall syndrome identified thus far, but Stickler
syndrome has also been shown previously to be caused by mutations leading to a
premature translation termination in the COL2A1 gene (see Kuivaniemi et al. 1997). A
clinical debate on the identity and diagnosis of Marshall and Stickler syndromes has been
going on for decades (O’Donnell et al. 1976, Baraitser 1982, Winter et al. 1983, Aymé &
Preus 1984, Stratton et al. 1991, Shanske et al. 1997, Shanske et al. 1998, Warman et al.
1998). To study the issue by a genetic approach, the COL11A1 and COL2A1 genes in
patients with Marshall or Stickler syndrome were screened for mutations by CSGE.
Altogether 23 mutations were identified, fifteen in the COL11A1 gene and eight in the
COL2A1 gene. The majority of those in the COL11A1 gene altered the splicing
consensus sequences, all of them affecting the 54 bp exons, as in the previously reported
case (Griffith et al. 1998). Furthermore, one patient had a genomic deletion resulting in
the loss of a 54 bp exon. Ten out of these eleven mutations were in a region spanning
exons 38 to 54 of the gene. Although more than one third of the exons in this region are
90 or 108 bp in size, no splicing mutations or deletions affecting them were found. The
only splicing mutation in the N terminal part of the α1(XI) triple helical region caused a
very different phenotype from the other splicing mutations, one in which only severe
ocular symptoms and shortness occurred, with no hearing deficit or facial features other
than the high arched palate typical of the Stickler and Marshall syndromes.
Six of the COL2A1 mutations resulted in a premature translation termination codon,
and two altered the splicing consensus sequences. Fairly good correlation has been
observed between the mutation type in the COL2A1 gene and the phenotype it causes
(see Kuivaniemi et al. 1997). The two patients who had a splicing mutation had features
typical of Stickler syndrome, with no signs of more severe Kniest dysplasia or SED,
which are known to be consequences of splicing mutations leading to in-frame deletions
in the COL2A1 gene. It is therefore likely that the mutations in the splicing consensus
sequences of these patients lead to cryptic splice sites, and thus to premature translation
termination codons, as was also reported in the original kindred described by Dr. Stickler
(Williams et al. 1996).
The finding that mutations in the COL11A1 and COL2A1 genes lead to similar
phenotypes is not surprising. Types II and XI are both fibril-forming collagens and form
components of the same fibril in cartilage. Furthermore, mutations in the COL11A2 gene,
which codes for the α2(XI) chain, have been reported to cause homozygous and
56
heterozygous OSMED, which has features similar to the Stickler and Marshall syndromes
(see Spranger 1998). The phenotypes caused by COL11A2 mutations nervertheless lack
ocular involvement, because the α2(V) chain rather than the α2(XI) chain is expressed in
the vitreous body (Mayne et al. 1993). Also, the correlation of the mutational types with
the severity of the phenotypes they cause fits well with the amounts of these collagen
types in the fibrils. Mutations leading to defective collagen molecules are known to cause
more severe phenotypes than mutations causing haploinsufficiency (Kuivaniemi et al.
1997). In the cases described here, the mutations leading to a premature translation
termination codon in a quantitatively more significant molecule in the fibrils generated a
similar phenotype to the mutations causing expression of defective molecules of a
quantitatively less significant fibril component.
The present results indicate that patients with a COL11A1 mutation, especially those
who had a splicing mutation or genomic deletion of a 54 bp exon in the C terminal half of
the α1(XI) molecule, had findings related to Marshall syndrome, while the patients with a
COL2A1 mutation had a more classical Stickler phenotype. The genotypic-phenotypic
correlation detected here supports the old clinical suspicion of two separate entities.
Other mutations in the COL11A1 gene, however, resulted in phenotypes overlapping
both the Marshall and Stickler syndromes, and it is these that possibly explain the
conflicting reports as to whether Stickler and Marshall syndromes are separate entities or
not.
The clinical distinction between these phenotypes provides important information on
the prognosis for the patient’s symptoms. Furthermore, if an analysis of the genetic defect
causing the phenotype is desired, the clinical distinction helps in choosing the right gene
for this analysis. The hearing deficit is probably the most outstanding feature
distinguishing between the phenotypes caused by mutations in the COL11A1 and
COL2A1 genes. Fourteen out of fifteen patients with a COL11A1 mutation had earlyonset hearing loss and required hearing aids, but the patients with a COL2A1 mutation
had normal hearing or only a mild hearing defect. Furthermore, ocular involvement was
less severe in the patients with COL11A1 mutations, although there were several
exceptions. The classification of the ocular symptoms suggested by Snead et al. (1994)
could not be performed, because the type of vitreal abnormality was not defined here.
Another interesting means of distinction could be the evaluation of facial characteristics.
The patients with Marshall syndrome were found to have more clearly pronounced
midfacial hypoplasia, a short nose and a flat nasal bridge. These features did not
disappear when the child grew older, as can be observed in individuals with Stickler
syndrome (Temple 1989, H. Kääriäinen, personal communication).
Cranial abnormalities including abnormal frontal sinuses, intracranial ossifications
and/or a thickened calvarium have been described in Marshall syndrome (Marshall 1958,
Aymé & Preuss 1984, O’Donnell et al. 1976, Griffith et al. 1998), and although cranial
radiographs were available for only a third of the present patients, the findings supported
the suggestion that a mutation in the COL11A1 gene may cause abnormalities in cranial
ossification. An attractive explanation for this would be the presence of high levels of
mRNA for the COL11A1 gene in the calvaria and brain but only low amounts of the
mRNAs for the COL11A2 and COL2A1 genes, as observed by Lui et al. (1995).
57
6.4. The association of a COL9A2 allele with intervertebral disc
disease
Six out of 157 patients with intervertebral disc disease carried the Trp for Gln/Arg
substitution in the central collagenous domain of the α2(IX) chain, while none of the 174
controls did so. Furthermore, the substitution co-segregated with the phenotype in the
families studied. The statistical analyses provided extremely strong evidence that the Trp
allele was associated with intervertebral disc disease.
Even though the Trp allele of the COL9A2 gene was tightly linked to the phenotype,
the results do not prove a direct causal role for it in the aetiology of the disease, as it is
possible that some other gene in close proximity to the COL9A2 gene that is in linkage
disequilibrium with the Trp allele is the true disease locus. A role for collagen IX
mutations in intervertebral disc disease has nevertheless been suggested in experiments
with transgenic mice, where long-term follow-up of the mice expressing a mutant Col9a1
gene showed intervertebral disc degeneration and herniations (Kimura et al. 1996). On
the other hand, the only known disease-causing collagen IX mutations in humans lead to
MED, which is characterized primarily by articular complaints. However, only two
mutations in the genes coding for collagen IX that cause MED have been reported, one in
the COL9A2 gene and one in the COL9A3 gene, and both lead to similar deletions of 12
amino acids at exactly same location in the COL3 domain of the molecule, suggesting the
importance of this domain for the pathogenesis of MED (Muragaki et al. 1996, Paassilta
et al. 1999a). This does not contradict the present results, as it is well known that
different mutations in the same collagen gene may lead to very different phenotypes. A
mutation in the COL2A1 gene, for example, may lead to a cartilage disorder without
ocular involvement, while another mutation in the same gene leads to a disorder with
only ocular manifestations (Ala-Kokko et al. 1990, Körkkö et al. 1993). Furthermore, a 9
bp deletion in the region coding for the COL1 domain of collagen IX that represents a
neutral variant without any phenotypic consequences has been identified in the COL9A3
gene (Paassilta et al. 1999b). Thus it would not be surprising for a splicing mutation in
the COL3 domain and a Trp substitution in the COL2 domain of the collagen IX
molecule to lead to different phenotypes.
The Trp substitution was the only putative disease-causing sequence variation in the
COL9A2 gene among the patients whose phenotype was linked to the gene. Even though
there might be another such mutation in an intron or in the promoter region of the gene,
or alternatively, the Trp substitution or the presumably neutral polymorphisms detected in
the gene might cause aberrant splicing, there are several arguments to suggest that the
Trp substitution itself is the cause of the disease. Trp is rarely found in the triple helices
of collagens, and there are no Trp residues in any of the collagenous domains of human
or mouse collagen IX (Muragaki et al. 1990, Perälä et al. 1993, Rokos et al. 1994, Perälä
et al. 1994, Brewton et al. 1995), which suggests that it fits poorly into the collagen triple
helix. Computerized molecular modelling of the sequence with Trp and the normal
sequence indicated that the substitution alters the conformation of the helix (A. Fertala,
unpublished data). It could also interfere with the covalent cross-link between the
collagen II and collagen IX molecules. In the triple helical heterotrimer, the Lys residue
in the α3(IX) chain that is involved in the cross-link is located only three amino acids
away from the Trp substitution in the α2(XI) chain.
58
Studies on disc tissue from a patient carrying the Trp allele could perhaps reveal the
mechanism by which the Trp causes the disease, but disc tissue from such patients is
difficult to obtain for this purpose, at least in sufficient amounts. Preliminary results of in
vitro studies performed with normal and Trp-carrying recombinant human collagen IX
expressed in insect cells and recombinant human collagen II expressed in a transfected
tumour cell line (HT1080) nevertheless indicate that collagen IX with the Trp allele has a
higher affinity for collagen II than does normal collagen IX (S. Annunen, A. Fertala, D.J.
Prockop, L. Ala-Kokko, unpublished data).
The co-existence of the Trp allele and two other sequence variants indicated that the
individuals with the Trp allele had inherited the same, relatively rare, ancestral haplotype.
Interestingly, the same haplotype was also found in a Chinese immigrant, but he was not
included in the series because an isolated Finnish population was thought to be a more
accurate target for linkage disequilibrium analysis. This finding does indicate, however,
that the haplotype with the Trp is not restricted to this genetically isolated Finnish
population.
Even though all the individuals with the Trp allele had intervertebral disc disease,
there were some family members who had the disease but not the Trp allele.
Intervertebral disc disease is common in the Finnish population, with a prevalence of
4.8%, and the Trp allele was found in about 4% of the patients, which leaves the
remaining 96% of the cases to be explained by other genetic and environmental factors.
Thus, it is not surprising that there are more than one aetiologies explaining the
phenotype, even within one family.
Although it is known that intervertebral disc disease has many environmental and
anthropometric risk factors (see Heliövaara 1989), the present results indicate a role for
genetic factors as well. This suggestion is supported by reports of familial intervertebral
disc disease in the literature (Varlotta et al. 1991, Scapinelli 1993, Battié et al. 1995,
Sambrook et al. 1999).
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