Type IX Collagen Gene Mutations Can Result in
Osteochondritis Dissecans and a Mild Myopathy
Gail C. Jackson,1,2Dominique Marcus-Soekarman,3Irene Stolte-Dijkstra,4Aad Verrips,5
Jacqueline A. Taylor,1and Michael D. Briggs1*
1Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, UK
2Regional Molecular Genetics Service, St. Mary’s Hospital, Manchester, UK
3Department of Clinical and Cytogenetics, University Hospital, Maastricht, the Netherlands
4Section Clinical Genetics, Department of Genetics, University Medical Center Groningen, Groningen, the Netherlands
5Neuromyologist Department of Child Neurology, Canisius-Wilhelmina Hospital, Nijmegen, the Netherlands
Received 23 March 2009; Accepted 16 November 2009
Multiple epiphyseal dysplasia (MED) is a clinically variable and
genetically heterogeneous disease that is characterized by mild
short stature and early onset osteoarthritis. Autosomal domi-
COL9A1, COL9A2, COL9A3, COMP, and MATN3, respectively.
an equivalent region of the COL3 domain in all three a(IX)
chains. MED has been associated with mild myopathy in some
families, in particular one family with a COL9A3 mutation and
also have osteochondritis dissecans and mild myopathy. This
study therefore extends the range of gene-mutations that can
cause MED-related myopathy. ? 2010 Wiley-Liss, Inc.
Key words: multiple epiphyseal dysplasia; myopthathy; type IX
collagen; cartilage; osteochondritis dissecans
Multiple epiphyseal dysplasia (MED) is a clinically and geneti-
cally heterogeneous disease that manifests with joint pain and
stiffness, mild short stature, and degenerative joint disease
allelic with the more classical forms of MED, including bilateral
hereditary micro-epiphyseal dysplasia (BHMED) [Mostert et al.,
2002]. The identification of the causative gene mutations has
allowed the classification of MED based on molecular genetic
criteria as well as clinical and radiographic features. It has become
clear that there is considerable allelic and non-allelic genetic
heterogeneity within MED, which is reflected in the extensive
clinical and radiographic variability of the MED phenotype.
Mutations identified in patients with MED have now been found
COL9A3) for the autosomal dominant forms, while mutations in
SLC26A2 cause autosomal recessive MED [Briggs and Chapman,
Several reports have described families with MED that
was associated with other complications such as osteochondritis
dissecans (i.e., the delamination of articular cartilage from the
Additional supporting information may be found in the online version of
Z/07/Z; Grant sponsor: European Commission FP6; Grant Number:
Michael D. Briggs, Wellcome Trust Centre for Cell Matrix Research,
Faculty of Life Sciences, University of Manchester, Michael Smith
Building, Oxford Road, Manchester M13 9PT, UK.
Published online 23 March 2010 in Wiley InterScience
How to Cite this Article:
Jackson GC, Marcus-Soekarman D, Stolte-
Dijkstra I, Verrips A, Taylor JA, Briggs MD.
2010. Type IX collagen gene mutations can
result in multiple epiphyseal dysplasia that is
a mild myopathy.
Am J Med Genet Part A 152A:863–869.
? 2010 Wiley-Liss, Inc.
underlying subchondral bone) [Versteylen et al., 1988] and mild
myopathy or muscle weakness [Bonnemann et al., 2000; Jakkula
MED, which was caused bya mutation in COL9A3,presented with
muscle weakness and a muscle biopsy showed mild myopathy that
was characterized by a variability in fiber size [Bonnemann et al.,
We had the opportunity to study two large families with MED
that was complicated by osteochondritis dissecans and myopathy.
We evaluated the clinical and molecular attributes of these dis-
orders in the two families to better understand the relationship of
these rare manifestations.
PATIENTS AND METHODS
This family has been reported previously [Versteylen et al., 1988],
an update and additional clinical and molecular data are reported
4 years [Patient B3 in Versteylen et al., 1988 and individual III-4 in
Fig. 1A]. At that time her height was 1.54m (?2.5 SD), arm span
1.53m, weight 84kg (>2 SD), OFC 56cm (0 to þ1 SD) and ICD
3.5cm (þ2 SD). Radiographs of the knees were available
and showed Blount’s disease (progressive varus deformity of the
proximal tibia associated with internal torsion of the tibia) and
in the thoracic region (Fig. 2). The patient’s family had been
reported in the past to have osteochondritis dissecans of the knees
suffered painfuland swollen kneesfromthe age of4yearsandlater
in life she also developed severe osteochondritis dissecans [Patient
B4 in Versteylen et al., 1988 and individual III-3 in Fig. 1A]. This
patient was not re-evaluated in this current study.
The eldest son of the proband (individual IV-6) was developing
startedtocomplain ofpaininthekneesduring walking.Hisheight
at age 5 years was 1.15m (0 SD), arm span 1.09m, weight 18.5kg
(?2 to 0 SD), OFC 53.5cm (þ1 SD), ICD 3.2cm (þ1 to þ2 SD),
and OCD 8.3cm (?1 SD). He had frontal bossing and a depressed
nasal bridge. The mother reported that he could not run like his
classmates and that he was easily fatigued. An examination by a
child neurologist showed weakness of the abdominal musculature.
longtime, but waspossible without theuseof hisarms. There were
both sides of the hip. A muscle biopsy taken at the age of 5 years
showed some variation in fiber size, but was otherwise normal.
of MED (Fig. 2).
age of 4 years he was also developing similar complaints as his
brother,mostnotably pain intheknees,clumsywalking, tiredness,
and easy exhaustion. He showed the same facial features as his
brother. His height was 1.02m (?1 SD), arm span 99cm, weight
16.9kg (0SD),OFC 52.5cm (0toþ1SD), ICD3cm (þ1SD), and
OCD 9cm (0 to þ1 SD). Radiographs of the knees at that age
showed similar abnormalities to his brother (data not shown).
FIG. 1. PedigreesoftwothefamilieswithMEDpresentedinthisstudy.AnarrowindicatesthoseindividualsinwhichaCOL9A2mutationwasidentified.
864AMERICAN JOURNAL OF MEDICAL GENETICS PART A
The index patient (individual III-1, Fig. 1B) was referred to the
Department of Clinical Genetics after itwasnoted on a radiograph
the age of 3 years because of an abnormal gait. Neurological
evaluation at that time showed normal development, with normal
FIG. 2. Radiographsoftheproband(individualIII-4,panelsA–D)inFamily1at33yearsofageandhereldestson(individualIV-6,panelsE,F)at3years
ofage. Themother’s radiographs showed (A)normal hand, (B) knees withbilateral genuvarus, irregular joint surfaces, anddecreased joint space,
especiallyof the right knee, (C)lateral spine withirregularendplates ofthe vertebral bodies in the thoracolumbal region and(D) relativelynormal
hips. Radiographs of her 3-year-old son showing (E) hips and (F) the left knee. The radiographs were consistent with a diagnosis of mild MED,
characterized bydelayed ossification ofthe epiphyses. The hipswere relatively spared but hadsmall proximal femoralepiphyses, while the knees
had small femoral and tibial epiphyses.
JACKSON ET AL.
up. Creatine kinase and lactate levels were normal. A left sided
developmental dysplasia of the hip was diagnosed, which was
surgically corrected. His gait however remained stiff and he had
years he had mild knee and foot pain after prolonged walking. His
height was 140.8cm (0 SD), weight 33.1kg (0 SD), sitting height
75.7cm (0 to þ1 SD), arm span 138.9cm (O SD), and head
circumference 54.8cm (þ1 SD). He had a slightly flat midface and
there was one tooth missing in the lower jaw. Ophthalmologic
examination was normal. His younger brother and sister
(individuals III-2 and III-3) did not have similar complaints. The
family history was positive for osteochondritis dissecans and
hypodontia. The father had osteochondritis dissecans of the right
elbow during puberty, but at the time of this report he was almost
puberty and she was reported to have had severe osteochondritis
her complaints subsided andat the time of this report she wasfully
mobile but there was some muscle weakness. Two of her children
(individuals III-4 and III-5) were also reported as affected and one
of her affected children has some teeth missing. The paternal
grandmother was noted to have asymptomatic osteochondritis
dissecans on radiographs taken when she 60 years of age (data not
shown). Only individuals II-1, II-2, III-1, III-2, and III-3 were seen
by a clinical geneticist (I-SD) since the rest of the family are
currently residing abroad.
Samples of DNA from affected members of both families were
collected for mutation analysis in the genes known to cause MED.
FIG. 3. Radiographs ofthe proband in Family2 (individual III-1) at age 9years and10 months of ageshowing (A) hand, (B) knee, (C) spine, and(D)
femoral epiphyses but they were relatively spared. There was some irregularity of the vertebral end plates (arrow).
866AMERICAN JOURNAL OF MEDICAL GENETICS PART A
Screening of the COMP and MATN3 genes was undertaken as
described previously [Jackson et al., 2004; Kennedy et al., 2005a;
Zankl et al., 2007]. Briefly, exons 8 to 19 of COMP and exon 2 of
MATN3 were amplified by PCR and subject to bi-directional DNA
sequencing, but this analysis did not reveal any mutations. We
and exon 8 of COL9A1 (NM_001851, NM_001852, and NM-
_001853, respectively) and the immediate splice donor and accep-
tor sites. Briefly, PCR amplifications (Primer Sequences; CO-
L9A1Ex8F 50-GGCCTCCTCTGGAAGGTAA-30, COL9A1EX8R
50-AGTCCTGCCCTTTCCTATTCT-3, COL9A2EX3F 50-TAGG-
GGACCTGGACAGAAGA-30, COL9A2EX3R 50-TCCCTTGAAA-
ACAGAGATGGA-30, COL9A3EX3F 50-GTTCTTGAGGGACCC-
CTGA-30, COL9A3EX3R 50-AATGACCCCTCTGTTCTGAG-30)
were performed in a total volume of 20ml using a custom PCR
ReadyMix? master mix (ABgene, Epsom, UK) containing 0.5mM
at 25?C), 16mM (NH4)2SO4, 3.7mM MgCl2, 0.085mg/ml BSA,
6.7mM EDTA, 0.75mM of each dNTP and 50–100ng of genomic
followed by a final extension at 72?C for 3min. Purified PCR
products were used as a template for bi-directional fluorescent
DNA sequencing and the sequence data were compared against a
reference trace and a negative control sequence to detect variants
using the Staden sequence analysis package [Staden et al., 2000].
Results of Genetic Analysis of
Type IX Collagen Genes
sons) were heterozygous for c.186þ2T>C in the splice donor
sequence of intron 3 of COL9A2. The index patient of Family 2
was heterozygous for c.186G>A in the splice donor sequence of
exon 3. Nucleotide numbering is according to mRNA sequence
with Genebank accession number for COL9A2¼NM_001852 and
nucleotide 1 has been counted as the first nucleotide of the
translation initiation codon.
Both of these mutations have been identified in families with
MED and shown to result in the skipping of exon 3 sequence from
COL9A2 mRNA [Muragaki et al., 1996; Holden et al., 1999].
Histological Analysis of a Muscle Biopsy
Microscopic examination (hematoxylin and eosin staining) of a
muscle biopsy from the m. vastus lateralis obtained at the age
of 5 years from the eldest son of the proband (individual IV-6,
Family 1) showed no significant morphological or histochemical
abnormalities (see supporting information Fig. 1 which may be
muscle fibers with central nuclei (i.e., indicative of fiber stress and
remodeling); however, some variation in fiber size was observed
(5–35mm with a mean diameter of 24mm), consistent with previ-
or necrosis was present and there was a normal fiber type distribu-
tion (ATPase staining). The periodic acid-Schiff, myophosphor-
ylase, naphtyl-esterase, NADH, SDH, and COX stains were all
normal. To evaluate the exercise-induced leg pain and fatigue, his
mitochondrial respiratory chain substrate oxidation rates, ATP
metabolism, and enzyme activities were measured in the biopsy.
The ATPþCrP production from pyruvate was diminished (22.3;
normal range 42.1–81.2nmol/hrmUCS), [1 -14C]pyruvate and
malate was 3.15nmolCO2/hr/UCS (normal range 3.61–7.48), [1,4
-14C]succinate and acetylcarnitine was 2.08nmolCO2/hr/UCS
(normal range 2.54–6.39). The other substrate oxidation rates and
theenzyme activities (complex I–V,citrate synthase)were normal.
3 in COL9A2 that results in MED in two unrelated families. These
data are consistent with previous studies showing that MED
mutations in COL9A2 reside exclusively in the splice donor se-
et al., 2000; Fiedler et al., 2002; Takahashi et al., 2006]. The strict
grouping of MED mutations in the splice donor and acceptor sites
of exon 3 of COL9A2 and COL9A3, respectively, and the splice
acceptor site of exon 8 of COL9A1, is yet to be fully explained.
The association of myopathy with MED has been noted before
and is associated with mutations in both COL9A3 [Bonnemann
et al., 2000; Lohiniva et al., 2000] and COMP, in particular
mutations in those exons of COMP that encode the C-terminal
domain of COMP [Jakkula et al., 2003; Kennedy et al., 2005b].
However, myopathy does not appear to be a consistent feature of
MED caused by COL9A3 mutations [Paassilta et al., 1999; Naka-
shima et al., 2005] and it has been suggested that the degree of
mutations in COL9A2 can also cause myopathy and suggests an
important role for type IX collagen in the musculoskeletal system.
feature of MED caused by COL9A2 mutations. Indeed, unrelated
MED families with the same mutations identified in this study do
not present with muscle weakness [Muragaki et al., 1996; Holden
et al., 1999]. It is becoming increasingly clear that there is consid-
1999; Bonnemann et al., 2000; Lohiniva et al., 2000; Nakashima
is interesting to speculate that the genetic modifiers of phenotypic
severity may becommonacross
pseudoachondroplasia-MED (PSACH-MED) bone dysplasia fam-
ily, regardless of which gene the causative mutation resides in.
The muscular component of the MED phenotype in these
patients is poorly understood, but has been characterized by either
increased protein kinase C levels or atrophic fibers observed in
muscle samples obtained through biopsy. In the patient reported
previous report [Bonnemann et al., 2000], the muscle at the site of
biopsy showedonlymild variationsin fiber size, even though there
were clinical symptoms of a mild myopathy in both families. The
thespectrum of the
JACKSON ET AL.
then it is possible that the muscle biopsy was obtained from the
medius and minimus muscles), or alternatively the Trendelenburg
mild PSACH-MED caused by a C-terminal COMP mutation
(p.Thr585Met) [Pirog-Garcia et al., 2007] also showed signs of
myopathy that was characterized by fibrosis, necrosis, and variable
fiber diameter in the muscle. However, these pathological changes
were restricted to the myotendinous junction, where COMP and
type IX collagen are expressed, and suggested that the observed
myopathy may be a secondary consequence of an underlying
tendinopathy [Pir? og et al., 2010]. It is therefore possible that the
muscle weakness seen with type IX collagen gene mutations may
conveyed during locomotion.
The clinical biochemistry of the eldest affected son of Family 1
mildly reduced pyruvate, malate, succinate, and acetylcarnitine
levels in muscle. These are all markers of the oxidative capacity of
mitochondrial dysfunction in the patient. There is an increasing
cell stress can directly trigger mitochondrial cell death pathways
[Guicciardi and Gores 2008]. For example, Hori et al.  have
shown that the suppression of protein synthesis, due to ER stress,
has a detrimental effect on the synthesis of mitochondrial-associ-
ated proteins. These can include the cytochrome oxidase (COX)
shown that the expression of mutant COMP and MATN3 muta-
tions can cause ER stress [reviewed in, Bateman et al., 2009], the
consequences of which have been reduced chondrocyte prolifera-
tion and increased apoptosis in vivo. This detrimental change in
chondrocyte phenotype is independent of whether the mutant
protein is retained in the rough ER [Leighton et al., 2007] or
efficiently secreted[Pirog-Garcia etal.,2007].Wehypothesizethat
the expression of mutant type IX collagen may elicit a cell stress
response that has down stream consequences with respects to cell
and mitochondrial function and viability. Type IX collagen is not
expressed in skeletal muscle [Irwin et al., 1985; Muller-Glauser
the bone-tendon interface and it is possible that the expression of
mutant type IX collagen in this tissue might cause an underlying
tendinopathy that ultimately affects a wide range of musculoskele-
tal tissues. This hypothesis is consistent with the myopathy in a
mouse model of mild PSACH-MED resulting from a Comp muta-
tion [Pir? og et al., 2010].
Further analysis of patients and mice models of PSACH-MED
will help clarify the pathology and disease mechanisms of mild
myopathy resulting from type IX collagen and COMP mutations.
This work was supported by grants from the Wellcome Trust
(Michael Briggs is the recipient of a Wellcome Trust Senior
Research Fellowship in Basic Biomedical Science; Grants 071161/
Z/03/Z and 084353/Z/07/Z) and European Commission FP6
(Grant No. 037471). The authors thank members of the European
Skeletal Dysplasia Network (ESDN) for confirming the clinical
diagnosis of these patients. In particular, Sheila Unger, Andrea
Superti-Furga, Jurgen Spranger (Freiburg), Michael Wright
(Newcastle), Geert Mortier (Gent), Valerie Cormier-Daire
(Paris), Christine Hall (London), and Andreas Zankl (Brisbane).
We also thank Simon Robben for his contribution.
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