American Journal of Medical Genetics 133A:61–67 (2005)
Dominant Negative Mutations in the C-Propeptide of
COL2A1 Cause Platyspondylic Lethal Skeletal Dysplasia,
Torrance Type, and Define a Novel Subfamily Within the
Type 2 Collagenopathies
Andreas Zankl,1* Luitgard Neumann,2Jaako Ignatius,3Peter Nikkels,4Connie Schrander-Stumpel,5
Geert Mortier,6Heymut Omran,7Michael Wright,8Katja Hilbert,9Luisa Bonafe ´,1Juergen Spranger,9
Bernhard Zabel,9and Andrea Superti-Furga1
1Division of Molecular Pediatrics, CHUV, Lausanne, Switzerland
2Institute of Human Genetics, Charite ´ University Hospital, Berlin, Germany
3Department of Clinical Genetics, Oulu University Hospital, Oulu, Finland
4Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands
5Department of Clinical Genetics, Academic Hospital Maastricht, Maastricht, The Netherlands
6Department of Medical Genetics, Ghent University Hospital, Ghent, Belgium
7Children’s Hospital, University of Freiburg, Freiburg, Germany
8Institute of Human Genetics, Newcastle-upon-Tyne, United Kingdom
9Children’s Hospital, University of Mainz, Mainz, Germany
Platyspondylic lethal skeletal dysplasia (PLSD)
Torrance type (PLSD-T) is a rare skeletal dyspla-
sia characterized by platyspondyly, brachydac-
tyly, and metaphyseal changes. Generally a
perinatally lethal disease, a few long-term survi-
vors have been reported. Recently, mutations in
the carboxy-propeptide of type II collagen have
ating that PLSD-T is a type 2 collagen-associated
disorder. We studied eight additional cases of
PLSD-T and found that all had mutations in the
C-propeptide domain of COL2A1. The mutational
spectrum includes missense, stop codon and fra-
meshift mutations. All non-sense mutations were
located in the last exon, where they would escape
PLSD-T is caused by mutations in the C-propep-
tide domain of COL2A1, which lead to biosynth-
esis of an altered collagen chain (as opposed to
a null allele). Similar mutations have recently
been found to be the cause of spondyloperipheral
dysplasia, a non-lethal dominant disorder whose
of the rare long-term survivors with PLSD-T.
Thus, spondyloperipheral dysplasia and PLSD-T
constitute a novel subfamily within the type II
collagenopathies, associated with specific muta-
tions in the C-propeptide domain and characteri-
zed by distinctive radiological features including
metaphyseal changes and brachydactyly that set
them apart from other type 2 collagenopathies
associated with mutations in the triple-helical
domain of COL2A1. The specific phenotype of
C-propeptide mutations could result from a com-
bination of diminished collagen fibril formation,
toxic effects through the accumulation of unfold-
ed collagen chains inside the chondrocytes, and
alteration of a putative signaling function of the
carboxy-propeptide of type 2 collagen.
? 2005 Wiley-Liss, Inc.
The platyspondylic lethal skeletal dysplasias (PLSDs) are a
heterogeneous group of chondrodysplasias characterized by
severe platyspondyly and shortening of limbs. The most
common form of PLSD is thanatophoric dysplasia (TD), with
its two variants, TD1 (OMIM 187600) and TD2 (OMIM
187610), both caused by mutations in the fibroblast growth
factor receptor 3 (FGFR3) [Tavormina et al., 1995]. In 1979,
two similar disorders have been distinguished, PLSD San
OMIM 151210). Each condition was felt to have specific histo-
logic findings, and in both, some collagen 2 abnormalities had
been observed but no molecular defect was identified. Several
years later, the San Diego variant was found to be caused by
FGFR3 mutations identical to those found in classical TD,
while no FGFR3 mutations were identified in the Torrance
variant [Brodie et al., 1999].
distinguish PLSD Torrance type from similar phenotypes
[Horton et al., 1979]. PLSD-T is characterized by varying
platyspondyly, short ribs with anterior cupping, hypoplasia of
the lower ilia with broad ischial and pubic bones, and short-
ening of the tubular bones with splayed and cupped meta-
physes. Histology of the growth plate typically shows focal
hypercellularity with slightly enlarged chondrocytes in the
Grant sponsor: European Skeletal Dysplasia Network (ESDN);
Grant number: QLGI-CT2001-02188 (EU grant number); Grant
sponsor: Swiss Federal Office for Science and Education (Grant
BBW); Grant number: 01.258; Grant sponsor: Swiss National
Science Foundation; Grant number: 3100A0-100485.
*Correspondence to: Dr. Andreas Zankl, Division of Molecular
Switzerland. E-mail: firstname.lastname@example.org
Received 13 August 2004; Accepted 1 November 2004
? 2005 Wiley-Liss, Inc.
ation and ossification at the chondroosseous junction [Horton
et al., 1979; Kaibara et al., 1983; Freisinger et al., 1996].
Though generally lethal in the perinatal period (and classified
amongst the letal platyspondylyc dysplasias), a few long-term
Neumann et al., 2003].
carboxy-propeptide of type II collagen in two patients with
PLSD-T, suggesting that PLSD-T may in fact be yet another
type 2 collagen-associated disorder. In this report, we present
eight additional cases with PLSD-T and mutations in the
C-propeptide region of COL2A1. Based on these observations
and the two cases from Nishimura et al., and in the light of
caused by specific COL2A1 mutations, review the genotype–
phenotype correlations of what seems to be a novel subfamily
within the type 2 collagenopathies, and discuss possible
pathogenetic mechanisms specific to type 2 collagen C-
DNA was isolated from peripheral blood lymphocytes
(patients 1, 3, 4, 5), skin fibroblasts (patients 7, 8) or cultured
amniocytes (patient 2). Exons 49-52 and flanking intronic
fragments were sequenced bidirectionally on an ABI 3100
Genetic Analyzer as described previously [Zankl et al., 2004].
Nucleotide numbering is based on RefSeq NM_001844, start-
ing with þ1 at the ATG translation initiation codon. In this
1,214, the C-propeptide comprises AA 1,215–1,488.
This is the second child of healthy parents. The older sib is
healthy. Mother reported rapid increase in her abdominal
diameter from 31 weeks of gestation. Ultrasound examination
edema of the fetus, short arms and legs, bowed radius, narrow
thorax and midface hypoplasia. Due to increasing polyhy-
dramnios, labor was induced and the patient was stillborn at
36 weeks. Post-mortem radiographs showed wafer-thin ver-
tebral bodies, short ribs with splayed ends, small and rounded
scapulae, shortened long bones with splayed metaphyseal
margins, hypoplasia of lower illia with medial spur, brachy-
dactyly with short metacarpals and phalanges, and splayed
metaphyseal margins (Fig. 1a–c). Histology of the rib reveal-
ed enlarged and vacuolized chondrocytes and slightly dis-
turbed columnar formation (Fig. 1d–e). In this patient, we
identified a 4423C>T non-sense mutation in exon 52, which
introduces a premature stop codon at position 1475 (Q1475X).
shortened long bones with splayed metaphyseal margins, hypoplasia of lower illia with medial spur, brachydactyly with short metacarpals and phalanges,
and splayed metaphyseal margins. d–e: Histology of the costo-chondral junction, showing slightly disturbed columnar formation; at higher magnifi-
cation, enlarged and vacuolized chondrocytes with relatively well preserved intercellular matrix.
a–c: Post-mortem radiographs of patient 1, showing wafer-thin vertebral bodies, short ribs with splayed ends, small and rounded scapulae,
62 Zankl et al.
equally absent in 50 normal control subjects.
This is the first child of non-consanguineous parents.
Prenatal ultrasound examination revealed polyhydramnios,
a small thorax with hypoplastic lungs and severe symmetrical
shortening of the limbs. The child was born after 39 weeks but
cleft palate and severe lung hypoplasia (body weight 2,753 g,
crown-heel length 36 cm, head circumference 37 cm). Post-
mortem radiographs (Fig. 2a,b) showed platyspondyly, short
long bones with splayed metaphyseal margins, hypoplasia of
lower illia with medial spur, and bowed tibiae. Histology of the
humeral chondroosseus junction, showed mild hypercellular-
ity with relatively well preserved columnar formation (Fig. 2c)
and vacuolization of chondrocytes (Fig. 2d). In this patient,
we identified a 12 bp in-frame deletion (4441_4452del,
I1481_V1484del). The mutation was not present in 50 normal
control subjects. The parents were unavailable for study.
Patients 3 and 4
Clinical and radiographic features of patients 3 and 4 have
been reported previously [Neumann et al., 2003]. In brief,
relatively large HC (56 cm), short fingers, lumbar lordosis and
complaints of pain in the hips and lower back. Her son showed
disproportionate short stature at birth with short limbs,
relatively large head and a narrow chest. At age 1.5 years, he
was 61.5 cm short, with a narrow chest, lumbar lordosis, genu
varum, and a waddling gait. Mental development was normal.
Based on radiographic findings, mother and son were diag-
nosed with PLSD-T. Figure 3 shows additional images, not
published in the original report, illustrating brachydactyly of
the hands and feet in patient 3. We have identified a missense
was not found in 50 normal control subjects.
Patients 5 and 6
Patient 5 and 6 have been published previously [Omran
et al., 2000; Neumann et al., 2003]. Patient 5 was born at term
with disproportionate short stature (44 cm), a relatively large
HC (38 cm), severe micromelia, and brachydactyly. She
required supplemental oxygen but was otherwise healthy. At
26 years (127 cm), she gave birth to patient 6 who presented
disproportionate short stature with a large head, a narrow
chest, severe micromelia, and brachydactyly at birth. The
infant suffered from lung hypoplasia and died after 22 days
both mother and son were diagnosed with PLSD-T. Figure 4
shows additional images of patient 5, not published in the
original report. We have identified a missense mutation
(4453T>G, C1485G) in the mother (DNA from the child was
not available for testing). The mutation was not found in 50
normal control subjects.
This female patient is the third child of healthy, non-
consanguineous parents. She was born after an uneventful
pregnancy. After birth, a disproportionate short stature with
short limbs and a relatively large head were noted. The face
was flat with micrognathia. The thorax was small with pro-
minent abdomen. Radiographs showed moderate platyspon-
dyly and shortened long bones with splayed metaphyseal
margins. The baby died due to respiratory insufficiency. We
identified a non-sense mutation (4335G>A, W1445X) in this
patient. The mutation was not found in 50 normal control
Patient 8is the second childofnon-consanguineous parents.
The previous baby had multiple congenital abnormalities with
absent thumb, lumbosacral segmentation defects, bilateral
diaphragmatic defects, horseshoe kidney, and ambiguous
genitalia. Long bones and ribs of this baby were normal. The
scapulae, shortened long bones with splayed metaphyseal margins, hypoplasia of lower illia with medial spur, bowed tibiae (c) histology of the humeral
chondroosseus junction, showing mild hypercellularity and relatively well preserved columnar formation (d) vacuolization of chondrocytes.
Mutations in PLSD-T63
second pregnancy developed normally until 21 weeks of
gestation, when prenatal ultrasound revealed shortening of
the pregnancy and the fetus was born at 21 weeks of gestation.
Post-mortem radiographs showed marked platyspondyly,
hypoplasia of lower illia with medial spur and shortened long
bones with splayed metaphyseal margins. We identified a
missense mutation (4342A>C, T1448P) in this patient. The
mutation was not found in 50 normal control subjects.
The identification of heterozygous mutations in the C-
propeptide region of COL2A1 in all patients with PLSD-T
PLSD-T among the type 2 collagenopathies. While most type 2
collagenopathies resulting in various forms of short-trunk
dwarfism like Achondrogenesis type 2, Hypochondrogenesis,
are very rare. The fact that all patients reported here have
mutations in this region suggests that PLSD-T is specifically
caused by mutations in this domain. This genotype–phenotype
correlation is so characteristic that it allowed us to identify an
additional patient from the literature who was initially
considered as achondrogenesis 2/hypochondrogenesis [Mortier
et al., 2000]: briefly, the patient was born at 36 weeks gestation
with a birth length of 39 cm. A small thorax and shortening of
died at 13 months from pneumonia. The diagnosis of
because of the presence of overmodified type 2 collagen in
cartilage specimens and radiographic abnormalities sugges-
tive for a type 2 collagen disorder. However, the phenotype of
this patient was considered remarkable because of quite
course. Also, the histology did not fit well with the diagnosis,
but no excess number of blood vessels and no prolongation
of hypertrophic chondrocytes into the primary trabeculae.
Nevertheless, a COL2A1 mutation analysis was performed
and revealed a missense mutation in exon 51 of COL2A1
(4169C>A, T1390N) [Mortier et al., 2000]. The finding of a
mutation in the C-propeptide region was unexpected as all
previously described mutations in Achondrogenesis II/Hypo-
chondrogenesis were identified in the triple helical domain.
Retrospectively, brachydactyly, metaphyseal involvement,
and the histology are more compatible with a diagnosis of
PLSD-T, as is the mutation in the C-propeptide domain.
The only other type 2 collagenopathy that is specifically
associated with mutations in the C-propeptide domain is
spondyloperipheral dysplasia (SPD) [Zabel et al., 1996;
Zankl et al., 2004]. In fact, SPD and the rare adult form of
are characterized by midface hypoplasia, lumbar hyperlordo-
sis, platyspondyly, epiphyseal dysplasia, and brachydactyly
E-like changes affecting mainly the distal and middle pha-
langes and the metacarpalia. PLSD-T may differ from SPD by
the absence of high-grade myopia and cleft palate, but these
features arevariable inothertype 2collagenopathies. Itmight
well be that the rare survivors of PLSD-T are in fact examples
Richards et al.  identified a missense mutation in the
C-propeptide domain of COL2A1 in a single large family with
(VPED). Affected family members exhibited extensive lattice
retinopathy and abnormal vitreal architecture, but no sig-
nificant myopia. Skeletal involvement consisted of premature
and middle phalanges. Spine involvement was minimal,
affected persons were of normal stature and did not show
It appears that brachydactyly, particularly of the distal and
middle phalanges and of the metacarpalia, is the most con-
sistent clinical feature of mutations in the C-propeptide
domain of type 2 collagen. This observation is particularly
relevant since brachydactyly is rare in the other type 2
collagenopathies, such as SEDC or Stickler syndrome, and is
an important differential diagnostic criterium.
Platyspondylic lethal skeletal dysplasia (PLSD)-T. In the hands, the distal
most severely affected (see radiographs in [Neumann et al., 2003]).
Brachydactyly in patient 3 with a non-lethal variant of
64 Zankl et al.
Histological studies of the growth plate in patients with
et al., 1983; Freisinger et al., 1996; Mortier et al., 2000] and
Figure 1. Electron microscopy demonstrated chondrocytes
with dilated cisternae of the rough endoplasmatic reticulum
containing a granular material [Freisinger et al., 1996].
Collagen content in PLSD-T cartilage is mildly reduced, and
the collagenfibrilsarethin [Freisingeretal.,1996].Freisinger
and Mortier observed overmodification of type 2 collagen in
fetal PLSD-T cartilage [Freisinger et al., 1996; Mortier et al.,
the abnormal a1(II) chains were not appropriately integrated
into stable cross-linked fibrils [Freisinger et al., 1996].
Freisinger et al.  attributed this overmodification to a
glycine substitution in the triple helix domain but failed to
identify such a mutation by SSCP. The radiographic appear-
and the failure to demonstrate a COL2A1 helical mutation,
lead to the interpretation of these collagen 2 findings as secon-
dary. Based on our findings, we propose a different pathoge-
C-propeptide region of COL2A1 and (2) result in the biosynth-
esis of an altered collagen chain (as opposed to mutations that
result in unstable mRNA and thus are functional null alleles).
The majority of mutations identified so far are missense
mutations that should result in the production of an altered
collagen chain (Fig. 5). Two of the mutations introduce
premature stop codons, but since both lie in the last exon of
COL2A1 they should escape non-sense mediated mRNA decay
[Schell et al., 2002; Zankl et al., 2004] and result in the
important role in triple-helix formation, assembly of the
altered a1(II) chains into trimeric collagen molecules is likely
to be impaired, as demonstrated by overmodification of the
resulting molecules. In addition, some of the mutated a1(II)
chains might not be integrated into collagen fibrils at all and
accumulate in the chondrocytes as free chains. The latter
would explain (1) the presence of granules in the dilated rough
endoplasmatic reticulum, (2) the higher content of abnormal
collagen in the salt-soluble fraction compared to the pepsin-
soluble fraction, and (3) the decreased collagen content and
small collagen fibril diameter in the extracellular matrix. We
have proposed a similar model to explain the pathogenetic
mechanisms underlying SPD [Zankl et al., 2004] and the
concept would also be compatible with the missense mutation
observed in the family with VEPD [Richards et al., 2002].
Since all three conditions share the unique feature of
brachydactyly, we could further speculate that the accumula-
tion of altered collagen chains in the chondrocytes somehow
causes slow proliferation of chondrocytes and/or premature
closure of specific growth plates resulting in abnormally short
tubular bones. Alternatively, the peculiar phenotype might be
related to a specific function of the C-propeptide that becomes
altered through a mutation in this region. The C-propeptide of
type 2 collagen is also known as chondrocalcin [Poole and
Rosenberg, 1986]. Chondrocalcin accumulates in the hyper-
trophic zones of the growth plate and seems to promote
shows hypoplasia of the lower ilium and dysplastic proximal femur. (see also radiographs in [Neumann et al., 2003; Omran et al., 2000]). The radiographic
findings beyond the neonatal period are identical to SPD (see Discussion).
Mutations in PLSD-T 65
mineralization in this zone, but little else is known about this
patients with brachydactyly type E [Johnson et al., 2003],
which shows some resemblance to the brachydactyly observed
in SPD and PLSD-T, but the relation to SPD and PLSD-T is
unclear. Maybe HOXD13 and Chondrocalcin act in a common
Support for a specific function of the C-propeptide comes
from collagen I. Most known mutations of collagen I lie within
the triple-helical region and produce osteogenesis imperfecta
(OI) in its remarkably wide phenotypic spectrum. Only a few
some cases, these mutations have resulted in a peculiar and
very rare variant, OI type IIC, characterized by fragile but
collagen I C-propeptide acts as a signaling molecule in the
extracellular matrix and that alteration of this activity
contributes to the unique features of OI IIC. In support of this
collagen synthesis in preosteoblasts [Mizuno et al., 2000] and
cells [Rushton et al., 1999].
In summary, our findings confirm and reinforce the original
observation by Nishimura et al. that PLSD-T is part of the
suggest that PLSD-T and SPD constitute a novel subfamily
within the type 2 collagenopathies, associated with specific
mutations in the C-propeptide domain and characterized
phenotypically by metaphyseal involvement and brachydac-
tyly in addition to the commonly observed spondyloepiphyseal
We would like tothank G. van Noort for the histology of case
2 and the members of the ESDN for helpful discussions.
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Mutations in PLSD-T67