Nature Genetics
23, 319 - 322 (1999)
doi:10.1038/15496
CACP, encoding a secreted proteoglycan, is mutated in camptodactyly-arthropathy-coxa
vara-pericarditis syndromeJose Marcelino1, 15, John D. Carpten2, 15, Wafaa M. Suwairi1, 4, 5, Orlando M. Gutierrez1, Stuart Schwartz1, Christiane Robbins2, Raman Sood2, Izabela Makalowska2, 3, Andy Baxevanis3, Brian Johnstone6, Ronald M. Laxer7, Lawrence Zemel8, Chong Ae Kim9, J. Kenneth Herd10, Johannes Ihle11, Cal Williams12, Mark Johnson12, Vidya Raman12, Luís Garcia Alonso13, Decio Brunoni13, Amy Gerstein14, Nickolas Papadopoulos14, Sultan A. Bahabri5, Jeffrey M. Trent2
& Matthew L. Warman11 Department of Genetics and Center for Human Genetics,
Case Western Reserve University and University Hospitals of Cleveland,
Cleveland, Ohio, USA. 2 Cancer Genetics Branch, National Human Genome Research
Institute, National Institutes of Health, Bethesda,
Maryland, USA. 3 Genome Technology Branch, National Human Genome Research
Institute, National Institutes of Health, Bethesda,
Maryland, USA. 4 Department of Pediatrics, Riyadh Armed Forces Hospital, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia. 5 Department of Pediatrics, King Faisal Specialist Hospital
and Research Center, Riyadh, Saudi Arabia. 6 Department of Orthopaedics, Case Western Reserve University
and University Hospitals of Cleveland, Cleveland, Ohio
, USA. 7 Division of Rheumatology and Department of Pediatrics,
The Hospital for Sick Children and the University of Toronto,
Toronto, Canada. 8 Division of Rheumatology, Newington Children's Hospital
, Hartford, Connecticut, USA. 9 Department of Genetics, University of São Paolo
, São Paolo, Brazil. 10 Department of Pediatrics, East Tennessee State University,
James H. Quillen College of Medicine, Johnson City,
Tennessee, USA. 11 Division of Rheumatology, University Children's Hospital
, Tuebingen, Germany. 12 Department of Pediatrics, Washington University and
St. Louis Children's Hospital, St. Louis, Missouri,
USA. 13 Medical Genetics Institute, São Paulo Federal
University-Paulista School of Medicine, São Paulo,
Brazil. 14 Institute of Cancer Genetics, Department of Pathology,
Columbia University, New York, New York, USA
. 15 These authors contributed equally to this work.
Correspondence should be addressed to Matthew L. Warman mlw14@po.cwru.edu or Jeffrey M. Trent jtrent@nhgri.nih.govAltered growth and function of synoviocytes, the intimal cells which line
joint cavities and tendon sheaths, occur in a number of skeletal diseases1. Hyperplasia of synoviocytes is found in both rheumatoid arthritis
and osteoarthritis, despite differences in the underlying aetiologies of the
two disorders. We have studied the autosomal recessive disorder camptodactyly-arthropathy-coxa
vara-pericarditis syndrome (CACP; MIM 208250) to identify biological pathways
that lead to synoviocyte hyperplasia, the principal pathological feature of
this syndrome. Using a positional-candidate approach, we identified mutations
in a gene (CACP) encoding a secreted proteoglycan as the cause of CACP.
The CACP protein, which has previously been identified as both 'megakaryocyte
stimulating factor precursor'2 and 'superficial zone protein'3, contains domains that have homology to somatomedin B, heparin-binding
proteins, mucins and haemopexins. In addition to expression in joint synovium
and cartilage, CACP is expressed in non-skeletal tissues including
liver and pericardium. The similarity of CACP sequence to that of other protein
families and the expression of CACP in non-skeletal tissues suggest
it may have diverse biological activities.Synovium is a specialized tissue that nourishes and lubricates joints and
tendons. Synovium also clears metabolites that accumulate in joint cavities4. Hyperplasia of synoviocytes in the context of inflammation is a
characteristic feature of rheumatoid arthritis5, in which synoviocyte
overgrowth may contribute to joint destruction by interfering with the normal
exchange of nutrients and waste products between the vascular/lymphatic plexus
and the joint cavity6. Hyperplastic synoviocytes may also directly
damage articular cartilage by producing degradative enzymes7
and by invading the articular cartilage surface8. Patients with
the heritable disorder CACP have synovial hyperplasia without evidence of
inflammation9,
10 (Fig. 1a). This
results in congenital or childhood-onset camptodactyly (flexion contractures
of the phalangeal joints of fingers and toes; Fig. 1b)
and childhood-onset arthropathy (pain, swelling and restricted range of motion
in the large joints; Fig. 1b,c). Thickening
of the pericardium can also occur in CACP (ref. 11)
and is associated with overgrowth of the intimal portion of the fibrous pericardium,
again without evidence of inflammation (Fig. 1d).
Fibrosing pleuritis has also been reported12. Pericarditis and
pleuritis, in the context of inflammation, occur in patients with rheumatoid
arthritis13,
14, suggesting that the protein product responsible
for causing CACP may also contribute to the pathogenesis of rheumatoid arthritis.
 | | |
The CACP locus has been mapped to a 1.9-cM genetic interval on human chromosome
1q25−q31 (ref. 15). Using an informative
simple sequence repeat polymorphism derived from an end-clone of CEPH mega-YAC
956-B9, we reduced the CACP candidate interval to less than 2 Mb (data not
shown). We constructed a complete BAC contig across the critical region (manuscript
in preparation) and performed sample sequencing to identify novel polymorphic
markers, as well as candidate genes within this interval. The assembled genomic
sample sequence of the human BAC clone b174L6 was BLAST searched to find homologous
sequences in the public databases using WebBLAST (16; http://genome.nhgri.nih.gov/webblast/). BLASTN identified
human EST90076, which is derived from synovial tissue cDNA and has 100% identity
to our query sequence. This EST is 98% identical to the human megakaryocyte
growth and stimulating factor precursor (MSF). The full-length cDNA coding
sequence that contains this EST is identical to that of MSF, leading us to
conclude that CACP and MSF are the same. A putative bovine orthologue of this
protein has been called 'superficial zone protein'3,
17 (SZP).
The protein is synthesized by chondrocytes in the superficial zone of articular
cartilage (closest to the joint cavity) and by joint synoviocytes18.
We used PCR and RT-PCR to amplify portions of CACP from patient-derived
genomic DNA and mRNA, respectively, and identified eight likely disease-causing
mutations. None of these mutations was observed in 100 control chromosomes.
Among affected offspring of consanguineous unions, all mutations co-segregate
with the phenotype and are present in the homozygous state. These include
four deletions (2805del5, 3240del7, 3023del2 and 3690del5) that alter the
reading frame and result in premature truncation of the full-length polypeptide
(Fig. 2b,e,g,h); one dinucleotide
transversion (4190CC AG) that creates a nonsense codon (
Fig. 2i); and one 41-bp insertion 14 nt residues upstream of
the intron 6 splice acceptor site (ins41-14IVS6) that disrupts the polypyrimidine
tract of the splice site (data not shown). In two unrelated patients whose
parents are non-consanguineous, we have been able to identify one mutant allele.
One patient is heterozygous for a single nucleotide transition (724CT)
that she inherited from her asymptomatic father. The second patient is heterozygous
for a frameshift mutation (3895del5) that he inherited from his asymptomatic
mother. We presume each of these patients has a second mutant allele that
we have not yet identified. We have also been unable to identify disease-causing
mutations in affected offspring from two other consanguineous kindreds (family
1 in ref. 15 and an unpublished case); all affected
individuals from these kindreds are homozygous for multiple polymorphic markers
flanking CACP, suggesting that our failure to find mutations is not
due to locus heterogeneity or phenocopy. Nearly 50% of CACP coding
sequence encodes the highly repetitive, mucin-like domain of the protein and
is contained in a single exon (exon 6). This region of the gene has been difficult
to sequence. Consequently, additional disease-causing mutations might be contained
in this exon. We have confirmed that the identified frameshift mutations in
exon 6 cause premature termination using the protein truncation test (data
not shown), but we have not found any additional frameshift or nonsense mutations
with this method. Our finding of eight different CACP mutations in
patients with CACP indicates that they cause the pathogenesis of the disorder.
| | Figure 2. Schematic of the CACP proteoglycan and the putative effects of each
mutation. | | |  | a, Full-length protein showing regions of homology to other protein
families. b,e,g,h,i, Predicted
protein products in affected offspring of consanguineous unions who are homozygous
for frameshift or nonsense mutations. Three frameshift mutations (b,
g,h) alter several polypeptides (filled segments) before causing
premature termination. c, Segregation of the 2805del5 mutation with
the CACP phenotype. Unaffected parents are heterozygous for mutant and wild-type
alleles, whereas the affected patients are homozygous for the mutant allele.
d, Chromotograms of wild-type and 2805del5 mutant alleles. f,
Chromatograms of wild-type and 3240del7 mutant alleles. d,f,
Boxed area indicates the nucleotide residues deleted in affected patients.
 Full Figure and legend (48K) |
| |
Thus far, all coding sequence mutations predict truncations in the protein.
The absence of heterozygote manifestations among carrier parents suggests
that the mutations cause a loss of protein function, rather than a gain of
new function. In one family, the CACP mutation truncates the polypeptide
by only eight amino acid residues (Fig. 2i).
This implies that full-length protein is essential to normal function; however,
in the absence of sequencing the entire gene we cannot preclude the possibility
that a more deleterious CACP mutation actually accounts for their disease.
The deleted carboxy-terminal residues are highly conserved across species
and the deleted amino acid motif (WXXCP) is also present at the C terminus
of vitronectin, a protein that shares several other regions of homology with
CACP (ref. 19).
The identification of CACP mutations should help delineate the protein's
normal function. CACP appears to encode a novel type of proteoglycan.
Its predicted peptide sequence does not contain membrane-spanning domains
found in cell-surface-receptor proteoglycans, such as syndecans, CD44 and
NG2 (refs 19,
20,
21,
22), nor does it appear to be covalently linked to membranes
like the glypicans23. Its secretion into the joint cavity distinguishes
it from cartilage-matrix-bound proteoglycans such as aggrecan and the small
leucine-rich proteoglycans decorin, fibromodulin and lumican, which are primarily
retained in the cartilage matrix through interactions with hyaluronan and
fibrillar collagens, respectively24. Due to its high glycosylation
content and mucin-like repeats, CACP may act as a joint/intimal cell
lubricant. Both synovial and pericardial cell hyperplasia may represent secondary
consequences of insufficient cell surface lubrication. The slowly progressive
nature of the arthropathy in patients affected with CACP and the incomplete
penetrance for symptomatic pericardial involvement support this hypothesis.
Cell overgrowth, however, may be primary to the pathogenesis of the disorder.
Two unrelated patients in our series had multiple small ganglion cysts (lesions
adjacent to tendon sheaths filled with mucinous material) that may result
from dysregulated synovial cell growth. Also, supporting a regulatory role
for the CACP protein product is the occurrence of coxa vara deformity25 (angular deformation of the hips), which is a developmental defect
of the femoral neck.
Another function of CACP protein may be to regulate intimal cell growth.
Because synoviocyte hyperplasia and, less commonly, hyperplasia of other intimal
cell layers (pericardium and pleura) occur in rheumatoid arthritis (RA), a
disease-associated disruption of the regulatory function of CACP protein may
contribute to disease progression in RA. CACP is expressed in synovial
tissue (Fig. 3). On a commercially available multi-tissue
northern blot, we detected CACP mRNA in several other tissues, including
liver (Fig. 3), and its 30-kD amino-terminal megakaryocyte
stimulating factor fragment is detectable in serum and urine19.
Therefore, although all sites of gene expression and protein secretion are
unknown, it is intriguing to speculate that CACP has widespread biological
activity.
Methods
Clinical material.
We obtained informed consent from
all study participants. Patients were clinically diagnosed as having CACP
using published criteria15. All patients had congenital or infancy-onset
camptodactyly, and developed large joint arthropathy during childhood. Coxa
vara deformity and pericarditis occurred in some patients. The kindred used
to reduce the CACP interval to less than 2 Mb has been described (family 4
in ref. 15), as have the clinical descriptions
of the two kindreds segregating the 7-bp deletion (families 2 and 3 in ref. 15). Bovine synovium and percardium were recovered
at the time of necropsy.
Histology.
We recovered patient-derived synovium and
pericardium following diagnostic synovial biopsy and therapeutic pericardectomy,
respectively; we fixed material in formalin and embedded in paraffin. Cross-sections
were stained with haemotoxylin and eosin.
DNA and RNA isolation.
Lymphocytes isolated from whole
blood were EBV-transformed as described26 and cultured in RPMI
containing 10% fetal bovine serum. We isolated human synoviocytes following
a brief incubation of synovial tissue with collagenase (Sigma). Synoviocytes
were cultured in DMEM containing 10% fetal bovine serum. We extracted DNA
with the Puregene kit (Puregene) and prepared human and bovine RNA using guanidine-HCl
and a CsCl step gradient27. We made cDNA with the superscript
pre-amplification system (Gibco BRL).
Reduction of the CACP candidate interval.
The centromeric
end of CEPH mega-YAC 956B9 was cloned using inverse PCR. This YAC contains
three completely linked simple sequence repeat polymorphisms (D1S191,
D1S2848, D1S444) and may contain the centromeric boundary of the
CACP interval (see family 4 from ref. 15). Using
the end-clone sequence, we designed a PCR primer pair to amplify a 113-bp
fragment from genomic DNA in family 4, which is consanguineous15.
Heterozygosity for SSCP alleles in the affected patient and his mother indicated
that the centromeric end of YAC956B9 lies outside of the CACP minimum interval,
which is homozygous by descent in the patient.
BAC DNA isolation.
We used a culture (40 ml) of BAC
b174L6 to isolate DNA for shotgun library construction using alkaline lysis
with an AutoGen 850 automated DNA isolation system following the manufacturer's
recommendation (Autogen). Subsequently, the BAC DNA was resuspended in distilled
water (600  l), treated with RNase (Ambion) and purified over a Microcon
100 column (Amicon).
Shotgun library construction and single-stranded DNA isolation.
Purified BAC DNA was sent for shotgun library construction in
M13 phage vector (SeqWright). Approximately 1,400 individual M13 plaques were
gridded into 96-well microtitre dishes and inoculated with Escherichia
coli strain JM101 in 2 YT media for single-stranded DNA isolation
and library storage. We isolated single-stranded DNA in a 96-well format using
the High-through Preparation of M13 DNA (THERMOMAX Prep) protocol from the
Washington University Sequencing Center.
Sample sequencing.
We sequenced single-stranded DNA
using the Energy Transfer fluorescently labelled M13 Forward sequencing primer
(Amersham Pharmacia). Briefly, 100 ng single-stranded template DNA was used
in a reaction for A/C (8 l) and 200 ng in a reaction for G/T (16 l)
with Thermo Sequenase (Amersham Pharmacia). Sequencing reactions were carried
out on an ABI CATALYST 800 Molecular Biology Lab Station (Perkin Elmer) using
the following protocol: 95 °C for 5 s, 55 °C for 10 s, 72 °C for
60 s, for 15 cycles. The four dye primer reactions were subsequently pooled
and precipitated with 95% ethanol (132 l) and glycogen (5 l; Boehringer),
dried by vacuum and resuspended in loading buffer (3 l). We electrophoresed
sequencing reactions in an ABI 377 XL Automated DNA Sequencer (PE Applied
Biosystems). We tracked and analysed the data with DNA Analysis Sequencing
Software 3.2 (PE Applied Biosystems).
Mutation detection.
We designed PCR primers to amplify
CACP from genomic DNA or lymphoblast-derived cDNA (Table 1, http://genetics.nature.com/supplementary_info/
). Typical cycling conditions consisted of a 4 min 95 °C initial
denaturation, followed by 35 cycles of 95 °C for 30 s, annealing temperature
(Table 1, http://genetics.nature.com/supplementary_info/) for 40
s, 72 °C for 1 min, and a final extension at 72 °C for 10 min. We
purified PCR products using Microcon-50 centrifugal filters (Millipore) and
sequenced them either with 33P-end-labelled primers using the
fmol DNA Sequencing System (Promega) or with an ABI 377 with labelled di-deoxy
terminators. We also screened 50 unaffected and unrelated control DNA samples
of United States origin for mutations.
Sequence analysis.
Data generated through systematic
BAC clone sequencing was analysed using WebBLAST (16). On generation of BAC clones giving sufficient coverage, data
was exported from WebBLAST and assembled using the PHRED/PHRAP/CONSED suite28,
29.
Protein truncation test.
We screened for CACP
exon 6 mutations as described30,
31. In brief, we PCR-amplified
template DNA (100−200 ng) using 5' PCR primers that introduce
consensus T7 promoter and Kozak sequences in-frame with the CACP sequence
(Table 1). We used PCR products in a coupled transcription-translation reaction
(Promega) in the presence of 35S-Met, and analysed the resultant
proteins by polyacrylamide gel electrophoresis. Gels were fixed and dried,
and autoradiography performed overnight. Three independent amplifications
of three different primer combinations showed the same result.
Northern-blot analysis.
We probed a bovine northern
blot and a human multiple-tissue northern blot (Clontech) with a 681-bp DNA
fragment generated from human synoviocyte cDNA using MFOR and NREV as primers
(Table 1, http://genetics.nature.com/supplementary_info/). We purified
the probe using a Microcon-50 Centrifugal Filter Device (Millipore) and
32P-dCTP labelled by random priming with the High Prime (Boehringer).
Hybridization was performed at 68 °C in ExpressHyb buffer (Clontech) and
washed at a final stringency of 0.1SSC at 50 °C for 40 min. We
exposed blots to a phosphor screen (Molecular Dynamics) and quantified using
the manufacturer's ImageQuant software. A control human actin probe was also
tested, following the manufacturer's recommended protocol (Clontech).
GenBank accession numbers
CACP, U70136; EST90076,
AA377436.
Note: supplementary information is available on the Nature Genetics web site http://genetics.nature.com/supplementary_info/).
Received 27 July 1999; Accepted 5 October 1999
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Acknowledgments
We thank the families for participating and S. Gregory, B. Lamb, E.
Eichler and members of their labs, J. Ivanovich, K. Gustashaw, J. Preston,
C. Williams, H. Kuivaniemi, G. Tromp, A. Superti-Furga, B. Athreya and I.
Simsek for sharing their clinical and scientific expertise. This work was
supported by a Biomedical Research Grant from the Arthritis Foundation and
NIH grant AR43827 (both to M.L.W.).
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