Identification of twelve mutations in cartilage oligomeric matrix protein (COMP) in patients with pseudoachondroplasia

Pseudoachondroplasia (PSACH) is an autosomal dominant dwarfing condition characterized by disproportionate short stature, joint laxity, and early-onset osteoarthrosis. PSACH is caused by mutations in the gene encoding cartilage oligomeric matrix protein (COMP). We are reporting on mutations in COMP in 12 patients with PSACH, including ten novel mutations. Eleven of the mutations are in exons 17A, 17B, and 18A, which encode the calcium-binding domains, and one mutation is in exon 19, which encodes part of the carboxy-terminal globular domain. Two of the mutations identified are the common delGAC(1430–1444) in exon 17B, which accounts for 36% of identified PSACH mutations. This report increases the range of mutations in COMP that cause PSACH and provides additional evidence for the importance of the calcium-binding domains and the globular domain to the function of COMP. Am. J. Med. Genet. 80:510–513, 1998. © 1998 Wiley-Liss, Inc.     

Identification of cartilage oligomeric matrix protein (COMP) gene mutations in patients with pseudoachondroplasia and multiple epiphyseal dysplasia

Mutations in the cartilage oligomeric matrix protein (COMP) gene are responsible for two dominantly inherited skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). Mutation analysis of the COMP gene in Korean patients with PSACH and MED was performed. All nine patients with PSACH had mutations in the COMP gene, while three of the five patients with MED had detectable COMP mutations. Eight mutations, including three novel mutations, were identified in the COMP gene in the patients with PSACH and MED. Six mutations were found within the calmodulin-like repeats (CLRs) domain, especially in the seventh CLR and the other two mutations were in exon 16 outside of CLRs, which encode the C-terminal globular domain. Among the three novel mutations, two were missense mutations (Asp473Tyr, Asp482His) and one was a consecutive two-codon deletion, delAspAsp(469–473) in the five consecutive aspartic acid residues. All three novel mutations produced the PSACH phenotype. Electronic Publication     

COMP (cartilage oligomeric matrix protein) is synthesized in ligament, tendon, meniscus, and articular cartilage

The presence of cartilage oligomeric matrix protein (COMP) in extracts of ligament, tendon, meniscus, and canine articular cartilage was demonstrated by Western blot analysis using anti-dog COMP antibody. When the tissues were cultured in the presence of [35-S]methionine/cysteine, metabolically labeled COMP was purified from the culture media and from tissue extracts by DEAE-cellulose gel chromatography. SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography and immunoblotting under reducing and non-reducing conditions revealed that COMP is synthesized by the cells of these connective tissues. Increased levels of COMP in samples of both synovial fluid and serum of patients with various joint diseases may not only be derived from cartilage but also from ligaments and tendons. COMP is not a highly tissue-specific cartilage molecule.     

A mutation in cartilage oligomeric matrix protein (COMP) causes early-onset osteoarthritis in a large kindred study

We performed a genome-wide linkage analysis to identify susceptibility loci in a large six-generation extended family previously reported with early-onset osteoarthritis (OA) DNA sequencing was performed to investigate involvement of the COMP (Cartilage oligomeric matrix protein) gene in this family. The region covering D19S884, D19S226, and D19S414 on chromosome 19p following genome-wide scan from 70 individuals of this kindred showed significant linkage, with a maximum point LOD (logarithm of the odds ratio) score of 2.51 at D19S226. Direct sequencing of the COMP gene, the most plausible candidate gene in the region, identified a c.2152C>T substitution in exon 18 which resulted in a substitution of tryptophan for arginine at position 718 located in the C terminal globular domain of the gene product. A total of 26 individuals were identified with this mutation of which 21 affected individuals had the mutation, and the other five younger individuals (18.6 ± 11.3 years of age) carried the mutation without symptoms. The results indicate that COMP is the disease susceptibility gene and the c.2152C>T mutation in exon 18 could cause early-onset OA phenotypes in this kindred, which is compatible with a previous report that this mutation also causes a mild form of multiple epiphyseal dysplasia (MED).     

Non-collagenous protein screening in the human chondrodysplasias: Link proteins,cartilage oligomeric matrix protein (COMP), and fibromodulin

A gel-electrophoretic screening for link proteins, cartilage oligomeric matrix protein (COMP), and fibromodulin abnormalities was performed in fetuses, newborn infants, and children with various types of chondrodysplasia. Microdissected freeze-dried sections of upper tibial growth cartilage were extracted with 4M guanidinium chloride in the presence of proteolysis inhibitors. After dialysis against 8M urea, the extracts were submitted to stepwise ion-exchange chromatography to separate the large proteoglycans (aggrecans) from the other components. The latter were analyzed by gel electrophoresis, electrotransferred onto nitrocellulose membranes, and reacted with specific antibodies. Control samples from individuals with apparently normal growth were analyzed in the same runs. Two link protein bands with abnormal electrophoretic migration were found in a sporadic case of spondylometaphyseal dysplasia, Kozlowski type. Three link protein bands with the same migration as in the control samples were found in thanatophoric dysplasia, homozygous achondroplasia, achondrogenesis type II, hypochondrogenesis, Goldblatt syndrome, Desbuquois dysplasia, pseudo-achondroplasia, and diastrophic dysplasia. In several pathologic cases with normal electrophoretic pattern of the link proteins, small link protein fragments appeared after reduction. The gel electrophoretic pattern of COMP was studied in thanatophoric dysplasia, diastrophic dysplasia, homozygous achondroplasia, fibrochondrogenesis, hypochondrogenesis, Goldblatt syndrome, and Kniest dysplasia. In all these cases the pattern was the same as in the control samples. The main band of fibromodulin had a normal migration rate in fibrochondrogenesis, Desbuquois dysplasia, Kniest dysplasia, and pseudoachondroplasia. It was delayed in diastrophic dysplasia. © 1994 Wiley-Liss, Inc.     

Identification of sequence polymorphisms of the COMP (cartilage oligomeric matrix protein) gene and association study in osteoarthrosis of the knee and hip joints

Osteoarthrosis (OA) is a common cause of musculoskeletal disability characterized by late-onset degeneration of articular cartilage. Although several candidate genes have been reported, susceptibility genes for OA remain to be determined. Hereditary osteochondral dysplasias produce severe, early-onset OA and hence are models for common idiopathic OA. Among them are pseudoachondroplasia and multiple epiphyseal dysplasia, both of which are caused by mutations in the cartilage oligomeric matrix protein (COMP) gene. Therefore, COMP may be a susceptibility gene for OA. We screened for polymorphisms by direct sequencing of all exons of the COMP gene with their flanking intron sequences and the promoter region. We identified 16 polymorphisms, of which 12 were novel. Using six polymorphisms spanning the entire COMP gene, we examined the association of COMP in Japanese patients with OA of the knee and hip joints. Genotype and allele frequencies of the polymorphisms were not significantly different between OA and control groups, and there was no significant difference in haplotypes. These results do not support an association between COMP and OA in the Japanese population. Received: March 30, 2001 / Accepted: May 7, 2001     

多发性骨骺发育不良患者软骨寡聚物基质蛋白基因新突变

目的 对1例多发性骨骺发育不良女性患者的软骨寡聚物基质蛋白(cartilage oligomeric matrix protein,COMP)基因进行突变分析,为遗传咨询和产前分子诊断提供指导.方法 采集患者血样,提取基因组DNA,用外显子序列捕获+第二代测序技术对COMP基因进行基因突变的检测并用聚合酶链反应结合Sanger法核苷酸序列测定进行突变位点验证.结果 该患者COMP基因第9外显子存在c.956A＞T错义突变,其COMP基因第319位密码子由原来编码天冬氨酸的密码子GAC突变为编码缬氨酸的密码子GTC(p.Asp319Val).SIFT功能预测该错义突变将影响蛋白结构.结论 该患者发病是由COMP基因c.956A＞T突变导致,该突变国内外未见报道.     

Multiple epiphyseal dysplasia and pseudoachondroplasia due to novel mutations in the calmodulin-like repeats of cartilage oligomeric matrix protein

A child with a mild form of pseudoachondroplasia was heterozygous for a deletion of 12 nucleotides from exon 10 of the cartilage oligomeric matrix protein (COMP) gene. It resulted in the deletion of valine 513 to lysine 516 from the eighth calmodulin-like repeat of COMP monomers. A child with the Fairbank's type of multiple epiphyseal dysplasia was also heterozygous for a COMP mutation. It substituted cysteine 371 by serine in the fourth calmodulin-like repeat. Both mutations were likely to alter the conformation and calcium binding of the mutant COMP protein chains. These findings support the proposal that deletions and insertions within the calmodulin-like domain produce pseudoachondroplasia, while amino acid substitutions with this domain may produce either pseudoachondroplasia or multiple epiphyseal dysplasia.     

Novel mutation in exon 18 of the cartilage oligomeric matrix protein gene causes a severe pseudoachondroplasia

Pseudoachondroplasia (PSACH) is a common skeletal dysplasia characterized by disproportionate short stature, early‐onset osteoarthrosis, and dysplasia of the spine, epiphysis, and metaphysis. Multiple epiphyseal dysplasia (MED) is a similar but less severe disorder characterized by dysplasia of the epiphysis. Both disorders are caused by mutations in the cartilage oligomeric matrix protein (COMP) gene. COMP mutations cluster in a region of the gene that encodes calmodulin‐like repeats (CLRs) and correlate closely with disease severity. Typically, mutations in exon 13 that composes the seventh CLR produce severe PSACH phenotypes, whereas mutations found elsewhere in the gene produce mild PSACH or MED phenotypes. We have identified a PSACH patient carrying a novel mutation in exon 18 of COMP that composes the C‐terminal globular domain. This mutation produced a severe PSACH phenotype with marked short stature and deformities of the spine and extremities. Our results extend the range of disease‐causing mutations within the COMP gene and demonstrate the importance of the additional domain of COMP protein in its in vivo function. © 2001 Wiley‐Liss, Inc.     

Identification of nine novel mutations in cartilage oligomeric matrix protein in patients with pseudoachondroplasia and multiple epiphyseal dysplasia

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1) are allelic disorders caused by mutations in the gene encoding cartilage oligomeric matrix protein (COMP). PSACH is a dominant condition characterized by disproportionate short stature, joint laxity, and early-onset osteoarthritis. EDM1 is a less severe skeletal dysplasia associated with average to mild short stature, joint pain, and early-onset osteoarthritis. COMP is an extracellular matrix protein present in cartilage, ligament, and tendon tissues. Here, we report on nine novel mutations in COMP causing PSACH and EDM1. Four of these mutations are in exons 13C and 14 where no previous mutations had been reported. One of those mutations was identified in two separate EDM1 families. In addition, we have identified the first case of PSACH resulting from an expansion of the five aspartates in exon 17B. We are also reporting a mutation in a third PSACH family with somatic/germline mosaicism. Therefore, this report increases the range of mutations that cause PSACH and EDM1 and provides additional regions to target for mutational analysis. Am. J. Med. Genet. 85:486–490, 1999. © 1999 Wiley-Liss, Inc.     

Novel mutation in exon 18 of the cartilage oligomeric matrix protein gene causes a severe pseudoachondroplasia.

Pseudoachondroplasia (PSACH) is a common skeletal dysplasia characterized by disproportionate short stature, early-onset osteoarthrosis, and dysplasia of the spine, epiphysis, and metaphysis. Multiple epiphyseal dysplasia (MED) is a similar but less severe disorder characterized by dysplasia of the epiphysis. Both disorders are caused by mutations in the cartilage oligomeric matrix protein (COMP) gene. COMP mutations cluster in a region of the gene that encodes calmodulin-like repeats (CLRs) and correlate closely with disease severity. Typically, mutations in exon 13 that composes the seventh CLR produce severe PSACH phenotypes, whereas mutations found elsewhere in the gene produce mild PSACH or MED phenotypes. We have identified a PSACH patient carrying a novel mutation in exon 18 of COMP that composes the C-terminal globular domain. This mutation produced a severe PSACH phenotype with marked short stature and deformities of the spine and extremities. Our results extend the range of disease-causing mutations within the COMP gene and demonstrate the importance of the additional domain of COMP protein in its in vivo function.     

Genotype to phenotype correlations in cartilage oligomeric matrix protein associated chondrodysplasias

Pseudoachondroplasia (PSACH) and autosomal dominant multiple epiphyseal dysplasia (MED) are chondrodysplasias resulting in short-limbed dwarfism, joint pain and stiffness and early onset osteoarthritis. All PSACH, and the largest proportion of MED, result from mutations in cartilage oligomeric matrix protein (COMP). The first mutations in COMP were identified in 1995 in patients with both PSACH and MED and subsequently there has been over 30 publications describing COMP mutations in at least 250 PSACH–MED patients. However, despite these discoveries, a methodical analysis of the relationship between COMP mutations and phenotypes has not been undertaken. In particular, there has, to date, been little correlation between the type and location of a COMP mutation and the resulting phenotype of PSACH or MED. To determine if genotype to phenotype correlations could be derived for COMP, we collated 300 COMP mutations, including 25 recently identified novel mutations. The results of this analysis demonstrate that mutations in specific residues and/or regions of the type III repeats of COMP are significantly associated with either PSACH or MED. This newly derived genotype to phenotype correlation may aid in determining the prognosis of PSACH and MED, including the prediction of disease severity, and in the long term guide genetic counselling and contribute to the clinical management of patients with these diseases.     

Identification of nine novel mutations in cartilage oligomeric matrix protein in patients with pseudoachondroplasia and multiple epiphyseal dysplasia.

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1) are allelic disorders caused by mutations in the gene encoding cartilage oligomeric matrix protein (COMP). PSACH is a dominant condition characterized by disproportionate short stature, joint laxity, and early-onset osteoarthritis. EDM1 is a less severe skeletal dysplasia associated with average to mild short stature, joint pain, and early-onset osteoarthritis. COMP is an extracellular matrix protein present in cartilage, ligament, and tendon tissues. Here, we report on nine novel mutations in COMP causing PSACH and EDM1. Four of these mutations are in exons 13C and 14 where no previous mutations had been reported. One of those mutations was identified in two separate EDM1 families. In addition, we have identified the first case of PSACH resulting from an expansion of the five aspartates in exon 17B. We are also reporting a mutation in a third PSACH family with somatic/germline mosaicism. Therefore, this report increases the range of mutations that cause PSACH and EDM1 and provides additional regions to target for mutational analysis.     

Thrombospondin-4 and cartilage oligomeric matrix protein form heterooligomers in equine tendon

Injuries of the equine superficial digital flexor tendon are common in racing horses. Knowledge of the tendon matrix composition is crucial to understand physiological and pathological processes in the tendon. The aim of this study was to analyze TSP-4 expressed in equine tendon. Equine tendons were extracted with 10 mM EDTA-containing buffer and TSP-4 purified with ion-exchange chromatography followed by heparin affinity chromatography. The purified TSP-4 was analyzed by one- and two-dimensional SDS-PAGE, immunoblotting, and MALDI-TOF mass spectrometry. Purified TSP-4 gave bands reacting with a TSP-4 specific antiserum, but also with an antiserum to COMP, when submitted to SDS-PAGE under nonreducing conditions. Two-dimensional SDS-PAGE (nonreducing followed by reducing conditions) and immunoprecipitation as well as MALDI-TOF mass spectrometry analysis showed that TSP-4 and COMP are both present in equine tendon and cannot be separated under nonreducing conditions despite significant differences in subunit size. This suggests that they are connected via disulfide bridges into heterooligomers.     

Changes in serum cartilage oligomeric matrix protein (COMP), plasma CPK and plasma hs-CRP in relation to running distance in a marathon (42.195 km) and an ultra-marathon (200 km) race

Marathon running is frequently associated with numerous cellular changes, but little information is available on the effects of exercise-mediated prolonged impact-stress on cartilage integrity. This study was undertaken to evaluate muscle and cartilage damage with different running distances. Twenty male marathoners and ultra-marathoners participated in the study. Serum COMP and plasma CPK and hs-CRP were measured as markers of cartilage and muscle damage and inflammation. Serum COMP was increased 1.6-fold at 10 km during a marathon race and declined to the pre-race level after 2 days recovery. In contrast, serum COMP was increased 1.9-fold after a 200-km race and maintained until day 3 of recovery, only returning to the pre-race level on day 6. Plasma CPK was increased at 10 km of the marathon race and up to threefold at the end of the race. This was further increased on day 1, only returning to pre-race level on day 6. Plasma CPK was increased 35-fold at the end of the 200-km race and remained increased until day 5. There was no change in plasma hs-CRP during the marathon race, but this was increased 3.4-fold by day 1, returning to the pre-race level on day 4. Plasma hs-CRP increased 40-fold by the end of the 200-km race and was still increased on day 6 of recovery. Therefore, longer distance running may induce more impact-stress both on muscle and cartilage. Further, the required time for recovery may vary with running distance and the tissue type, e.g. cartilage or skeletal muscle as in this case.     

软骨寡聚基质蛋白在SD大鼠肺动脉平滑肌表型转化中的作用

目的探讨软骨寡聚基质蛋白(COMP)对肺动脉平滑肌细胞(PASMCs)表型转化的影响。方法利用常氧和缺氧条件培养的SD大鼠(21%O_2或10%O_2饲养21d)与PASMCs(21%O_2或3%O_2培养48h),采用免疫组织化学染色、Western blot和q-PCR方法检测大鼠肺组织和PASMCs COMP表达;qPCR法检测特异性siRNA敲低COMP条件下,SM22α-、SMα-actin-和calponin mRNA水平的表达;Western blot法检测缺氧、siRNA-COMP或腺病毒过表达COMP后,SM22α、SMα-actin、calponin、osteopontin和proliferating cell nuclear antigen(PCNA)蛋白的表达。结果缺氧条件下,大鼠肺组织和PASMCs中COMP表达水平降低;COMP缺失引起PASMCs收缩表型蛋白SM22α和calponin的表达减少,osteopontin和PCNA表达增加;过表达COMP可以逆转缺氧引起的肺动脉平滑肌细胞SM22α和calponin表达的减少。结论 COMP有助于维持缺氧条件下肺动脉平滑肌处于收缩表型。     

Different mechanical loading protocols influence serum cartilage oligomeric matrix protein levels in young healthy humans

The purpose of the study was to investigate whether a relationship between the loading mode of physical activity and serum cartilage oligomeric matrix protein (COMP) concentration exists and whether the lymphatic system contributes to COMP release into the serum. Serum COMP levels were determined in healthy male subjects before, after and at 18 further time points within 7 h at four separate experimental days with four different loading interventions. The loading intervention included high impact running exercise, slow but deep knee bends, and lymphatic drainage of 30 min duration, respectively, and a resting protocol. The serum COMP levels were measured using a commercially available quantitative enzyme-linked immunosorbent assay. An increase (p < 0.001) in serum COMP concentration was detected immediately after 30 min running exercise. Slow but deep knee bends did not cause any significant changes in serum COMP levels. Lymphatic drainage also had no effect on the serum COMP concentration. After 30 min of complete rest the serum COMP level was significantly (p = 0.008) reduced. The elevation of COMP serum concentration seems to depend on the loading mode of the physical activity and to reflect the extrusion of COMP fragments from the impact loaded articular cartilage or synovial fluid.     

Identification and characterization of cartilage oligomeric matrix protein as a novel pathogenic factor in keloids

To elucidate pathogenic molecules in keloids, microarray analysis was performed using RNAs extracted from keloid-derived fibroblasts and normal skin-derived fibroblasts from the same patient with a typical keloid. Among 11 up-regulated extracellular matrix genes, cartilage oligomeric matrix protein (COMP) was most prominently increased. Up-regulation of COMP mRNA and protein was confirmed in the keloid tissue by quantitative RT-PCR and Western blot. Using immunohistochemistry, we compared 15 keloids and 6 control normal tissues using a COMP-specific antibody and found that COMP stained positively in 10 keloids (66.7%), whereas no staining was observed in normal tissues, demonstrating the ectopic expression of COMP in keloids. Comparing keloids smaller or larger than 10 cm(2), the larger keloids were significantly more intensely stained with the COMP-specific antibody. Because COMP reportedly accelerates collagen type I fibril assembly, we examined whether extracellular type I collagen deposition is altered by silencing COMP mRNA by small interfering RNA (siRNA). Immunocytochemistry showed at 96 hours after transfection with COMP siRNA that the extracellular deposition of type I collagen was decreased compared to that observed with control siRNA. Further, COMP knockdown decreased amount collagens type I to V in the medium and on the cell surfaces. Our data suggest that COMP facilitates keloid formation by accelerating collagen deposition, thus providing a new therapeutic target.     

Mutation (D472Y) in the type 3 repeat domain of cartilage oligomeric matrix protein affects its early vesicle trafficking in endoplasmic reticulum and induces apoptosis

Cartilage oligomeric matrix protein (COMP) is a large pentameric extracellular glycoprotein found in cartilage, tendon, and synovium, and plays structural roles in cartilage as the fifth member of the thrombospondin family. Familial mutations in type 3 repeats of COMP are known to cause pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). Although such mutations induce enlarged rough endoplasmic reticulum (rER) as a morphological change, the metabolic trafficking of mutated COMP remains unclear. In transfected COS7 cells, wild-type COMP was rapidly secreted into culture medium, while the great majority of COMP with the type 3 repeats mutation (D472Y) remained in the cells and a small portion of mutated COMP was secreted. This finding was followed up with a confocal study with an antibody specific to COMP, which demonstrated mutated COMP tightly associated with abnormally enlarged rER. Phosphorylated eIF2alpha, an ER stress protein, was expressed as a pathological reaction in virtually all COS7 cells expressing mutated but not wild-type COMP. Moreover, COS7 cells expressing mutated COMP exhibited significantly more apoptotic reaction than those expressing wild-type COMP. Pathological accumulation of COMP in rER and apoptosis in COS7 cells that were induced by the mutation (D472Y) in COMP imply that COMP mutations play a role in the pathogenesis of PSACH.