A novel COMPmutation in a pseudoachondroplasia family of Chinese origin
© Dai et al; licensee BioMed Central Ltd. 2011
Received: 13 December 2010
Accepted: 21 May 2011
Published: 21 May 2011
Pseudoachondroplasia (PSACH) is caused exclusively by mutations in the gene for cartilage oligomeric matrix protein (COMP). Only a small number of studies have documented the clinical phenotype and genetic basis in Chinese PSACH patients.
We investigated a four-generation PSACH pedigree of Chinese Han origin. Two patients and two unaffected individuals were recruited for clinical evaluation and molecular genetic analysis. The genomic DNA was extracted from peripheral blood leukocytes. Polymerase chain reaction (PCR) was adopted to amplify the 8-19 exons of COMP gene. Then the products were sequenced bi-directionally for screening mutation. Clinical evaluation revealed that PSACH patients in this pedigree had a severe disproportionate short stature (-10SD). A heterozygous TGTCCCTGG insertion in exon 13, between nucleotide 1352T and 1353G, were identified in the patients except the unaffected individuals, which resulted in a three-amino-acid insertion (451V_452P ins VPG) in the sixth calmodulin-like repeat of the COMP protein.
This c. 1352_1353ins TGTCCCTGG is a novel mutation responsible for severe familial PSACH.
Pseudoachondroplasia (PSACH, OMIM 177170) is a rare autosomal dominant osteochondrodysplasia characterized by typical disproportionate short stature, brachydactyly, lower limbs anomalies, joint laxity, scoliosis, early onset osteoarthritis, epiphyseal and metaphyseal abnormalities. It is estimated to affect at least 1 in 20,000 persons [1, 2]. The affected individual has a normal length at birth,and growth retardation generally will not be recognized until two or three years of age. Notably, all patients have normal craniofacial appearance and intelligence [1, 3–5].
The PSACH gene was initially localized to chromosome 19 in 1993 [6, 7]. Mutations on the Cartilage Oligomeric Matrix Protein (COMP) gene were subsequently found to cause PSACH and another skeletal dysplasia, multiple epiphyseal dysplasia [8, 9]. COMP protein is a large secreted pentameric glycoprotein of the thrombospondin family, expressed in the extracellular matrix (ECM) surrounding the cells that make up ligaments and tendons. COMP monomer consists of an amino-terminal domain, four type IIepidermal growth like repeats, eight type III calmodulin-like repeats (CLRs), and a carboxyl-terminal globular domain. Numerous mutations of COMP gene have been found to date, most of them occur in the CLR regions (~85%), while others in the C-terminal globular domain (~15%) (the Human Gene Mutation Database, http://www.hgmd.cf.ac.uk/ac/index.php). Although the normal function is not fully known, the pathological link between gene mutation, protein alteration, abnormal cell growth and PSACH has been established [1, 10–17]. More than 60 COMP mutations have been identified to be with PSACH [10, 18], but only a few in Chinese patients [19, 20]. Severe short stature (-6 SD) in PSACH has been associated with mutations in exon 13, particularly in the region encoding CLR7 of COMP protein . Elliott AM et al recently detected the c.812A→T in exon 8 in a sporadic Inuit PSACH patient with severe short stature (-7.5 SD) . In the present study, we examined the COMP gene for mutations in a Chinese family affected by severe PSACH.
Subjects and methods
Using a commercially available kit (Bio Teke, Beijing, China), genomic DNA was isolated from blood samples utilizing the standard protocol provided by the manufacturer of the kit. The procedure used for amplifying COMP exons 8-19 (accession number NM000095) and PCR is outlined previously . The resulting product was purified, separated by PAGE, and sequenced bi-directionally using an ABI 3730xl sequencer.
Clinical features and skeletal radiographic findings
Mutational analysis of the COMP gene
Up to now, COMP is the only gene known to be associated with pseudoachondroplasia. COMP mutations and pathological consequences have been extensively studied in Caucasians, but a few in the Chinese population. Besides the c.815C→T in exon 8  and the c.1447-1455del  in exon 13 of COMP gene reported lately in Chinese PSACH patients, we identified the c. 1352_1353insTGTCCCTGG within exon 13 of COMP that contributes to severe PSACH phenotype in a Han Chinese pedigree. This novel trinucleotide expansion was located in the region coding the sixth CLR (p. 451V_452PinsVPG) and expands the knowledge of COMP mutations leading to PSACH.
PSACH mutations are located in exons 8-14 of COMP gene encoding the type III CLRs of COMP protein [3, 18, 28]. The most frequent mutation, present in one third of PSACH patients, is the deletion of GAC (c.1405-1419delGAC) in a very short triplet repeat (GAC5), which encodes five consecutive aspartic acid residues within the CLR7 of the protein, usually referred as p.469delD, p.473 delD or p.del D469-473 [3, 8, 29, 30]. Most other COMP mutations involve the substitution of one amino acid for another in the COMP protein. Trinucleotide expansion at the region encoding type III CLRs of the protein has now been reported in this study and one other previous study . Genotype-phenotype studies revealed that mutations in CLR7 were associated with more severe PSACH phenotypes than those in elsewhere in the CLRs . The early onset characteristics of PSACH, severe short stature, generalized dysplasia of epiphyses and metaphyses, in these individuals with mutations in other CLRs continues to enforce the importance of the COMP gene.
COMP is a multifunctional structural protein that affects cellular attachment, proliferation and chondrogenesis. It also participates in the ECM assembly through interactions with numerous proteins including types I, II, IV, XI, XII collagen, decorin, fibronectin and matrilin-3 [32, 33]. According to the newly published crystal structure of the COMP protein , the p.451V_452PinsVPG was located to a pocket structure in the end of the sixth CLR of the COMP protein, where a calcium ion is embedded. Calcium supports the binding of COMP to fibronectin which reportedly influences the conformation of COMP . Specifically, alterations in CLRs make calcium-dependant protein folding malfunction [11, 30, 32, 34–37], lead to the assembly of mutant COMP and other proteins in the rER of affected chondrocytes . This intracellular retention is cytotoxic and prevents efficient secretion of these proteins, alters the extracellular matrix and results in premature chondrocyte death. Eventually, the upregulated apoptosis of chondrocytes from the growth plate diminishes linear growth, and the abnormal ECM causes early onset osteoarthritis observed in PSACH patients [1, 38–40]. In PSACH, only a small fraction of COMP pentamers (3% or less) contains all wild-type COMP subunits, most of the COMP pentamers contain one or more mutant subunits that exert dominant negative effects [8, 30]. A mouse model built by Posey et al. recapitulated this cellular pathology of human PSACH, including the retention of ECM proteins, intracellular matrix formation in the rER cisternae, and increased apoptosis of chondrocyte . Previous studies have shown that calcium-binding is critical in the function of the COMP protein, and missense mutations or in-frame deletions in the calmodulin-like repeats region reduces this ability . The longer amino acids sequence in CLR6 due to this novel mutation may have similar negative effects on the function of the COMP protein, the proliferation and apoptosis of chondrocytes. This study suggests that the 9 bp insertion can be responsible for the severe phenotype in the Chinese family in despite no direct functional evidence. In conclusion, the c.1352_1352insTGTCCCTGG within exon 13 of COMP expands the spectrum of mutations that causes PSACH. It could be of value to the molecular diagnosis, genotype-phenotype relationship understanding and pathogenesis of this disorder.
cartilage oligomeric matrix protein
calmodulin-like repeat domain
We are very grateful to the persons who participated in this work. This work was supported in part by the National Natural Science Foundation of China (Grant ID: 30700679, mutation screening), by the State Key Basic Research Project (Grant ID: 2007CB511905, specimen collection), and by the National Institutes of Health Fogarty training grants 1D43TW008323-01 and 1D43TW007864-01.
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