Biomedical subjects
Richard Haynes
Publications and source records attributed to Richard Haynes.
Expression of mutant cartilage oligomeric matrix protein in human chondrocytes induces the pseudoachondroplasia phenotype.
Over 70 mutations in the cartilage oligomeric matrix protein (COMP), a large extracellular pentameric glycoprotein synthesized by chondrocytes, have been identified as causing two skeletal dysplasias: multiple epiphyseal dysplasia (MED/EDM1), and a dwarfing condition, pseudoachondroplasia (PSACH). These mutations induce misfolding of intracellular COMP, resulting in retention of the protein in the rough endoplasmic reticulum (rER) of chondrocytes. This accumulation of COMP in the rER creates the phenotypic enlarged rER cisternae in the cells, which is believed to compromise chondrocyte function and eventually cause cell death. To study the molecular mechanisms involved with the disease, we sought to develop an in vitro model that recapitulates the PSACH phenotype. Normal human chondrocytes were transfected with wildtype (wt-) COMP or with mutant COMP (D469del; mt-) recombinant adenoviruses and grown in a nonattachment redifferentiating culture system that provides an environment allowing formation of a differentiated chondrocyte nodule. Visualization of normal cells expressing COMP suggested the hallmarks of the PSACH phenotype. Mutant COMP expressed in normal cells was retained in enlarged rER cisternae, which also retained IX collagen (COL9) and matrilin-3 (MATN3). Although these proteins were secreted normally into the ECM of the wt-COMP nodules, reduced secretion of these proteins was observed in nodules composed of cells transfected with mt-COMP. The findings complement those found in chondrocytes from PSACH patient growth plates. This new model system allows for production of PSACH chondrocyte pathology in normal costochondral chondrocytes and can be used for future mechanistic and potential gene therapy studies.
In vivo human Cartilage oligomeric matrix protein (COMP) promoter activity.
Cartilage oligomeric matrix protein (COMP) is a large extracellular matrix protein whose function is unknown. Mutations in COMP cause pseudoachondroplasia and multiple epiphyseal dysplasia, two skeletal dysplasias which are associated with intracellular retention of COMP in chondrocytes. In contrast, COMP null mice are normal suggesting gene redundancy or that the detrimental effect is associated with mutant COMP rather than the absence of functional COMP. To define the elements that regulate COMP transcription and tissue-specificity, we have evaluated the human COMP promoter driving fusion gene expression in vitro and in vivo. COMP promoter activity is higher in rat chondrosarcoma cells (RCS) than in a fibroblast cell line. In RCS cells, expression of a reporter gene containing 1.7 kb of the human COMP promoter was three-fold higher than all shorter COMP promoter constructs. In transgenic mice, 1.7 kb of the human COMP promoter is active early in development in the limbs, spine, and eye. As development progresses, promoter activity diminishes in the eye and migrates from the center to the ends of the long bones. On the other hand, while 375 bp of the human COMP promoter is sufficient for proper tissue-specific expression, levels are less than those found with the 1.7-COMP promoter. The expression pattern of both promoters recapitulates endogenous cartilage COMP expression in mice. Our findings indicate that the elements required for chondrocyte-specific expression lie within 375 bp of the translational start site, while DNA enhancer elements are located between 1.0 to 1.7 kb.
Differentiation-induced loss of heparan sulfate in human exostosis derived chondrocytes.
An exostosis or osteochondroma is an aberrant bony growth occurring next to the growth plate either as an isolated growth abnormality or as part of the Hereditary Multiple Exostosis (HME) syndrome. Mutations in either exostosin 1 (EXT1) or exostosin 2 (EXT2) gene cause the HME syndrome and also some isolated osteochondromas. The EXT1 and EXT2 genes are glycosyltransferases that function as hetero-oligomers in the Golgi to add repeating glycosaminoglycans (GAGs) to heparan sulfate (HS) chains. Previously, we demonstrated that HS is markedly diminished in the exostosis cartilage cap and that the HS proteoglycan, perlecan, has an abnormal distribution in these caps. The present studies were undertaken to evaluate which chondrocyte-specific functions are associated with diminished HS synthesis in human chondrocytes harboring either EXT1 or EXT2 mutations. Systematic evaluation of exostosis cartilage caps and chondrocytes, both in vitro and in vivo, suggests that chondrocyte-specific cell functions account for diminished HS levels. In addition, we provide evidence that perichondrial cells give rise to chondrocytes that clonally expand and develop into an exostosis. Undifferentiated EXT chondrocytes synthesized amounts of HS similar to control chondrocytes; however, EXT chondrocytes displayed very poor survival in vitro under conditions that promote normal chondrocyte differentiation with high efficiency. Collectively, these observations suggest that loss of one copy of either the EXT1 or EXT2 gene product compromises the perichondrial chondrocytes' ability to differentiate normally and to survive in a differentiated state in vitro. In vivo, these compromised responses may lead to abnormal chondrocyte growth, perhaps from a perichondrial stem cell reserve.
COMP mutations, chondrocyte function and cartilage matrix.
Cartilage oligomeric matrix protein (COMP) is a large extracellular pentameric glycoprotein found in the territorial matrix surrounding chondrocytes. More than 60 unique COMP mutations have been identified as causing two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED/EDM1). Recent studies demonstrate that calcium-binding and calcium induced protein folding differ between wild type and mutant COMP proteins and abnormal processing of the mutant COMP protein causes the characteristic large lamellar appearing rough endoplasimic reticulum (rER) cisternae phenotype observed in PSACH and EDMI growth plate chondrocytes. To understand the cellular events leading to this intracellular phenotype, PSACH chondrocytes with a G427E, D469del and D511Y mutations were grown in 3-D culture to produce cartilage nodules. Each nodule was assessed for the appearance and accumulation of cartilage-specific proteins within the rER and for matrix protein synthesis. All three COMP mutations were associated with accumulation of COMP in the rER cisternae by 4 weeks in culture, and by 8 weeks the majority of chondrocytes had the characteristic cellular phenotype. Mutations in COMP also affect the secretion of type IX collagen and matrilin-3 (MATN3) but not the secretion of aggrecan and type II collagen. COMP, type IX collagen and MATN3 were dramatically reduced in the PSACH matrices, and the distribution of these proteins in the matrix was diffuse. Ultrastructural analysis shows that the type II collagen present in the PSACH matrix does not form organized fibril bundles and, overall, the matrix is disorganized. The combined absence of COMP, type IX collagen and MATN3 causes dramatic changes in the matrix and suggests that these proteins play important roles in matrix assembly.
Role of TSP-5/COMP in pseudoachondroplasia.
Pseudoachondroplasia (PSACH) is a well-characterized dwarfing condition associated with disproportionate short stature, abnormal joints and osteoarthritis requiring joint replacement. PSACH is caused by mutations in cartilage oligomeric matrix protein (COMP). COMP, the fifth member of the thrombospondin (TSP) gene family, is a pentameric protein found primarily in the extracellular matrix of musculoskeletal tissues. Functional studies have shown that COMP binds types II and IX collagens but the role of COMP in the extracellular matrix remains to be defined. Mutations in COMP interfere with calcium-binding and protein conformation. PSACH growth plate and growth plate chondrocytes studies indicate that COMP mutations have a dominant negative effect with both COMP and type IX collagen being retained in large rER cisternae. This massive retention causes impaired chondrocyte function with little COMP secreted into the matrix and premature loss of chondrocytes. Deficiency of linear growth results from loss of chondrocytes from the growth plate. Secondarily, the matrix contains minimal COMP, which may be normal and/or mutant, and little type IX collagen. This deficiency results in abnormal joints that are easily eroded and cause painful osteoarthritis. Unlike other misfolded proteins that are targeted for degradation, much of the retained COMP escapes degradation, compromises cell function, and causes cell death. Gene therapy will need to target the reduction of COMP in order to restore normal chondrocyte function and longevity.
Chondrocyte cell death and intracellular distribution of COMP and type IX collagen in the pseudoachondroplasia growth plate.
Cartilage oligomeric matrix protein (COMP) is a large extracellular matrix protein expressed in cartilage, ligament and tendon. Mutations in the COMP gene cause two dominantly inherited skeletal dysplasias, pseudoachondroplasia (PSACH) and Multiple Epiphyseal Dysplasia (MED/EDM1). We report on a novel point mutation D511Y in the seventh calcium-binding repeat of the COMP gene and the resulting iliac crest growth plate pathology. The PSACH iliac crest growth plate is comprised of a large region of resting chondrocytes above a narrow region composed of clusters of disorganized proliferative and hypertrophic chondrocytes. Chondrocytes in all zones show massive intracellular retention of COMP and the surrounding extracellular matrix is deficient in COMP. Moreover, the 511Y COMP mutation selectively affects type IX collagen as little is found in the growth plate matrix whereas type II collagen and aggrecan are abundant in the matrix. Chondrocyte remnants are observed in the chondrocyte clusters and dead cells are found throughout the growth plate. Apoptosis studies demonstrate an unusual pattern of TUNEL staining in the PSACH chondrocytes compared to the control growth plate. These in vivo findings support our previous observation that retention of COMP leads to chondrocyte death. These results also add to the increasing evidence that PSACH and EDM1 are rER storage diseases and that impaired linear growth and joint erosion are caused by the disruptive effect of massive amounts of COMP within the chondrocytes.
Reevaluation of a genetic model for the development of exostosis in hereditary multiple exostosis.
EXT1 and EXT2 are genes that have been shown to cause hereditary multiple exostosis (HME), a syndrome marked by the formation of bony growths juxtaposed to the growth plate. These genes are members of a growing family of proteins with glycosyltransferase activity required for the synthesis of heparan sulfate chains. This protein activity is predicted to play a role in the expression of proteoglycans on the cell surface and in the extracellular matrix. We and others have previously suggested that a two-hit mutational model applies to the development of an exostosis where a germline mutation coupled with a somatic mutation results in the loss of EXT1 or EXT2 function and subsequent tumor formation. We report the direct sequencing and loss of heterozygosity (LOH) analysis of 12 exostoses from 10 HME families, 4 solitary exostoses, and their corresponding constitutional DNA. Of the 16 exostoses screened, we find only one solitary case in which two somatic mutations, a deletion and an LOH, are present. This provides limited support for the two-hit hypothesis involving the EXT1 and EXT2 genes for the development of an exostosis. Alternative models are developed based on the functional significance of EXT proteins in heparan sulfate biosynthesis.
Ultrastructural abnormalities in cultured exostosis chondrocytes.
Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by inappropriate chondrocyte proliferation and bone growth arising at the juxtaepiphyseal region of the long bones. HME is caused by mutations in the EXT 1 and EXT 2 genes, which have glycosyltransferase activity. These genes are responsible for synthesis of heparan sulfate (HS) chains, which are important signaling molecules in chondrocyte differentiation. HME chondrocytes in monolayer culture have been shown by transmission electron and deconvolution microscopy to contain enormous bundles of actin, cross-linked with muscle specific alpha-actinin. Here additional ultrastructural anomalies in HME chondrocytes are reported, including lobulated nuclei, shortened channels of rER, large numbers of cell processes and podosomes, nontypical junctions, elongated, bulbous-ended mitochondria, and reduced extracellular matrix. Microfilaments are present throughout the cytoplasm, compartmentalizing it, and isolating organelles. The excess microfilaments, attributed to increased cell adhesiveness, are likely to interfere with secretion and cytokinesis, and sterically hinder intracellular organelle differentiation. The observed surface modifications and cytoskeletal abnormalities are proposed to play a role in development of the mutant phenotype, via changes in cell adhesiveness and/or binding of signals to receptors, which results in loss of the unidirectionality of growth in the epiphyseal plate.
A prospective evaluation of the WeeFIM in patients with cerebral palsy undergoing orthopaedic surgery.
PURPOSE: Although frequently used in pediatric rehabilitation settings, the WeeFIM has not been tested in surgical pediatric orthopaedic patients. METHODS: The WeeFIM was administered to patients with surgical cerebral palsy at defined intervals preoperatively and at both 6 and 12 months postoperatively. The age-adjusted change scores from baseline to follow-up were tested both parametrically and nonparametrically. RESULTS: Four hundred sixty-eight patients had baseline evaluations. There were 161 six-month follow-up assessments and 108 twelve-month follow-up assessments. The baseline WeeFIM was able to separate children with different patterns of cerebral palsy. Hemiplegic patients had higher scores than diplegic and tetraplegic patients. Overall age-adjusted scores were improved at both 6 (mean increase 2.0) and 12 months (mean increase 2.2). The instrument showed significant ceiling effects for diplegic and hemiplegic patients with lower or upper extremity surgery and limited responsiveness for lower extremity surgery in tetraplegic patients. Parametrically, it showed improvements in mobility for both rhizotomy and tetraplegic upper extremity surgery. Nonparametric tests were not significant for rhizotomy mobility improvement. CONCLUSIONS: Although the WeeFIM adequately reflects the severity of neurological involvement in pediatric orthopaedic patients with cerebral palsy, it has a significant ceiling effect in diplegic and hemiplegic patients limiting responsiveness and lacks content validity for tetraplegic patients. The instrument may have some use in tetraplegic patients with upper extremity surgery and in rhizotomy patients. We recommend against its general use for orthopaedic surgery in patients with cerebral palsy lower extremity or spine surgery and in hemiplegic patients with upper extremity surgery.
Rectus and hamstring surgery in cerebral palsy: a gait analysis study of results by functional ambulation level.
The purpose of this study is to determine if children more severely involved with cerebral palsy respond as well to rectus transfer and hamstring surgery as those with less severe involvement. Ninety-nine children were classified as independent community ambulators, crutch/walker-dependent community ambulators, or household/exercise ambulators. Maximum knee extension in stance and total range of knee motion in gait increased following surgery in all groups. Peak knee flexion in swing was maintained in the independent group only, but timing of knee flexion in swing improved in all groups. All groups showed increases in stride length, and the household/exercise group also showed an increase in walking speed. Four of 39 crutch/walker-dependent community ambulators and 13 of 21 household/exercise ambulators progressed to the next higher functional ambulation group.