Identification of quantitative trait loci for osteoarthritis of hip joints in dogs.

OBJECTIVE: To identify quantitative trait loci (QTL) associated with osteoarthritis (OA) of hip joints of dogs by use of a whole-genome microsatellite scan. ANIMALS: 116 founder, backcross, F1, and F2 dogs from a crossbred pedigree. PROCEDURES: Necropsy scores and an optimized set of 342 microsatellite markers were used for interval mapping by means of a combined backcross and F2 design module from an online statistical program. Breed and sex were included in the model as fixed effects. Age of dog at necropsy and body weight at 8 months of age were also included in the model as covariates. The chromosomal location at which the highest F score was obtained was considered the best estimate of a QTL position. Chromosome-wide significance thresholds were determined empirically from 10,000 permutations of marker genotypes. RESULTS: 4 chromosomes contained putative QTL for OA of hip joints in dogs at the 5% chromosome-wide significance threshold: chromosomes 5, 18, 23, and 31. CONCLUSIONS AND CLINICAL RELEVANCE: Osteoarthritis of canine hip joints is a complex disease to which many genes and environmental factors contribute. Identification of contributing QTL is a strategy to elucidate the genetic mechanisms that underlie this disease. Refinement of the putative QTL and subsequent candidate gene studies are needed to identify the genes involved in the disease process.

A random model for mapping imprinted quantitative trait loci in a structured pedigree: an implication for mapping canine hip dysplasia.

Genetic imprinting may have played a more notable role in shaping embryonic development of plants, animals, and humans than previously appreciated. Quantitative trait loci that are imprinted (iQTL) exert monoallelic effects, depending on the parent of origin, which is an exception to the laws of Mendelian genetics. In this article, we present a modified random effect-based mapping model to use in a genome-wide scan for the distribution of iQTL that contribute to genetic variance for a complex trait in a structured pedigree. This model, implemented with the maximum likelihood method, capitalizes on a network of relatedness for maternally and paternally derived alleles through identical-by-descent sharing, thus allowing for the discrimination of the genetic variances due to alleles derived from maternal and paternal parents. The model was employed to map iQTL responsible for canine hip dysplasia in a multihierarchical canine pedigree, founded with seven greyhounds and six Labrador retrievers. Of eight significant QTL detected, three, located on CFA1, CFA8, and CF28, were found to trigger significant parent-of-origin effects on the age of femoral capital ossification measured at the left and right hips of a canine. The detected iQTL provide important candidate regions for fine-mapping of imprinted genes and for studying their structure and function in the control of complex traits.

Quantitative genetics of secondary hip joint osteoarthritis in a Labrador Retriever-Greyhound pedigree.

OBJECTIVE: To evaluate the quantitative inheritance of secondary hip joint osteoarthritis in a canine pedigree. ANIMALS: 137 Labrador Retrievers, Greyhounds, and mixed-breed dogs. PROCEDURES: Necropsy scores ranging from 0 to 4 were obtained for each hip joint. Seven unaffected Greyhounds with normal hip joint conformation were also used for genetic modeling, but were not euthanized. Sixty-six male and 71 female dogs were allocated to 2 groups (< or = 12 months of age and > 12 months of age). Statistical models were developed to establish the inheritance pattern of hip joint osteoarthritis that developed secondary to hip dysplasia. RESULTS: 62 dogs had evidence of osteoarthritis in a hip joint, and 75 had no evidence of osteoarthritis. After sex was adjusted for, the necropsy score was found to be inherited additively but without dominance. Each Labrador Retriever allele increased the necropsy score by 0.7 to 0.9 points, compared with the Greyhound allele, and male sex increased the necropsy score 0.74 over female sex. Approximately 10% of the variation in necropsy score was attributable to the litter of puppies' origin. CONCLUSIONS AND CLINICAL RELEVANCE: Because secondary hip joint osteoarthritis is inherited additively, selection pressure could be applied to reduce its incidence. Similar statistical models can be used in linkage and association mapping to detect the genes in the underlying quantitative trait loci that contribute to hip joint osteoarthritis.

Modeling extent and distribution of zygotic disequilibrium: implications for a multigenerational canine pedigree.

Unlike gametic linkage disequilibrium defined for a random-mating population, zygotic disequilibrium describes the nonrandom association between different loci in a nonequilibrium population that deviates from Hardy-Weinberg equilibrium. Zygotic disequilibrium specifies five different types of disequilibria simultaneously that are (1) Hardy-Weinberg disequilibria at each locus, (2) gametic disequilibrium (including two alleles in the same gamete, each from a different locus), (3) nongametic disequilibrium (including two alleles in different gametes, each from a different locus), (4) trigenic disequilibrium (including a zygote at one locus and an allele at the other), and (5) quadrigenic disequilibrium (including two zygotes each from a different locus). However, because of the uncertainty on the phase of the double heterozygote, gametic and nongametic disequilibria need to be combined into a composite digenic disequilibrium and further define a composite quadrigenic disequilibrium together with the quadrigenic disequilibrium. To investigate the extent and distribution of zygotic disequilibrium across the canine genome, a total of 148 dogs were genotyped at 247 microsatellite markers located on 39 pairs of chromosomes for an outbred multigenerational pedigree, initiated with a limited number of unrelated founders. A major portion of zygotic disequilibrium was contributed by the composite digenic and quadrigenic disequilibrium whose values and numbers of significant marker pairs are both greater than those of trigenic disequilibrium. All types of disequilibrium are extensive in the canine genome, although their values tend to decrease with extended map distances, but with a greater slope for trigenic disequilibrium than for the other types of disequilibrium. Considerable variation in the pattern of disequilibrium reduction was observed among different chromosomes. The results from this study provide scientific guidance about the determination of the number of markers used for whole-genome association studies.

Assessment of bone mineral density of the femoral head in dogs with early osteoarthritis.

OBJECTIVE: To compare the bone mineral density (BMD) of the proximal portion of the femur in dogs with and without early osteoarthritis secondary to hip dysplasia. ANIMALS: 24 dogs (3 Greyhounds, 6 Labrador-Greyhound crossbreeds, and 15 Labrador Retrievers). PROCEDURE: Computed tomography (CT) of the pelvis, including a bone-density phantom, was performed for each dog. Centrally located transverse CT slices and a computer workstation were used to identify 16 regions of interest (ROIs) in the proximal portion of the femur. For each ROI, the mean Hounsfield unit value was recorded; by use of the bone-density phantom and linear regression analysis, those values were converted to equivalent BMD (eBMD). Mean eBMD values for the subchondral and nonsubchondral ROIs in dogs with and without osteoarthritis (determined at necropsy) were compared. A mixed-model ANOVA and post hoc linear contrasts were used to evaluate the effects of osteoarthritis, breed, and sex on the BMD value. RESULTS: At necropsy, osteoarthritis was detected in 14 hip joints in 9 dogs; all lesions included early cartilage fibrillation. After adjusting for breed and sex, eBMD in subchondral ROIs 8 and 12 (adjacent to the fovea) were 8% and 6% higher, respectively, in osteoarthritis-affected dogs, compared with unaffected dogs; in the nonsubchondral ROIs, eBMD was 10% higher in osteoarthritis-affected dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Compared with findings in unaffected dogs, increased eBMD in hip joints of dogs with early osteoarthritis supports a strong relationship between the subchondral and epiphyseal regions and articular cartilage in the pathogenesis and progression of osteoarthritis.

Quantitative trait loci for hip dysplasia in a cross-breed canine pedigree.

Canine hip dysplasia is a common developmental inherited trait characterized by hip laxity, subluxation or incongruity of the femoral head and acetabulum in affected hips. The inheritance pattern is complex and the mutations contributing to trait expression are unknown. In the study reported here, 240 microsatellite markers distributed in 38 autosomes and the X chromosome were genotyped on 152 dogs from three generations of a crossbred pedigree based on trait-free Greyhound and dysplastic Labrador Retriever founders. Interval mapping was undertaken to map the QTL underlying the quantitative dysplastic traits of maximum passive hip laxity (the distraction index), the dorsolateral subluxation score, and the Norberg angle. Permutation testing was used to derive the chromosome-wide level of significance at p<0.05 for each QTL. Chromosomes 4, 9, 10, 11 (p<0.01), 16, 20, 22, 25, 29 (p<0.01), 30, 35, and 37 harbor putative QTL for one or more traits. Successful detection of QTL was due to the cross-breed pedigree, multiple-trait measurements, control of environmental background, and marked advancement in canine mapping tools.

Increased MIG-6 mRNA transcripts in osteoarthritic cartilage.

The biochemical mechanism for initiation of cartilage destruction in osteoarthritis (OA) is unknown but may involve as yet unidentified cartilage genes. The first evidence that MIG-6, a protein involved in signal transduction, is expressed in articular cartilage came from our recent in vitro microarray experiments using the Affymetrix canine GeneChip. Quantitative RT-PCR (q RT-PCR) confirmed a fourfold increase in MIG-6 mRNA in cartilage in response to mechanical impact in vitro. Our goal is to determine if MIG-6, which responds to mechanical impact, could have a role in the initiation of OA. We determined that mRNA transcript levels of MIG-6 were fourfold higher in degenerated cartilage from dogs with hip osteoarthritis than in disease-free cartilage from unaffected dogs and twofold higher than in the cartilage surrounding the lesion. This is the first report associating MIG-6 with OA. Additional studies will determine what role MIG-6 has in the origin of cartilage degeneration.

Caspase-3/7 inhibition alters cell morphology in mitomycin-C treated chondrocytes.

Apoptosis may play a role in osteoarthritis (OA). Apoptosis can proceed via two different pathways depending on the stimulus. However, both pathways converge upon the effector caspases, caspases-3 and -7. In some systems inhibition of caspases-3 and -7 can prevent apoptosis and may therefore have important therapeutic implications. To confirm this, apoptosis was induced in canine chondrocytes with mitomycin-c (MMC), either in the presence or absence of the general caspase inhibitor, Z-VAD FMK, or a specific caspase-3/7 inhibitor. Z-VAD FMK prevented MMC induced cell death. In contrast, inhibition of caspases-3 and -7 in the presence of MMC induced morphological changes that could be described as necrotic-like or paraptotic-like but did not prevent cell death. The addition of an inhibitor of caspase-8 or caspase-9 along with inhibitor of caspase-3/7 was required to reduce cell death. The morphological changes did not occur in the presence of the caspase-3/7 inhibitor alone and could be prevented by addition of Z-VAD FMK. These data lead to the conclusion that, if the apoptotic program cannot be completed, the cells are pushed into a necrotic or other nonapoptotic mode of death which may involve caspase-8 and/or caspase-9.

The potential and limitations of cartilage-specific (V+C)(-) fibronectin and cartilage oligomeric matrix protein as osteoarthritis biomarkers in canine synovial fluid.

OBJECTIVE: To determine if levels of the cartilage-specific (V+C)(-) fibronectin isoform in the synovial fluid is associated with cartilage change during osteoarthritis. DESIGN: Synovial fluid was collected from 26 healthy dogs presenting to the Orthopedic Surgery Clinic with unilateral cranial cruciate rupture, 22 control dogs, and 13 dogs from a colony maintained for the study of canine hip dysplasia. Total fibronectin, (V+C)(-) fibronectin, and cartilage oligomeric matrix protein (COMP) were quantitated by ELISA assays. Statistical analysis used Wilcoxon's signed-rank and rank-sum tests and Spearman's rank correlation. RESULTS: The concentration of total fibronectin was increased in affected (P<0.0001) and contralateral (P=0.0005) knees of the clinic population (compared to unaffected knees in colony controls). Both (V+C)(-) fibronectin and COMP concentrations were elevated in the contralateral knees in clinical patients relative to unaffected knees in the colony controls (P=0.03 and P=0.04, respectively), and relative to the affected knees (P=0.003); however, corrections for joint effusions suggest elevated totals in the affected knees. (V+C)(-) fibronectin and COMP concentrations were correlated (r(sp)=0.74; P<0.0001) in 30 unaffected knees of patients and colony controls. Total fibronectin was correlated negatively with months since the initial injury (r(sp)=-0.44; P=0.03) in the affected joints. The intraoperative lesion severity score did not correlate with total fibronectin or (V+C)(-) fibronectin (P>or=0.35). CONCLUSIONS: Concentration of total fibronectin in synovial fluid might be a useful biomarker for cross-sectional studies in osteoarthritis, but only (V+C)(-) fibronectin provides information specifically about cartilage damage. Elevated concentrations of (V+C)(-) fibronectin and COMP seen in the contralateral knees of patients with cranial cruciate rupture might indicate cartilage changes early in the disease process (pre-clinical). However, the wide range of values obtained limits the diagnostic value for any one individual. Joint effusions obscure the total amount of biomarkers in affected synovial joints.

The spread of cell death from impact damaged cartilage: lack of evidence for the role of nitric oxide and caspases.

UNLABELLED: Over 21 days in culture, cell death spreads, both radially and transversely, from loaded to surrounding cartilage. This spread was prevented by physical separation and separate culture post-impact. OBJECTIVE: One aim was to determine if nitric oxide (NO) is the intercellular signal mediating cell death. Another aim was to clarify the nature of the cell death, whether caspase mediated apoptosis or necrosis. DESIGN: Cyclic impacts were applied to the central 2 mm core of 4 mm canine articular cartilage discs. Post-impact culturing was for 21 days in the presence or absence of the iNOS inhibitor, L-NAME, or the broad-spectrum caspase inhibitor, Z-VAD FMK. Cell death was quantified using the TUNEL assay. Culture media were collected every 2 days for measurements of glycosaminoglycan (GAG) and NO release. RESULTS: Cell death spread from the loaded core into the surrounding ring over 21 days in culture. Although L-NAME significantly reduced nitrite release into the culture media of both loaded and control cartilage, the spread of cell death was not prevented. Neither was the spread of cell death prevented by Z-VAD FMK. CONCLUSIONS: These data indicate that NO is not acting as an intercellular signalling factor in this in vitro system and that the cell death post-impact is not caspase mediated.

Evaluation of multiple radiographic predictors of cartilage lesions in the hip joints of eight-month-old dogs.

OBJECTIVE: To determine the radiographic methods that best predict the development of osteoarthritis in the hip joints of a cohort of dogs with hip dysplasia and unaffected dogs. ANIMALS: 205 Labrador Retrievers, Greyhounds, and Labrador Retriever-Greyhound crossbred dogs. PROCEDURE: Pelvic radiography was performed when the dogs were 8 months old. Ventrodorsal extended-hip, distraction, and dorsolateral subluxation (DLS) radiographs were obtained. An Orthopedic Foundation for Animals-like hip score, distraction index, dorsolateral subluxation score, and Norberg angle were derived from examination of radiographs. Osteoarthritis was diagnosed at the time of necropsy in dogs > or = 8 months of age on the basis of detection of articular cartilage lesions. Multiple logistic regression was used to determine the radiographic technique or techniques that best predicted development of osteoarthritis. RESULTS: A combination of 2 radiographic methods was better than any single method in predicting a cartilage lesion or a normal joint, but adding a third radiographic method did not improve that prediction. A combination of the DLS score and Norberg angle best predicted osteoarthritis of the hip joint or an unaffected hip joint. All models that excluded the DLS score were inferior to those that included it. CONCLUSIONS AND CLINICAL RELEVANCE: A combination of the DLS score and Norberg angle was the best predictor of radiographic measures in 8-month-old dogs to determine whether a dog would have normal or osteoarthritic hip joints.

Effect of dorsal hip loading, sedation, and general anesthesia on the dorsolateral subluxation score in dogs.

OBJECTIVE: To determine whether dorsal loading of the pelvis and type of chemical restraint affected the dorsolateral subluxation (DLS) score of dog hips. STYDY DESIGN: In vivo testing of diagnostic method. ANIMALS: Labrador retrievers, Greyhounds, and crosses between both breeds (n = 119 dogs). METHODS: Dorsal load was applied to the hips through a strap that was placed over the hips, and the peak vertical and steady-state vertical, ground-reaction forces exerted on the stifles were measured in the DLS position. The DLS score was measured with their hips under dorsal load and compared with the DLS score without load. For 24 dogs, the DLS score was measured both under chemical restraint (medetomidine) and under general anesthesia. Wilcoxon-signed rank test and paired t test was used to compare effects of dorsal load and restraint method on the left and right DLS hip score, and P <.05 was considered significant. RESULTS: Both stifles together accepted median 15% body weight in the vertical plane in the unloaded steady state. Loading the hips increased the steady-state, vertical ground-reaction force to 34% body weight. At 8 months of age, loading significantly decreased the DLS score from 65% +/- 11% (mean +/- SD) to 60% +/- 12% for left hips and 66% +/- 11% to 63% +/- 13% for right hips. The left hip DLS score was affected by load more consistently than the right hip. In general, loading the hips decreased the DLS score in all breeds by 4% to 6% except for the greyhounds, in which the DLS score was unchanged by dorsal loading. When grouped by their DLS scores, load significantly decreased DLS scores in dogs with unloaded scores greater than 55% (nondysplastic), whereas DLS scores of dogs with unloaded scores between 55% and 45% (dysplastic) and less than 45% did not change significantly with load. Unloaded scores did not change significantly when compared under sedation versus general anesthesia. However, load significantly decreased the DLS score under general anesthesia for the left hip but not for the right hip. CONCLUSIONS: Less than half of the vertical ground-reaction force normally exerted by the hindlegs of a standing dog was sufficient to induce hip subluxation in the DLS position. Although dorsal loading approximately doubled the steady-state, vertical ground-reaction force, the decrease in the DLS score under load was never greater than 6%. Furthermore, the DLS scores of dogs most likely to be dysplastic (based on DLS scores <45%) were not affected by load, whereas load slightly decreased DLS cores of dogs with DLS scores greater than 55%. CLINICAL RELEVANCE: External influences can change the DLS score significantly, but the magnitude of change is unlikely to be of clinical importance, making the method useful as a field screening test for both dysplastic and nondysplastic hips in young dogs.

Spontaneous and experimental osteoarthritis in dog: similarities and differences in proteoglycan levels.

The unilateral canine model is the most commonly used model of experimental osteoarthritis (OA). In this model, the anterior cruciate ligament (ACL) of one knee is transected and the contralateral joint is usually used as a control. However, dogs, similar to humans, can develop OA spontaneously with old age. Additionally, certain breeds of dogs are genetically predisposed to OA and can develop symptoms at a young age. The goal of this study was to compare the pathological changes of proteoglycans in OA cartilage from dogs that developed OA spontaneously to those that underwent ACL transection. For this reason, biglycan, decorin and fibromodulin levels and degradation patterns were compared by Western blot hybridization, and aggrecan contents were quantified by dimethylmethylene blue assay. The changes in proteoglycan levels in the cartilage of dogs with spontaneous OA, regardless of their age, were very similar to those published for human OA cartilage. However, when OA developed as a result of ACL-surgery, the changes in proteoglycans were different from those of slowly developing spontaneous OA. Therefore, these differences should be taken into consideration when the ACL-transection model is used.

Power of a Labrador Retriever-Greyhound pedigree for linkage analysis of hip dysplasia and osteoarthritis.

OBJECTIVE: To estimate the number of dogs required to find linkage to heritable traits of hip dysplasia in dogs from an experimental pedigree. ANIMALS: 147 Labrador Retrievers, Greyhounds, and their crossbreed offspring. PROCEDURE: Labrador Retrievers with hip dysplasia were crossed with unaffected Greyhounds. Age at detection of femoral capital ossification, distraction index (DI), hip joint dorsolateral subluxation (DLS) score, and hip joint osteoarthritis (OA) were recorded. Power to find linkage of a single marker to a quantitative trait locus (QTL) controlling 100% of the variation in a dysplastic trait in the backcross dogs was determined. RESULTS: For the DI at the observed effect size, recombination fraction of 0.05, and heterozygosity of 0.75, 35 dogs in the backcross of the F1 to the Greyhound generation would yield linkage at a power of 0.8. For the DLS score, 35 dogs in the backcross to the Labrador Retriever generation would be required for linkage at the same power. For OSS, 45 dogs in the backcross to the founding Labrador Retrievers would yield linkage at the same power. Fewer dogs were projected to be necessary to find linkage to hip OA. Testing for linkage to the DLS at 4 loci simultaneously, each controlling 25% of the phenotypic variation, yielded an overall power of 0.7 CONCLUSIONS AND CLINICAL SIGNIFICANCE: Based on this conservative single-marker estimate, this pedigree has the requisite power to find microsatellites linked to susceptibility loci for hip dysplasia and hip OA by breeding a reasonable number of backcross dogs.

The cartilage-specific fibronectin isoform has a high affinity binding site for the small proteoglycan decorin.

Binding of fibronectin to the small proteoglycan decorin plays an important role in cell differentiation and cell migration. The cartilage-specific (V+C)(-) fibronectin isoform, in which nucleotides that normally encode the protein segments V, III(15), and I(10) are spliced out, is one of the major splice variants present in cartilage matrices. Full-length and truncated cDNA constructs were used to express recombinant versions of fibronectin. Results demonstrated that the (V+C)(-) isoform has a higher affinity for decorin. Dissociation constants for decorin and fibronectin interaction were calculated to be 93 nm for the V(+)C(+) isoform and 24 nm and 223 nm for (V+C)(-) fibronectin. Because heparin competed with decorin competitively, binding of decorin to fibronectin likely occurs at a heparin-binding region. We propose that alternative splicing of the V and C regions changes the global conformation of fibronectin in such a way that it opens an additional decorin-binding site. This conformational change is responsible for the higher affinity of the (V+C)(-) fibronectin isoform for decorin.

Specific immunological detection of the (V+C)(-) fibronectin isoform.

The population of fibronectins in adult mammalian cartilage includes high levels of a cartilage-specific (V+C)(-) isoform which lacks the V, III-15, and I-10 segments and thus contains a novel junction between protein segments III-14 and I-11. We report production of a monoclonal antibody specific for (V+C)(-) fibronectin without cross-recognition of V(+)C(+) and V(-)C(+) isoforms found in plasma and other tissues. Presentation of epitope to this antibody requires the III-14/I-11 junction, but the epitope itself extends beyond 14 amino acids immediately surrounding the junction site and involves a conformational change in III-14 and/or the N-terminal portion of I-11. The antibody, designated Mab 5D10 anti (V+C)(-), displays specificity for (V+C)(-) fibronectin from multiple mammalian species including humans and utility in immunoblots, immunohistochemistry, and ELISA.

Quantitative genetics of traits associated with hip dysplasia in a canine pedigree constructed by mating dysplastic Labrador Retrievers with unaffected Greyhounds.

OBJECTIVE: To determine the genetic influence on expression of traits associated with canine hip dysplasia. ANIMALS: 193 dogs from an experimental canine pedigree. PROCEDURE: An experimental canine pedigree was developed for linkage analysis of hip dysplasia by mating dysplastic Labrador Retrievers with nondysplastic Greyhounds. A statistical model was designed to test the effects of Labrador Retriever and Greyhound alleles on age at detection of femoral capital epiphyseal ossification, 8-month distraction index, and 8-month dorsolateral subluxation score. RESULTS: The additive effect was significant for age at detection of femoral capital epiphyseal ossification. Restricted maximum likelihood estimates (+/-SD) for this trait were 6.4+/-1.95, 10.2+/-2.0, 10.8+/-3.1, 11.4+/-2.1, and 13.6+/-4.6 days of age for Greyhounds, Greyhound backcross dogs, F1 dogs, Labrador Retriever backcross dogs, and Labrador Retrievers, respectively. The additive effect was also significant for the distraction index. Estimates for this trait were 0.21+/-0.07, 0.29+/-0.15, 0.44+/-0.12, 0.52+/-0.18, and 0.6+/-0.17 for the same groups, respectively. For the dorsolateral subluxation score, additive and dominance effects were significant. Estimates for this trait were 73.5+/-4.1, 71.3+/-6.5, 69.1+/-6.0, 50.6+/-12.9, and 48.4+/-7.7%, respectively, for the same groups. CONCLUSIONS: In this canine pedigree, traits associated with canine hip dysplasia are heritable. Phenotypic differences exist among founder dogs of each breed and their crosses. This pedigree should be useful for identification of quantitative trait loci underlying the dysplastic phenotype.

Absence of the I-10 protein segment mediates restricted dimerization of the cartilage-specific fibronectin isoform.

The cartilage-specific (V + C)(-) fibronectin isoform does not efficiently heterodimerize with other V-region splice variants of fibronectin. To understand better the structural elements that determine this restricted dimerization profile, a series of truncated fibronectin expression constructs with various internal deletions in the V, III-15, or I-10 segments were constructed and co-transfected into COS-7 cells with either the V(+)C(+) or the (V + C)(-) isoform. SDS-PAGE and immunoblot analyses of the resulting conditioned media suggest that the I-10 segment must either be present in both monomeric subunits of fibronectin or absent from both subunits for efficient dimerization to occur. Further studies suggest that the I-10 segment specifically, not simply a balanced number of type I repeats at the carboxyl terminus of each monomeric subunit, plays an important role in determining different fibronectin dimerization patterns. Neither I-11 nor I-12 could be substituted for segment I-10 without significantly reducing the formation of heterodimers. Therefore, absence of segment I-10 explains why (V + C)(-) fibronectin is not found in heterodimeric configurations with other native V-region splice variants in cartilage. The unique dimerization pattern of (V + C)(-) fibronectin does not prevent matrix formation yet is consistent with this isoform having specialized properties in situ that are important for either the structural organization and biomechanical properties of cartilage or the regulation of a chondrocytic phenotype.