Manon C. Zweers

Haploinsufficiency of TNXB Is Associated with Hypermobility Type of Ehlers-Danlos Syndrome

To the Editor: Ehlers-Danlos syndrome (EDS) is a heterogeneous group of heritable connective-tissue disorders, generally affecting skin, joints, and blood vessels. The most recent classification recognizes six subtypes (Beighton et al. 1998), of which the hypermobility type (HT-EDS [formerly EDS type III] [MIM 130020]) is the most common. This type of EDS is similar to benign joint hypermobility syndrome (BJHS), and both are often considered to represent the same hyperlaxity syndrome, since no clear clinical distinction can be made (Grahame 1999). Although various causative genes have been found in all other types of EDS, the genetic basis of HT-EDS or BJHS remains unexplained (Steinmann et al. 2002). One family has been described that has a missense mutation in COL3A1 (Narcisi et al. 1994), resulting in a phenotype that resembles HT-EDS, without obvious vascular complications. Mutations in COL3A1 generally result in the severe vascular type of EDS (MIM 130050). To our knowledge, no other cases of COL3A1 mutations in HT-EDS have been reported. Recently, we showed that deficiency of the extracellular-matrix protein tenascin-X (TNX), encoded by the TNXB gene, causes a new type of recessively inherited EDS (Schalkwijk et al. 2001). Patients with complete deficiency of TNX showed marked joint hypermobility, skin hyperextensibility, and easy bruising. The absence of atrophic scars and recessive inheritance distinguishes TNX deficiency from the classical type of EDS. In our initial report (Schalkwijk et al. 2001), only a few heterozygous family members were available for examination. Here, we have examined all 20 heterozygous family members (individuals from families A–D in table 1) who were available for further study, regardless of clinical symptoms; in all of these individuals, we have found significantly reduced serum TNX levels (56% ± 6% vs. 100% ± 14% in the control population; P<.001, by Students t test) (fig. 1f), and, in 17 of them, we have confirmed heterozygosity for a truncating TNXB mutation (table 1). Clinical examination revealed generalized joint hypermobility in nine family members (45%), using the Beighton score (Beighton et al. 1973), for HT-EDS, or the Brighton criteria (Grahame et al. 2000), for BJHS (table 1 and fig. 1e). Skin hyperextensibility and easy bruising, frequently seen in the individuals with complete TNX deficiency, were absent. A number of patients with haploinsufficiency had recurring joint dislocations and chronic joint pain, as are seen in HT-EDS and BJHS. Only four family members carrying two normal TNXB alleles were available for study, of whom none had hypermobility. The local medical ethics committee (CMO Regio Arnhem-Nijmegen) approved the study protocol, and informed consent was obtained from all patients. Figure  1 TNXB haploinsufficiency and generalized joint hypermobility. a–d, Pedigrees of families A–D (also see table 1). e, Joint hypermobility in individual III9 from family A. f, Distribution of serum TNX levels in control population, population ... Table 1 Clinical and Molecular Findings in Individuals with TNXB Haploinsufficiency/Reduced Serum TNX Levels A striking finding is that 0 of the 6 males with haploinsufficiency fulfilled the clinical criteria for HT-EDS or BJHS, whereas 9 of 14 (64%) females were positive. This finding is in accordance with previous population-based studies that show a female preponderance in joint hypermobility syndromes (Larsson et al. 1987; Rikken-Bultman et al. 1997). In a control group of 30 unaffected females of the same age as the females with haploinsufficiency in the present study, we found no individuals with a Beighton score >4. This indicates that the prevalence of generalized joint hypermobility in a population of females with haploinsufficiency is significantly higher than in a control population (P<.001, by χ2 test). No sex differences in serum TNX levels in unaffected individuals and individuals with haploinsufficiency were found (not shown). Because our observations in families carrying previously described TNXB mutations suggested an association between TNXB haploinsufficiency and joint hypermobility, we wondered about the prevalence of TNXB haploinsufficiency in patients with HT-EDS. We measured serum TNX levels (by ELISA) in an unselected cohort of 80 patients with HT-EDS who were recruited through the Dutch organization for patients with EDS. All patients were diagnosed with HT-EDS by a medical specialist, and ∼90% were female. Although the mean serum TNX level was not different in the cohort with HT-EDS overall (99.4%±19.7%) (fig. 1f), six of these patients (7.5% [all female]) had serum TNX levels >2.5 SDs (65%) below the mean for unaffected individuals. On the basis of the normal distribution of serum TNX levels, only 0.6% of individuals would be expected to have such low serum TNX levels, which is significantly less than the frequency found in the population with HT-EDS described in the present study (P<.001, by Fishers exact test). Clinically, patients with reduced TNX levels showed hypermobile joints, often associated with joint subluxations and chronic musculoskeletal pain (table 1). The clinical findings in these patients differ from those with complete TNX deficiency. Patients with haploinsufficiency do not have skin hyperextensibility and lack the easy bruising seen in patients with TNX deficiency. In addition, TNXB haploinsufficiency is expected to be an autosomal dominant trait, which is in accordance with the observed mode of inheritance of HT-EDS and BJHS. On screening for the presence of a 30-kb deletion described previously (Burch et al. 1997; Schalkwijk et al. 2001), we found that this deletion was present in one of these six patients. The 30-kb deletion creates a fusion gene of TNXB and XA, a partial duplicate of TNXB. The XA gene has an internal deletion that truncates its ORF, rendering XA and the fusion gene nonfunctional (Gitelman et al. 1992). The deleted allele also lacks CYP21, so this individual is also a carrier for congenital adrenal hyperplasia. Subsequently, we PCR amplified and directly sequenced the coding regions and the intron-exon boundaries of TNXB in the other five patients presumed to have haploinsufficiency (for primers used, see Schalkwijk et al. 2001). One patient (individual F in table 1) was heterozygous for a 2-bp deletion, [AA56063] del, in exon 8, resulting in a premature stop codon at the position of amino acid 1231. In the other four patients, we were unable to identify mutations in TNXB. These patients may have mutations, in regulatory sequences or in exons of the TNXB gene, that have not yet been identified, or they may represent the extreme in normal variation of TNX expression. In conclusion, in the present study, we have reported a genetic defect associated with HT-EDS or BJHS. On the basis of the observed phenotype in patients with complete TNX deficiency and the high prevalence of generalized joint hypermobility in heterozygous females, this is likely to be a causative relationship. Reduced TNX expression could disturb deposition of collagen (Mao et al. 2002) and the elastic fiber network (Burch et al. 1997), as has been shown for complete TNX deficiency, resulting in increased laxity of ligaments and tendons. TNXB haploinsufficiency is dominantly inherited and appears to produce clinical findings primarily in women, consistent with clinical descriptions of HT-EDS. Although we identified inactivating TNX mutations in only 2.5% of this cohort with HT-EDS, 7.5% had serum TNX levels low enough to affect collagen metabolism. The present study demonstrates that TNXB haploinsufficiency is associated with HT-EDS and suggests that locus heterogeneity exists for this disorder, as it does for other types of EDS (Byers 1994).

Interactions of primary fibroblasts and keratinocytes with extracellular matrix proteins: contribution of α2β1 integrin

The α2β1 integrin is a collagen-binding protein with very high affinity for collagen I. It also binds several other collagens and laminins and it is expressed by many cells, including keratinocytes and fibroblasts in the skin. In the past, α2β1 integrin was suggested to be responsible for cell attachment, spreading and migration on monomeric collagen I and contraction of three-dimensional collagen lattices. In view of these functions, normal development and fertility in integrin α2-deficient mice, which we generated by targeting the integrin α2 gene, came as a surprise. This suggested the existence of compensatory mechanisms that we investigate here using primary fibroblasts and keratinocytes isolated from wild-type and α2-deficient mice, antibodies blocking integrin function and downregulation of integrin α2 expression. The results show that the α2β1 integrin is absolutely required for keratinocyte adhesion to collagens whereas for fibroblasts other collagen-binding integrins partially back-up the lack of α2β1 in simple adhesion to collagen monomers. A prominent requirement for α2β1 integrins became apparent when fibroblasts executed mechanical tasks of high complexity in three-dimensional surroundings, such as contracting free-floating collagen gels and developing isometric forces in tethered lattices. The deficits observed for α2-deficient fibroblasts appeared to be linked to alterations in the distribution of force-bearing focal adhesions and deregulation of Rho-GTPase activation.

Defective granulation tissue formation in mice with specific ablation of integrin-linked kinase in fibroblasts – role of TGFβ1 levels and RhoA activity

Wound healing crucially relies on the mechanical activity of fibroblasts responding to TGFβ1 and to forces transmitted across focal adhesions. Integrin-linked kinase (ILK) is a central adapter recruited to integrin β1 tails in focal adhesions mediating the communication between cells and extracellular matrix. Here, we show that fibroblast-restricted inactivation of ILK in mice leads to impaired healing due to a severe reduction in the number of myofibroblasts, whereas inflammatory infiltrate and vascularization of the granulation tissue are unaffected. Primary ILK-deficient fibroblasts exhibit severely reduced levels of extracellular TGFβ1, α-smooth muscle actin (αSMA) production and myofibroblast conversion, which are rescued by exogenous TGFβ1. They are further characterized by elevated RhoA and low Rac1 activities, resulting in abnormal shape and reduced directional migration. Interference with RhoA–ROCK signaling largely restores morphology, migration and TGFβ1 levels. We conclude that, in fibroblasts, ILK is crucial for limiting RhoA activity, thus promoting TGFβ1 production, which is essential for dermal repair following injury.

Collagen XXIII, Novel Ligand for Integrin α2β1 in the Epidermis

Cellular receptors for collagens belong to the family of β1 integrins. In the epidermis, integrin α2β1 is the only collagen-binding integrin present. Its expression is restricted to basal keratinocytes with uniform distribution on the cell surface of those cells. Although α2β1 receptors localized at the basal surface interact with basement membrane proteins collagen IV and laminin 111 and 332, no interaction partners have been reported for these integrin molecules at the lateral and apical membranes of basal keratinocytes. Solid phase binding and surface plasmon resonance spectroscopy demonstrate that collagen XXIII, a member of the transmembrane collagens, directly interacts with integrin α2β1 in an ion- and conformation-dependent manner. The two proteins co-localize on the surface of basal keratinocytes. Furthermore, collagen XXIII is sufficient to induce adhesion and spreading of keratinocytes, a process that is significantly reduced in the absence of functional integrin α2β1.

Abdominal aortic aneurysm is associated with high serum levels of tenascin-X and decreased aneurysmal tissue tenascin-X.

Background— Tenascin-X is a large extracellular matrix protein that is abundantly expressed in several connective tissues. A 140-kDa C-terminal fragment of tenascin-X is present in human serum. Complete deficiency of tenascin-X is associated with Ehlers-Danlos syndrome, and these patients show major connective tissue alterations in their skin, as well as blood vessel fragility. In this study, we investigated whether tenascin-X is present in normal human aorta and abdominal aortic aneurysm (AAA) tissues and whether an association exists between serum tenascin-X levels and AAA. Methods and Results— Five normal aortas and 5 AAA tissues were immunostained for tenascin-X and elastin. Tenascin-X was present throughout the entire aorta and was especially abundant near the elastic lamellae, whereas tenascin-X expression was strongly decreased in AAA tissue. Measurement of tenascin-X serum concentration by enzyme-linked immunosorbent assay (ELISA) in 87 AAA patients and 86 controls demonstrated an increasing risk for AAA with increasing tenascin-X serum concentrations. After adjustment for established risk factors, tenascin-X serum concentrations in the highest quartile were associated with a 5-fold increase in risk of AAA (odds ratio, 5.3; 95% confidence interval, 2.0 to 13.8). Conclusions— Tenascin-X expression is markedly decreased in AAA tissue, and AAA is associated with high serum concentrations of tenascin-X.

Transplantation of reconstructed human skin on nude mice: a model system to study expression of human tenascin-X and elastic fiber components

Tenascin-X is a large extracellular matrix protein that is widely expressed in connective tissues during development and in the adult. Genetically determined deficiency of tenascin-X causes the connective tissue disease Ehlers–Danlos syndrome. These patients show reduced collagen density and fragmentation of elastic fibers in their skin. In vitro studies on the role of tenascin-X in elastic fiber biology are hampered because monolayers of fibroblasts do not deposit tenascin-X and elastic fibers into the extracellular matrix. Here, we applied an organotypic culture model of fibroblasts and keratinocytes to address this issue. We investigated the deposition of tenascin-X and elastin into skin-equivalent in vitro and also in vivo after transplantation onto immunodeficient mice. Whereas tenascin-C and fibrillin-1 were readily expressed in the skin-equivalents before transplantation, tenascin-X and elastin were not present. Three weeks post-grafting, a network of elastin was observed that coincided with the appearance of tenascin-X. At the ultrastructural level, microfibrils were observed, some of which were associated with elastin. Transplanted skin-equivalents containing tenascin-X-deficient fibroblasts showed deposition of immunoreactive elastin in similar quantities and distribution as those containing control fibroblasts. This suggests that tenascin-X is important for the stability and maintenance of established elastin fibers, rather than for the initial phase of elastogenesis. Thus, the transplantation of reconstructed skin on nude mice allows the study of tenascin-X and elastin expression and could be used as a model system to study the potential role of tenascin-X in matrix assembly and stability.

Integrin α2β1 deficiency does not affect contact hypersensitivity

Collagens in the extracellular matrix are thought to play an important role in regulating inflammatory responses by affecting cell adhesion and migration. The contact between collagens and cells is established mainly by α1β1, α2β1 and α11β1integrin receptors. Here, we analyzed the contact hypersensitivity (CHS) reaction in mice that were genetically deficient in the collagen receptor α2β1. Integrin α2β1 is widely expressed and has been suggested to play an important role in mediating inflammatory responses. CHS was induced by applying dinitrofluorobenzene to abdominal skin and challenging with the same reagent on ear skin. Macroscopically and histologically, ear swelling in α2β1-deficient mice did not differ from that in wild-type control mice. Immunohistological detection of infiltrated T lymphocytes, neutrophils and mast cells in inflamed ear skin revealed similar numbers in controls and integrin α2β1-deficient animals. Our results suggest that the adhesive functions of integrin α2β1 are dispensable for the CHS response; they may be compensated for by the collagen receptor α1β1 or other collagen receptors.