Rolf Brenner

Ehlers-Danlos syndrome type VI: lysyl hydroxylase deficiency due to a novel point mutation (W612C)

Abstract Ehlers-Danlos syndrome type VI (EDS VI) is a rare autosomal recessively inherited disease of connective tissue. The characteristic symptoms are hyperflexibility of joints and hyperelasticity of skin together with marked scoliosis, ocular manifestations and involvement of the vascular system. The underlying biochemical defect in EDS VI is a deficiency in lysyl hydroxylase (PLOD) activity resulting from mutations in the PLOD gene causing a low hydroxylysine content in various tissues. We found that two out of three patients showed a recently described duplication of about 800 bp in their LH mRNA. In the third patient we identified a new point mutation (2036 G→C) resulting in a substitution of tryptophan by cysteine in the highly conserved C-terminal region of the enzyme (W612C). In addition, this mutation destroys a restriction site of MwoI. Restriction analysis of the patient’s cDNA with MwoI showed the sole occurrence of the mutated transcript, while one allele in his genomic DNA contained the MwoI restriction site. Restriction analysis of the genomic DNA of the unaffected parents displayed a heterozygous loss of the restriction site for MwoI in the mother while the DNA of the father appeared normal. Our study demonstrates that the new point mutation (W612C) in conjunction with a functionless allele, most probably a null allele, for the LH gene may explain the functional deficiencies seen in this patient.

Different Regulation of Clonal Growth by Transforming Growth Factor-β 1 in Human Fetal Articular and Costal Chondrocytes

ABSTRACT: The variable affection of rib and limb growth in human skeletal dysplasias suggests the presence of sitespecific regulatory mechanisms for chondrocyte proliferation. We therefore studied the clonal growth of normal human costal and articular chondrocytes from the same four fetuses (15 to 30 wk of gestation) in a semisolid medium (0.8% methylcellulose) with a basal supplementation of 5% heat-inactivated FCS. IGF-I [0.3-12.5 ng/mL (0.04-1.6 nmol/L)], IGF-II [0.3-12.5 ng/mL (0.04-1.7 nmol/L)], and hGH [0.5-25 ng/mL (0.02-1.1 nmol/L)] stimulated clonal growth of articular and costal chondrocytes without site-specific difference. In contrast, a significant difference was found for transforming growth factorβ1, which proved to be a potent growth factor for fetal articular chondrocytes but did not stimulate or only minimally stimulated fetal costal chondrocytes [p < 0.05 for 0.3 ng/mL (0.01 nmol/L) TGF- β 1 andp < 0.01 for 1.25 ng/mL (0.05 nmol/L) TGF-β 1 using paired t test]. Preincubation with an IGF-I receptor antibody ( αIR-3) completely prevented the proliferative effect of IGF-I, IGF-II, and hGH, indicating that hGH acts via autocrine or paracrine induction of IGF. The antibody partly reduced TGF- β1 action on articular chondrocytes [p < 0.05 for 0.3 ng/mL (0.01 nmol/L) TGF- β1, NS for 1.25 ng/mL (0.05 nmol/L) TGF-β1 using paired t test]. These results indicate that TGF- β 1 is involved in the regulation of human fetal growth and has a different effect in ribs and limbs.

Expression of osteoblastic markers in cultured human bone and fracture callus cells

We compared the expression of osteoblastic markers in cultured human cells isolated from fracture calluses of various histological states of development with that in cells from adult and fetal bone. Adult osteoblasts and all callus cells produced almost exclusively type I collagen, whereas fetal osteoblasts produced also considerable amounts of type III collagen in vitro. 1,25-Dihydroxyvitamin D3 induced the synthesis of osteocalcin in all bone and callus cells but to varying extents. Fetal bone cells and early-stage callus cells synthesized less than 10% the amount of osteocalcin produced by adult bone cells. Late-stage callus cells produced intermediate levels of osteocalcin. Fetal bone cells and early-stage callus cells responded to parathyroid hormone with a less pronounced increase in intracellular cAMP than did adult bone cells. Late-stage callus cells showed the best response to parathyroid hormone. The activity of alkaline phosphatase was highest in fetal bone cells. These observations show that cells isolated from fetal bone and from fracture callus tissues express a pattern of markers clearly relating them to the osteoblastic lineage. On the basis of the different patterns of osteoblastic markers expressed in vitro we conclude that functionally distinct subtypes of osteoblasts do exist in different mineralized tissues and at different developmental stages.

Increased Cell Surface Expression of Receptors for Transforming Growth Factor-β on Osteoblasts from Patients with Osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder usually characterized by either a reduction in the production of normal collagen I or the synthesis of abnormal collagen. The variability in the clinical phenotype is not in each case sufficiently explained by the underlying mutation in the collagen I genes. Also, biochemical differences between mutant collagen from different tissues suggest additional regulatory mechanisms possibly involved in matrix deposition and maturation, two processes in which transforming growth factor-β (TGF-β) plays an important role. We, therefore, studied the cell surface expression and functional properties of TGF-β receptors I, II and III on osteoblasts from a group of OI patients compared to healthy controls. Receptor number and affinity were determined by Scatchard analysis of binding data and TGF-β receptor II gene expression was assessed by RT-PCR. Ligand-induced downregulation of TGF-β receptors was analyzed to demonstrate the dynamic response to exogenous stimuli. All experiments were performed in parallel in human osteoblastic cells from OI patients and from age-matched controls. TGF-β receptors I, II and III (betaglycan) were present on osteoblasts from both healthy donors and OI patients. The receptor numbers were significantly higher (29,000 per cell) on OI osteoblasts than on age-matched control osteoblasts (12,000 per cell) in spite of similar steady state levels for TGF-β receptor II mRNA in OI and control cells. Furthermore, receptor affinity was not significantly different in OI osteoblasts (181 vs. 177 nM–1), and the receptor number did not depend on the culture substrate. With respect to dynamic adaption, ligand-induced downregulation of TGF-β receptors was reduced in OI osteoblasts. In conclusion, the human osteoblastic cells from patients with OI investigated all have an elevated number of cell surface receptors for TGF-β, without any evidence for a transcriptional regulation of TGF-β receptor II. On the functional level, there is some evidence for an impaired adaptive behavior of receptor presentation, whereas receptor affinity is unchanged.

Ehlers-Danlos-Syndrom

ZusammenfassungDas Ehlers-Danlos-Syndrom (EDS) umfaßt eine heterogene Gruppe von 9 hereditären Bindegewebserkrankungen, die in unterschiedlichem Ausmaß durch die Symptome einer Hyperelastizität der Haut und einer Überstreckbarkeit der Gelenke gekennzeichnet sind. Die Haut ist leicht verletzbar und zeigt abnorme Wundheilung. Der größte Teil der EDS-Patienten entfällt auf die Typen I–III, bei denen die Genese der Erkrankung noch nicht geklärt ist, auch wenn neuere Daten eine Rolle für Kollagen V in der Pathogenese nahelegen. Bei den Typen IV, VI und VII hingegen konnte die Ursache verifiziert werden: Während das EDS IV durch Mutationen im Gen für Kollagen III ausgelöst wird, liegt beim EDS VI eine Mutation im Gen der Lysylhydroxylase vor. Beim EDS VII liegt die Mutation im Gen für Kollagen I. Die EDS-Typen V, VIII und X sind sehr selten und ähneln in ihrer Symptomatik dem EDS II.SummaryThe Ehlers-Danlos syndrome (EDS) comprises a heterogenous group of nine hereditary connective tissue disorders, characterized by hyperelasticity of skin and hypermobility of joints to differing extents. The skin is easily injured and wound healing is delayed. The majority of EDS patients belong to EDS-types I–III. The pathogenesis in these cases is not known, although recent data suggest a role for collagen V. In contrast, the etiology of EDS-types IV, VI and VII has been found. While EDS IV is caused by a mutation in the collagen III gene, in EDS VI a mutation in the lysyl hydroxylase gene is present. In EDS VII, the underlying defect is a mutation in the collagen I gene. The EDS-types V, VII and X are very rare; their symptoms resemble those of EDS-type II.

In vitro studies on clonal growth of chondrocytes in thanatophoric dysplasia

Thanatophoric dysplasia (TD) is characterized by a disorganized growth plate with markedly reduced proliferative and hypertrophic cartilage zones. Therefore, we studied in vitro the proliferation rates of articular chondrocytes from five TD patients and age-matched controls in response to bFGF, IGF-I, IGF-II, and TGF-beta 1. In human fetal controls bFGF was the most potent growth factor. Clonal growth the articular chondrocytes in response to bFGF was reduced in two of five TD patients and slightly below the range of controls in a third case. Stimulation of chondrocyte proliferation by IGF I and II was reduced in the patient whose response to bFGF was most markedly impaired. The effect of TGF-beta 1 ranged from normal to slightly elevated values in TD fetuses. These results indicate heterogeneity of the underlying defects in TD. Low proliferative responses of chondrocytes to bFGF and IGF-I/II are likely to play a key role in the pathogenesis of some cases. In two of five patients studied, the mechanisms of bFGF and IGF-signal transduction are candidates for the primary molecular defect.

Femurosteosynthesen mit dem Teleskopnagel nach Bailey-Dubow bei Osteogenesis imperfecta

OperationsprinzipDeformierungen des Femurs, Achsfehlstellungen oder Femurfrakturen bei Deformation des Knochens werden durch segmentale Osteotomien korrigiert und nach Aufbohren der Markhöhle mit dem Teleskopnagel nach Bailey u. Dubow [1] stabilisiert.Der mit dem Wachstum sich elongierende Nagel wird transartikulär durch die interkondyläre Grube des distalen Femurs verankert.T-förmige Verbreiterung der Nagelenden sorgen dafür, daß mit dem Längenwachstum des Knochens der Nagel auseinandergezogen wird.

Surgical treatment of the femur in osteogenesis imperfecta with intramedullary elongating rods

Surgical Principles Deformation of the femur or fractures of the femur with displacement are corrected with segmental osteotomies and are stabilized with elongating rods according to Bailey-Dubow [1] following reaming the medullary cavity. The rods, which elongate during growth, are anchored transarticularly through the intercondylar notch of the distal femur.

146 PROLIFERATION AND COLLAGEN BIOSYNTHESIS OF CHONDROCYTES AND OSTEOBLASTS IN LETHAL SKELETAL DYSPLASIAS

We studied in vitro proliferation and collagen biosynthesis of chondrocytes and osteoblasts in thanatophoric dysplasia (TD), chondroectodermal dysplasia (CED), short rib polydaktyly-syndrome type III (SRP-III), short rib syndrome type Beemer (SR-Beemer) and osteogenesis imperfecta type II (OI-II). In TD morphologically proliferation zone is markedly reduced and in 2 out of 3 cases studied clonal growth of articular chondrocytes in methylcellulose was not or only slightly stimulated by IGF-I (1,25 ng/ml:.0%; 31% of control) and IGF-II (1,25 ng/ml:10%; 22% of control) but almost normally by TGF-β (1,25 ng/ml: 153%; 63% of control). In CED and SR-Beemer we found persistent hypertrophic cartilage islands in metaphyseal bone and in vitro elevated sensitivity of chondrocyte proliferation to TGF-β (1,25 ng/ml:376%; 213% of control). In OI-II osteoblasts synthesised electro-phoretically slower migrating collagen α1(I)-chains indicating posttranslational overmodification.We conclude that both, defects in matrix synthesis and regulation of chondrocyte proliferation play an important role in the pathogenesis of lethal skeletal dysplasias.

144 OSTEOGENESIS IMPERFECTA (OI): COLLAGEN METABOLISH OF OSTEOBLASTS IN VITRO

OI is characterized by disorders in collagen I metabolism. Biochemical aspects of OI have been studied mostly with skin fibroblasts although bone is the most affected tissue in OI. We studied collagen metabolism of osteoblasts in culture (OBC) derived from 30 OI patients (types I-IV). All OI OBC produced low lewels of collagen. Qualitative abnormal collagen I as determined by PAGE was found among all OI types. In single cases procollagen processing was slow. Many OI OBC showed a decreased pericellular accumulation of collagen I (coll.I).Conclusion: Decreased synthesis and pericellular accumulation of collagen was found associated with, but also irrespective of, obvious structural defects in collagen I and may play a crucial role in the phenotype of OI osteoblasts.