Perlecan is a large heparan sulfate (HS) proteoglycan present in all basement membranes and in some other tissues such as cartilage, and is implicated in cell growth and differentiation. Mice lacking the perlecan gene (Hspg2) have a severe chondrodysplasia with dyssegmental ossification of the spine and show radiographic, clinical and chondro-osseous morphology similar to a lethal autosomal recessive disorder in humans termed dyssegmental dysplasia, Silverman-Handmaker type (DDSH; MIM 224410). Here we report a homozygous, 89-bp duplication in exon 34 of HSPG2 in a pair of siblings with DDSH born to consanguineous parents, and heterozygous point mutations in the 5' donor site of intron 52 and in the middle of exon 73 in a third, unrelated patient, causing skipping of the entire exons 52 and 73 of the HSPG2 transcript, respectively. These mutations are predicted to cause a frameshift, resulting in a truncated protein core. The cartilage matrix from these patients stained poorly with antibody specific for perlecan, but there was staining of intracellular inclusion bodies. Biochemically, truncated perlecan was not secreted by the patient fibroblasts, but was degraded to smaller fragments within the cells. Thus, DDSH is caused by a functional null mutation of HSPG2. Our findings demonstrate the critical role of perlecan in cartilage development.

Perlecan, a large heparan sulfate proteoglycan, is a component of the basement membrane and other extracellular matrices and has been implicated in multiple biological functions. Mutations in the perlecan gene (HSPG2) cause two classes of skeletal disorders: the relatively mild Schwartz-Jampel syndrome (SJS) and severe neonatal lethal dyssegmental dysplasia, Silverman-Handmaker type (DDSH). SJS is an autosomal recessive skeletal dysplasia characterized by varying degrees of myotonia and chondrodysplasia, and patients with SJS survive. The molecular mechanism underlying the chondrodystrophic myotonia phenotype of SJS is unknown. In the present report, we identify five different mutations that resulted in various forms of perlecan in three unrelated patients with SJS. Heterozygous mutations in two patients with SJS either produced truncated perlecan that lacked domain V or significantly reduced levels of wild-type perlecan. The third patient had a homozygous 7-kb deletion that resulted in reduced amounts of nearly full-length perlecan. Unlike DDSH, the SJS mutations result in different forms of perlecan in reduced levels that are secreted to the extracellular matrix and are likely partially functional. These findings suggest that perlecan has an important role in neuromuscular function and cartilage formation, and they define the molecular basis involved in the difference in the phenotypic severity between DDSH and SJS.

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Heparan sulfate proteoglycans are thought to mediate the action of growth factors. The heparan sulfate-containing proteoglycans in extracts of the bovine fetal rib growth plate were detected using the monoclonal antibody 3G10, which recognizes a neoepitope generated by heparitinase digestion (David, G., Bai, X. M., Van der Schueren, B., Cassiman, J. J., and Van den Berghe, H. (1992) J. Cell Biol. 119, 961-975). The heparan sulfate proteoglycans that react with this antibody were identified using antisera to known proteoglycans; purified using CsCl density gradient centrifugation, molecular sieve, and ion exchange chromatography; and then characterized. The major heparan sulfate proteoglycans in the growth plate had core proteins of 200 kDa and larger and were identified as perlecan and aggrecan. These two heparan sulfate proteoglycans could be effectively separated from each other by CsCl density gradient centrifugation alone. Perlecan contained 25% heparan sulfate and 75% chondroitin sulfate. The heparan sulfate chains on growth plate perlecan were considerably smaller than the chondroitin sulfate chains, and the heparan sulfate disaccharide content was different than that found for heparan sulfate from either kidney, tumor tissue, or growth plate aggrecan. Aggrecan contained only 0.1% heparan sulfate, which was localized to the CS-1 domain of aggrecan. These results indicate that perlecan and aggrecan would be the principal candidate proteoglycans involved in the action of heparan sulfatebinding proteins in the developing growth plate.

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