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.

Human bone marrow stromal cells (HBMSC) are pluripotent cells with the potential to differentiate into osteoblasts, chondrocytes, myelosupportive stroma, and marrow adipocytes. We used high-throughput DNA sequencing analysis to generate 4258 single-pass sequencing reactions (known as expressed sequence tags, or ESTs) obtained from the 5Ј (97) and 3Ј (4161) ends of human cDNA clones from a HBMSC cDNA library. Our goal was to obtain tag sequences from the maximum number of possible genes and to deposit them in the publicly accessible database for ESTs (dbEST of the National Center for Biotechnology Information). Comparisons of our EST sequencing data with nonredundant human mRNA and protein databases showed that the ESTs represent 1860 gene clusters. The EST sequencing data analysis showed 60 novel genes found only in this cDNA library after BLAST analysis against 3.0 million ESTs in NCBI's dbEST database. The BLAST search also showed the identified ESTs that have close homology to known genes, which suggests that these may be newly recognized members of known gene families. The gene expression profile of this cell type is revealed by analyzing both the frequency with which a message is encountered and the functional categorization of expressed sequences. Comparing an EST sequence with the human genomic sequence database enables assignment of an EST to a specific chromosomal region (a process called digital gene localization) and often enables immediate partial determination of intron/exon boundaries within the genomic structure. It is expected that high-throughput EST sequencing and data mining analysis will greatly promote our understanding of gene expression in these cells and of growth and development of the skeleton.Key Words: human bone marrow stromal cells, EST sequencing analysis, novel genes, gene expression profile, data mining them potentially useful for cell and gene therapy [2][3][4]. After extensive proliferation in vitro, the HBMSC population includes precursor cells for at least four types of connective tissue: bone, cartilage, hematopoiesis-supporting stroma, and associated adipocytes [5][6][7][8]. When single bone marrow stromal cells develop into individual HBMSC colonies, they show different morphologies and rates of proliferation. HBMSC strains derived from individual colonies also vary widely in their ability to form bone and the hematopoietic microenvironment after in vivo transplantation [9]. Despite efforts to understand the biology of HBMSC through studies 8Article doi:10.1006/geno.2001.6683, available online at http://www.idealibrary.com on IDEAL at different levels, the study of genes and proteins important to the biological phenotypes of HBMSC is still in its infancy. Well-known exceptions include STRO1 [10,11], THY1 [12,13], and SCA1 [14]. Determining the genetic expression profile of this specific cell type is key to rapid advances in understanding skeletal growth and development. The ability to peer into HBMSC and read their molecular signature will enable us to identify more preci...