New insights into ADPKD molecular pathways using combination of SAGE and microarray technologies.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the PKD1 or PKD2 gene, but cellular mechanisms of cystogenesis remain unclear. In an attempt to display the array of cyst-specific molecules and to elucidate the disease pathway, we have performed comprehensive high-throughput expression analysis of normal and ADPKD epithelia in a two-step fashion. First, we generated expression profiles of normal and cystic epithelia derived from kidney and liver using serial analysis of gene expression (SAGE). We found 472 and 499 differentially expressed genes with fivefold difference in liver and kidney libraries, respectively. These genes encode growth factors, transcription factors, proteases, apoptotic factors, molecules involved in cell-extracellular matrix interactions, and ion channels. As a second step, we constructed a custom cDNA microarray using a subset of the differentially regulated genes identified by SAGE and interrogated ADPKD patient samples. Subsequently, a set of differentially expressed genes was refined to 26 up-regulated and 48 down-regulated genes with ap value of <0.01. This study may provide valuable insights into the pathophysiology of ADPKD and suggest potential therapeutic targets.

Crystallization and preliminary X-ray analysis of recombinant human acid beta-glucocerebrosidase, a treatment for Gaucher's disease.

Acid beta-glucocerebrosidase (N-acylsphingosyl-1-O-beta-D-glucoside:glucohydrolase) is a lysosomal glycoprotein that catalyzes the hydrolysis of the glycolipid glucocerebroside to glucose and ceramide. Inadequate levels of this enzyme underly the pathophysiology of Gaucher's disease. Cerezyme (Genzyme Corporation, Cambridge, MA, USA) is a partially deglycosylated form of recombinant human acid beta-glucocerebrosidase that is used in the treatment of Gaucher patients. Although acid beta-glucocerebrosidase belongs to a large family of glycosidases, relatively little is known regarding its structural biology. Here, the crystallization and the initial diffraction analysis of Cerezyme are reported. The crystals are C-centered orthorhombic, with unit-cell parameters a = 285.0, b = 110.2, c = 91.7 A. A 99.9% complete data set has been collected to 2.75 A with an R(sym) of 8.8%.

Tumors associated with oncogenic osteomalacia express genes important in bone and mineral metabolism.

Oncogenic osteomalacia (OOM) is associated with primitive mesenchymal tumors that secrete phosphaturic factors resulting in low serum concentrations of phosphate and calcitriol, phosphaturia, and defective bone mineralization. To identify overexpressed genes in these tumors, we compared gene expression profiles of tumors resected from patients with OOM and histologically similar control tumors using serial analysis of gene expression (SAGE). Three hundred and sixty-four genes were expressed at least twofold greater in OOM tumors compared with control tumors. A subset of 67 highly expressed genes underwent validation with an extended set of OOM and control tumors using array analysis or reverse-transcription polymerase chain reaction (RT-PCR). Ten of these validated genes were consistently overexpressed in all OOM tumors relative to control tumors. Strikingly, genes with roles in bone matrix formation, mineral ion transport, and bone mineralization were highly expressed in the OOM tumors.

Canine PKD1 is a single-copy gene: genomic organization and comparative analysis.

Most cases of autosomal dominant polycystic kidney disease are caused by mutations in the gene PKD1, encoding polycystin-1. To gain insight into the role of polycystin-1 in tubulogenesis and cystogenesis using the well-characterized canine kidney epithelial cell line MDCK, we have now cloned and characterized the exon/intron structure of the canine gene PKD1. FISH analysis showed that the dog genome lacks the multiple PKD1 homologs present in human. Intron 21 of dog PKD1 lacked the polypyrimidine tract characteristic of the human gene, whereas pyrimidine-rich elements were identified in canine intron 30. Canine polycystin-1 showed a higher degree of homology with the human counterpart and lower homology with mouse and rat. A striking degree of conservation (97% identity) was determined for the leucine-rich repeat domain between dog and human. Also, the homology analysis indicated that 4 of 16 Ig-like repeats (IgIII, IgVII, IgX, and IgXV) are likely to be functionally significant. This is particularly important in light of our recent findings demonstrating that Iglike domains form strong homophilic interactions and can mediate cell-cell adhesion. These data enable detailed analysis of the role of polycystin-1 in cystogenesis and tubulogenesis using the canine MDCK cell line.