Pan-tumor landscape of fibroblast growth factor receptor 1-4 genomic alterations.

BACKGROUND: Selective tyrosine kinase inhibitors targeting fibroblast growth factor receptor (FGFR) 1-4 genomic alterations are in development or have been approved for FGFR-altered cancers (e.g. bladder cancer and advanced intrahepatic cholangiocarcinoma). Understanding FGFR inhibitor-resistance mechanisms is increasingly relevant; we surveyed the pan-tumor landscape of FGFR1-4 genomic alterations [short variants (SVs), gene rearrangements (REs), and copy number alterations (CNAs)], including their association with tumor mutational burden (TMB) and the genomic comutational landscape. PATIENTS AND METHODS: Comprehensive genomic profiling of 355 813 solid tumor clinical cases was performed using the FoundationOne and FoundationOne CDx assays (Foundation Medicine, Inc.) to identify genomic alterations in >300 cancer-associated genes and TMB (determined on ≤1.1 megabases of sequenced DNA). RESULTS: FGFR1-4 SVs and REs occurred in 9603/355 813 (2.7%), and CNAs in 15 078/355 813 (4.2%) samples. Most common FGFR alterations for bladder cancer, intrahepatic cholangiocarcinoma, and glioma were FGFR3 SVs (1051/7739, 13.6%), FGFR2 REs (618/6641, 9.3%), and FGFR1 SVs (239/11 550, 2.1%), respectively. We found several, potentially clinically relevant, tumor-specific associations between FGFR1-4 genomic alterations and other genomic markers. FGFR3 SV-altered bladder cancers and FGFR1 SV-altered gliomas were significantly less likely to be TMB-high versus unaltered samples. FGFR3 SVs in bladder cancer significantly co-occurred with TERT and CDKN2A/B alterations; TP53 and RB1 alterations were mutually exclusive. In intrahepatic cholangiocarcinoma, FGFR2 REs significantly co-occurred with BAP1 alterations, whereas KRAS, TP53, IDH1, and ARID1A alterations were mutually exclusive. FGFR1 SVs in gliomas significantly co-occurred with H3-3A and PTPN11 alterations, but were mutually exclusive with TERT, EGFR, TP53, and CDKN2A/B alterations. CONCLUSIONS: Overall, our hypothesis-generating findings may help to stratify patients in clinical trials and guide optimal targeted therapy in those with FGFR alterations.

Downmodulation of key inflammatory cell markers with a topical Janus kinase 1/2 inhibitor.

BACKGROUND: INCB018424 is a novel, potent Janus kinase (JAK)1/JAK2 inhibitor that blocks signal transduction of multiple proinflammatory cytokines. OBJECTIVES: To evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics and preliminary efficacy of topical INCB018424 phosphate cream in patients with plaque psoriasis. METHODS: Topical INCB018424 phosphate 1·0% or 1·5% cream was applied once daily (QD) or twice daily (BID) for 4 weeks to 2-20% body surface area in five sequential cohorts of five patients aged 18-65 years. Target lesions were scored on a scale of 0-4 for erythema, scaling and thickness. Additionally, the overall disease activity in each patient was measured using Physician's Global Assessment. INCB018424 concentrations were measured in plasma, and cytokine stimulated phosphorylated signal transducer and activator of transcription 3 phosphorylation (pSTAT3) levels in peripheral blood cells were evaluated. Pretreatment and post-treatment skin biopsies were compared with healthy skin, including evaluation of histopathology, immunohistochemistry and mRNA expression. RESULTS: Treatment with INCB018424 phosphate cream either 1·0% QD or 1·5% BID resulted in improvements in lesion scores. No significant inhibition of pSTAT3 in peripheral blood cells was observed following topical application, consistent with the generally low steady-state plasma concentrations of INCB018424 measured. Transcriptional markers of immune cell lineage/activation in lesional skin were reduced by topical INCB018424, with correlations observed between clinical improvement and decreases in markers of T helper 17 lymphocyte activation, dendritic-cell activation and epidermal hyperplasia. INCB018424 treatment reduced epidermal hyperplasia and dermal inflammation in most patient samples, with reductions in CD3, CD11c, Ki67 and keratin 16 observed by immunohistochemical analysis. CONCLUSIONS: Topical INCB018424 dosed for 28 days QD or BID is pharmacologically active in patients with active psoriasis and modulates proinflammatory cytokines in the pathogenesis of psoriatic lesions.

A comprehensive system for protein purification and biochemical analysis based on antibodies to c-myc peptide.

The genomics revolution has created a need for increased speed and generality for recombinant protein production systems as well as general methods for conducting biochemical assays with the purified protein products. 9E10 is a well-known high-affinity antibody that has found use in a wide variety of biochemical assays. Here we present a standardized system for purifying proteins with a simple epitope tag based on c-myc peptide using an antibody affinity column. Antibodies with binding parameters suitable for protein purification have been generated and characterized. To purify these antibodies from serum-containing medium without carrying through contaminating immunoglobulin G, a peptide-based purification process was developed. A fluorescence polarization binding assay was developed to characterize the antigen-antibody interaction. Protein purification protocols were optimized using a fluorescein-labeled peptide as a surrogate "protein." Binding and elution parameters were evaluated and optimized and basic operating conditions were defined. Several examples using this procedure for the purification of recombinant proteins are presented demonstrating the generality of the system. In all cases tested, highly pure final products are obtained in good yields. The combination of the antibodies described here and 9E10 allow for almost any biochemical application to be utilized with a single simple peptide tag.

Human BIN3 complements the F-actin localization defects caused by loss of Hob3p, the fission yeast homolog of Rvs161p.

The BAR adaptor proteins encoded by the RVS167 and RVS161 genes from Saccharomyces cerevisiae form a complex that regulates actin, endocytosis, and viability following starvation or osmotic stress. In this study, we identified a human homolog of RVS161, termed BIN3 (bridging integrator-3), and a Schizosaccharomyces pombe homolog of RVS161, termed hob3+ (homolog of Bin3). In human tissues, the BIN3 gene was expressed ubiquitously except for brain. S. pombe cells lacking Hob3p were often multinucleate and characterized by increased amounts of calcofluor-stained material and mislocalized F-actin. For example, while wild-type cells localized F-actin to cell ends during interphase, hob3Delta mutants had F-actin patches distributed randomly around the cell. In addition, medial F-actin rings were rarely found in hob3Delta mutants. Notably, in contrast to S. cerevisiae rvs161Delta mutants, hob3Delta mutants showed no measurable defects in endocytosis or response to osmotic stress, yet hob3+ complemented the osmosensitivity of a rvs161Delta mutant. BIN3 failed to rescue the osmosensitivity of rvs161Delta, but the actin localization defects of hob3Delta mutants were completely rescued by BIN3 and partially rescued by RVS161. These findings suggest that hob3+ and BIN3 regulate F-actin localization, like RVS161, but that other roles for this gene have diverged somewhat during evolution.

Aspartyl beta -hydroxylase (Asph) and an evolutionarily conserved isoform of Asph missing the catalytic domain share exons with junctin.

The mouse aspartyl beta-hydroxylase gene (Asph, BAH) has been cloned and characterized. The mouse BAH gene spans 200 kilobase pairs of genomic DNA and contains 24 exons. Of three major BAH-related transcripts, the two largest (6,629 and 4,419 base pairs) encode full-length protein and differ only in the use of alternative polyadenylation signals. The smallest BAH-related transcript (2,789 base pairs) uses an alternative 3' terminal exon, resulting in a protein lacking a catalytic domain. Evolutionary conservation of this noncatalytic isoform of BAH (humbug) is demonstrated in mouse, man, and Drosophila. Monoclonal antibody reagents were generated, epitope-mapped, and used to definitively correlate RNA bands on Northern blots with protein species on Western blots. The gene for mouse junctin, a calsequestrin-binding protein, was cloned and characterized and shown to be encoded from the same locus. When expressed in heart tissue, BAH/humbug preferably use the first exon and often the fourth exon of junctin while preserving the reading frame. Thus, three individual genes share common exons and open reading frames and use separate promoters to achieve differential expression, splicing, and function in a variety of tissues. This unusual form of exon sharing suggests that the functions of junctin, BAH, and humbug may be linked.

The thrombospondin motif of aggrecanase-1 (ADAMTS-4) is critical for aggrecan substrate recognition and cleavage.

Aggrecanase-1 (ADAMTS-4) is a member of the a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) protein family that was recently identified. Aggrecanase-1 is one of two ADAMTS cartilage-degrading enzymes purified from interleukin-1-stimulated bovine nasal cartilage (Tortorella, M. D., Burn, T. C., Pratta, M. A. , Abbaszade, I., Hollis, J. M., Liu, R., Rosenfeld, S. A., Copeland, R. A., Decicco, C. P., Wynn, R., Rockwell, A., Yang, F., Duke, J. L., Solomon, K., George, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Ross, H., Wiswall, B. H., Murphy, K., Hillman, M. C., Jr., Hollis, G. F., and Arner, E.C. (1999) Science 284, 1664-1666; 2 Abbaszade, I., Liu, R. Q., Yang, F., Rosenfeld, S. A., Ross, O. H., Link, J. R., Ellis, D. M., Tortorella, M. D., Pratta, M. A., Hollis, J. M., Wynn, R., Duke, J. L., George, H. J., Hillman, M. C., Jr., Murphy, K., Wiswall, B. H., Copeland, R. A., Decicco, C. P., Bruckner, R., Nagase, H., Itoh, Y., Newton, R. C., Magolda, R. L., Trzaskos, J. M., and Burn, T. C. (1999) J. Biol. Chem. 274, 23443-23450). The aggrecan products generated by this enzyme are found in cartilage cultures stimulated with cytokines and in synovial fluid from patients with arthritis, suggesting that aggrecanase-1 may be important in diseases involving cartilage destruction. Here we demonstrate that the thrombospondin type-1 (TSP-1) motif located within the C terminus of aggrecanase-1 binds to the glycosaminoglycans of aggrecan. Data from several studies indicate that this binding of aggrecanase-1 to aggrecan through the TSP-1 motif is necessary for enzymatic cleavage of aggrecan. 1) A truncated form of aggrecanase-1 lacking the TSP-1 motif was not effective in cleaving aggrecan. 2) Several peptides representing different regions of the TSP-1 motif effectively blocked aggrecanase-1 cleavage of aggrecan by preventing the enzyme from binding to the substrate. 3) Aggrecanase-1 was not effective in cleaving glycosaminoglycan-free aggrecan. Taken together, these data suggest that the TSP-1 motif of aggrecanase-1 is critical for substrate recognition and cleavage.

Sites of aggrecan cleavage by recombinant human aggrecanase-1 (ADAMTS-4).

Aggrecan, the major proteoglycan of cartilage that provides its mechanical properties of compressibility and elasticity, is one of the first matrix components to undergo measurable loss in arthritic diseases. Two major sites of proteolytic cleavage have been identified within the interglobular domain (IGD) of the aggrecan core protein, one between amino acids Asn(341)-Phe(342) which is cleaved by matrix metalloproteinases and the other between Glu(373)-Ala(374) that is attributed to aggrecanase. Although several potential aggrecanase-sensitive sites had been identified within the COOH terminus of aggrecan, demonstration that aggrecanase cleaved at these sites awaited isolation and purification of this protease. We have recently cloned human aggrecanase-1 (ADAMTS-4) (Tortorella, M. D., Burn, T. C., Pratta, M. A., Abbaszade, I., Hollis, J. M., Liu, R., Rosenfeld, S. A., Copeland, R. A., Decicco, C. P., Wynn, R., Rockwell, A., Yang, F., Duke, J. L., Solomon, K., George, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Ross, H., Wiswall, B. H., Murphy, K., Hillman, M. C., Jr., Hollis, G. F., Newton, R. C., Magolda, R. L., Trzaskos, J. M., and Arner, E. C. (1999) Science 284, 1664-1666) and herein demonstrate that in addition to cleavage at the Glu(373)-Ala(374) bond, this protease cleaves at four sites within the chondroitin-sulfate rich region of the aggrecan core protein, between G2 and G3 globular domains. Importantly, we show that this cleavage occurs more efficiently than cleavage within the IGD at the Glu(373)-Ala(374) bond. Cleavage occurred preferentially at the KEEE(1667-1668)GLGS bond to produce both a 140-kDa COOH-terminal fragment and a 375-kDa fragment that retains an intact G1. Cleavage also occurred at the GELE(1480-1481)GRGT bond to produce a 55-kDa COOH-terminal fragment and a G1-containing fragment of 320 kDa. Cleavage of this 320-kDa fragment within the IGD at the Glu(373)-Ala(374) bond then occurred to release the 250-kDa BC-3-reactive fragment from the G1 domain. The 140-kDa GLGS-reactive fragment resulting from the preferential cleavage was further processed at two additional cleavage sites, at TAQE(1771)-(1772)AGEG and at VSQE(1871-1872)LGQR resulting in the formation of a 98-kDa fragment with an intact G3 domain and two small fragments of approximately 20 kDa. These data elucidate the sites and efficiency of cleavage during aggrecan degradation by aggrecanase and suggest potential tools for monitoring aggrecan cleavage in arthritis.

Possible role of valvular serotonin 5-HT(2B) receptors in the cardiopathy associated with fenfluramine.

Dexfenfluramine was approved in the United States for long-term use as an appetite suppressant until it was reported to be associated with valvular heart disease. The valvular changes (myofibroblast proliferation) are histopathologically indistinguishable from those observed in carcinoid disease or after long-term exposure to 5-hydroxytryptamine (5-HT)(2)-preferring ergot drugs (ergotamine, methysergide). 5-HT(2) receptor stimulation is known to cause fibroblast mitogenesis, which could contribute to this lesion. To elucidate the mechanism of "fen-phen"-associated valvular lesions, we examined the interaction of fenfluramine and its metabolite norfenfluramine with 5-HT(2) receptor subtypes and examined the expression of these receptors in human and porcine heart valves. Fenfluramine binds weakly to 5-HT(2A), 5-HT(2B), and 5-HT(2C) receptors. In contrast, norfenfluramine exhibited high affinity for 5-HT(2B) and 5-HT(2C) receptors and more moderate affinity for 5-HT(2A) receptors. In cells expressing recombinant 5-HT(2B) receptors, norfenfluramine potently stimulated the hydrolysis of inositol phosphates, increased intracellular Ca(2+), and activated the mitogen-activated protein kinase cascade, the latter of which has been linked to mitogenic actions of the 5-HT(2B) receptor. The level of 5-HT(2B) and 5-HT(2A) receptor transcripts in heart valves was at least 300-fold higher than the levels of 5-HT(2C) receptor transcript, which were barely detectable. We propose that preferential stimulation of valvular 5-HT(2B) receptors by norfenfluramine, ergot drugs, or 5-HT released from carcinoid tumors (with or without accompanying 5-HT(2A) receptor activation) may contribute to valvular fibroplasia in humans.

Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family.

Aggrecan is responsible for the mechanical properties of cartilage. One of the earliest changes observed in arthritis is the depletion of cartilage aggrecan due to increased proteolytic cleavage within the interglobular domain. Two major sites of cleavage have been identified in this region at Asn(341)-Phe(342) and Glu(373)-Ala(374). While several matrix metalloproteinases have been shown to cleave at Asn(341)-Phe(342), an as yet unidentified protein termed "aggrecanase" is responsible for cleavage at Glu(373)-Ala(374) and is hypothesized to play a pivotal role in cartilage damage. We have identified and cloned a novel disintegrin metalloproteinase with thrombospondin motifs that possesses aggrecanase activity, ADAMTS11 (aggrecanase-2), which has extensive homology to ADAMTS4 (aggrecanase-1) and the inflammation-associated gene ADAMTS1. ADAMTS11 possesses a number of conserved domains that have been shown to play a role in integrin binding, cell-cell interactions, and extracellular matrix binding. We have expressed recombinant human ADAMTS11 in insect cells and shown that it cleaves aggrecan at the Glu(373)-Ala(374) site, with the cleavage pattern and inhibitor profile being indistinguishable from that observed with native aggrecanase. A comparison of the structure and expression patterns of ADAMTS11, ADAMTS4, and ADAMTS1 is also described. Our findings will facilitate the study of the mechanisms of cartilage degradation and provide targets to search for effective inhibitors of cartilage depletion in arthritic disease.

Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins.

We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis.

Multiple control elements are required for expression of the human CD34 gene.

Two cis regulatory elements of the human CD34 gene, the promoter and a 3' enhancer, have previously been described. In transient transfection assays, the promoter was not sufficient to direct cell type specific expression. In contrast, the 3' enhancer was active only in CD34+ cell lines, suggesting that this element might be responsible for stem cell-restricted expression of the CD34 gene. In the current work, through deletion and transient transfection experiments, we delineated the core enhancer sequence. We examined the role of this element upon stable integration. Our data suggested the presence of additional control elements. In order to identify them, using DNaseI hypersensitivity and methylation studies, we determined the chromatin structure of the entire CD34 locus. Amongst a number of DNaseI hypersensitive sites, we detected a strong CD34+ cell type-specific site in intron 4. This region, however, did not work as an enhancer by itself. By analyzing stable transfectants and transgenic animals, we demonstrated that the 3' enhancer and intron 4 hypersensitive regions, either alone or together, did not function as a locus control region upon chromosomal integration. In contrast, a 160kb genomic fragment encompassing the entire CD34 gene contained regulatory elements sufficient for high-level CD34 mRNA expression in murine stable lines. Our data indicate that combinatorial action of multiple, proximal and long-range, cis elements is necessary for proper regulation of CD34 expression.

A 2.5-Mb transcript map of a tumor-suppressing subchromosomal transferable fragment from 11p15.5, and isolation and sequence analysis of three novel genes.

11p15.5 is an important tumor-suppressor gene region, showing loss of heterozygosity in Wilms tumor, rhabdomyosarcoma, adrenocortical carcinoma, and lung, ovarian, and breast cancer. We previously mapped directly by genetic complementation a subtransferable fragment (STF) harboring an embryonal tumor-suppressor gene and spanning about 2.5 Mb. We have now mapped the centromeric end of this STF between D11S988 and D11S12 and its telomeric end between D11S1318 and TH. We have isolated a complete contig of PAC, P1, BAC, and cosmid genomic clones spanning the entire 2.5-Mb region defined by this STF, as well as more than 200 exons from these genomic clones using exon trapping. We have isolated genes in this region by directly screening DNA libraries as well as by database searching for ESTs. Nine of these genes have been reported previously by us and by others. However, the initial mapping of most of those genes was based on FISH or somatic cell hybrid analysis, and here we precisely define their physical location. These genes include RRM1, GOK (D11S4896E), Nup98, CARS, hNAP2 (NAP1L4), p57KIP2 (CDKN1C), KVLQT1 (KCNA9), TAPA-1, and ASCL2. In addition, we have identified several novel genes in this region, three of which, termed TSSC1, TSSC2, and TSSC3, are reported here. TSSC1 shows homology to Rb-associated protein p48 and chromatin assembly factor CAF1, and it is located between GOK and Nup98. TSSC2 is homologous to Caenorhabditis elegans beta-mannosyl transferase, and it lies between Nup98 and CARS. TSSC3 shows homology to mouse TDAG51, which is implicated in FasL-mediated apoptosis, and it is located between hNAP2 and p57KIP2. Thus, these genes may play a role in malignancies that involve this region.

Polycystin: in vitro synthesis, in vivo tissue expression, and subcellular localization identifies a large membrane-associated protein.

The primary structure of polycystin predicts a large integral membrane protein with multiple cell recognition motifs, but its function remains unknown. Insight into polycystin's normal function and its role in the development of autosomal dominant polycystic kidney disease (PKD1) requires the assembly of an extensive collection of molecular reagents to examine its expression and create model systems for functional studies. Development of these crucial reagents has been complicated due to the presence of transcriptionally active homologous loci. We have assembled the authentic full-length PKD1 cDNA and demonstrated expression of polycystin in vitro. Polyclonal antibodies directed against distinct extra- and intracellular domains specifically immunoprecipitated in vitro translated polycystin. The panel of antibodies was used to determine localization of polycystin in renal epithelial and endothelial cell lines and tissues of fetal, adult, and cystic origins. In normal adult kidney and maturing fetal nephrons, polycystin expression was confined to epithelial cells of the distal nephron and vascular endothelial cells. Expression in the proximal nephron was only observed after injury-induced cell proliferation. Polycystin expression was confined to ductal epithelium in liver, pancreas, and breast, and restricted to astrocytes in normal brain. We report clear evidence for the membrane localization of polycystin by both tissue sections and by confocal microscopy in cultured renal and endothelial cells. Interestingly, when cultured cells made cell-cell contact, polycystin was localized to the lateral membranes of cells in contact. These data suggest that polycystin is likely to have a widespread role in epithelial cell differentiation and maturation and in cell-cell interactions.

The NTN2L gene encoding a novel human netrin maps to the autosomal dominant polycystic kidney disease region on chromosome 16p13.3.

The netrins define a family of chemotropic factors that have been shown to play a central role in axon guidance. We identified two exon traps encoding netrin-like sequences during the assembly of a transcriptional map for the genomic interval surrounding the polycystic kidney disease type 1 and tuberous sclerosis type 2 genes. We describe the characterization of a novel human netrin-2-like gene, designated NTN2L, and its transcript. The genomic interval containing the NTN2L gene was sequenced, and the coding region was predicted based on computer analysis. The structure of the NTN2L gene has been confirmed utilizing nested RT-PCR. The NTN2L gene is predicted to encode a 580-amino-acid protein having homology to the chicken and Drosophila netrins and to Caenorhabditis elegans UNC-6. The NTN2L gene has a restricted pattern of expression; its transcript is undetectable by Northern analysis in all tissues examined, but can be recovered from spinal cord RNA by RT-PCR. This report represents the first description and characterization of a human netrin.

The cloning of a human ABC gene (ABC3) mapping to chromosome 16p13.3.

The ATP binding cassette (ABC) transporters, or traffic ATPases, constitute a large family of proteins responsible for the transport of a wide variety of substrates across cell membranes in both prokaryotic and eukaryotic cells. We describe a human ABC protein with regions of strong homology to the recently described murine ABC1 and ABC2 transporters. The gene for this novel protein, human ABC3, maps near the polycystic kidney disease type 1 (PKD1) gene on chromosome 16p13.3. The ABC3 gene is expressed at highest levels in lung compared to other tissues.

The tuberin (TSC2), autosomal dominant polycystic kidney disease (PKD1), and somatostatin type V receptor (SSTR5) genes form a synteny group in the Fugu genome.

The tuberous sclerosis 2 (TSC2) and polycystic kidney disease 1 (PKD1) genes are adjacent on human chromosome 16p13.3 and form part of a conserved synteny group with mouse chromosome 17. We have determined that the PKD1 gene is evolutionarily conserved, single copy, and linked to TSC2 in the Fugu genome. A short cosmid contig has been identified containing both genes based on hybridization, exon trapping, and random sequence data. In addition sequences homologous to the somatostatin type V receptor (SSTR5) were identified 5' to PKD1, defining a larger syntenic region, as this gene has also been mapped to human chromosome 16p13.3. As in mammalian genomes, the Fugu TSC2 and PKD1 genes are adjacent in a tail-to-tail orientation.

A novel ribosomal protein L3-like gene (RPL3L) maps to the autosomal dominant polycystic kidney disease gene region.

A full-length cDNA encoding a novel ribosomal protein L3 gene was isolated and sequenced. The deduced protein sequence is 407 amino acids long and shows 77% identity to other known mammalian ribosomal protein L3 genes, which are themselves highly conserved. Southern blot analysis of human genomic DNA suggests that this novel gene is single copy. While the previously identified human ribosomal protein L3 gene has ubiquitous expression in all tissues surveyed, the novel gene described herein is strongly expressed in skeletal muscle and heart tissue, with low levels of expression in the pancreas. This novel gene, RPL3L, is located in a gene-rich region near the PKD1 and TSC2 genes on chromosome 16p13.3.

Generation of a transcriptional map for a 700-kb region surrounding the polycystic kidney disease type 1 (PKD1) and tuberous sclerosis type 2 (TSC2) disease genes on human chromosome 16p3.3.

A 700-kb region of DNA in human chromosome 16p13.3 has been shown to contain the polycystic kidney disease 1 (PKD1) and the tuberous sclerosis type 2 (TSC2) disease genes. An estimated 20 genes are present in this region of chromosome 16. We have initiated studies to identify transcribed sequences in this region using a bacteriophage P1 contig containing 700 kb of DNA surrounding the PKD1 and TSC2 genes. We have isolated 96 unique exon traps from this interval, with 23 of the trapped exons containing sequences from five genes known to be in the region. Thirty exon traps have been mapped to additional transcription units based on data base homologies, Northern analysis, or their presence in cDNA or reverse transcriptase (RT)-PCR products. We have mapped the human RNPS gene to the cloned interval. We have obtained cDNAs or RT-PCR products from eight novel genes, with sequences from seven of these genes having homology to sequences in the data bases. Two of the newly identified genes represent human homologs for rat and murine genes identified previously. We have isolated three exon traps with homology to sequences in the data bases but have been unable to confirm the presence of these exon traps in expressed sequences. In addition, we have isolated 43 exon traps that do not map to our existing cDNAs or PCR products and have no homology to sequences in the data bases. In this report we present a transcriptional map for the 700 kb of DNA surrounding the PKD1 and TSC2 genes.

Practicing managed care in the academic medical center.

The purpose of this article is to outline the contrasts between the traditional AMC and an organization oriented toward the delivery of population-based managed care. Academic medical centers differ from one another considerably in the extent to which they serve as quaternary care community resources, the degree to which they emphasize primary care in training and care delivery, and the amount of research undertaken. Nor is there a single organizational structure for managed care; successful managed care is practices in IPAs, multispecialty groups, PHOs, and staff-model HMOs. Nonetheless, the contrasts outlined here between AMCs and managed care organizations (MCOs) are valid in most cases.