Deregulation of the platelet-derived growth factor B-chain gene via fusion with collagen gene COL1A1 in dermatofibrosarcoma protuberans and giant-cell fibroblastoma.

Dermatofibrosarcoma protuberans (DP), an infiltrative skin tumour of intermediate malignancy, presents specific features such as reciprocal translocations t(17;22)(q22;q13) and supernumerary ring chromosomes derived from the t(17;22). In this report, the breakpoints from translocations and rings in DP and its juvenile form, giant cell fibroblastoma (GCF), were characterised on the genomic and RNA level. These rearrangements fuse the platelet-derived growth factor B-chain (PDGFB, c-sis proto-oncogene) and the collagen type I alpha 1 (COL1A1) genes. PDGFB has transforming activity and is a potent mitogen for a number of cell types, but its role in oncogenic processes is not fully understood. COL1A1 is a major constituent of the connective tissue matrix. Neither PDGFB nor COL1A1 have so far been implicated in any tumour translocations. These gene fusions delete exon 1 of PDGFB, and release this growth factor from its normal regulation.

Association of a novel constitutional translocation t(1q;3q) with familial renal cell carcinoma.

Four cases of late onset clear cell renal cell carcinoma (RCC), a case of gastric cancer, and a case of exocrine pancreatic cancer were identified in a Japanese family. In order to elucidate the underlying mechanism for tumorigenesis in this family, extensive genetic studies were performed including routine and spectral karyotyping (SKY), fluorescence in situ hybridisation (FISH), comparative genomic hybridisation (CGH), loss of heterozygosity studies (LOH), and VHL mutation analysis. A germline translocation t(1;3)(q32-q41;q13-q21) was identified by karyotyping in five members of the family including all three RCC cases tested. The translocation was refined to t(1;3)(q32;q13.3) by FISH analysis using locus specific genomic clones, and the two breakpoints were mapped to a 5 cM region in 3q13.3 and a 3.6 cM region in 1q32. Both CGH and allelotyping using microsatellite markers showed loss of the derivative chromosome 3 carrying a 1q segment in the three familial RCCs analysed. Additional chromosomal imbalances were identified by CGH, including amplifications of chromosomes 5 and 7 and loss of 8p and 9. No germline VHL mutation was found but two different somatic mutations, a splice (IVS1-2A>C) and a frameshift (726delG), were identified in two RCCs from the same patient confirming their distinct origin. Taken together, these results firmly support a three step model for tumorigenesis in this family. A constitutional translocation t(1q;3q) increased the susceptibility to loss of the derivative chromosome 3 which is then followed by somatic mutations of the RCC related tumour suppressor gene VHL located in the remaining copy of chromosome 3.

Characterization of the human NIPSNAP1 gene from 22q12: a member of a novel gene family.

Rapid progress in sequencing of human and other genomes allows high-resolution analysis of their gene content on the basis of comparison between species. We have used a combined computer and biochemical approach to characterize 135 kb of human genomic sequence from 22q12 and discovered a new 10 exon gene, termed NIPSNAP1, located between the neurofibromatosis type 2 and the pK1.3 genes. The NIPSNAP1 gene spans 26 kb of genomic sequence and shows to large introns in the 5'-region. All exon-intron junctions contain the gt/ag consensus splice site. The putative promoter of the NIPSNAP1 gene is TATA-less and resides in a GC-rich island characteristic of housekeeping genes. The NIPSNAP1 mRNA is 2.1 kb, is expressed ubiquitously at variable levels, with the highest expression in liver, is terminated by an uncommon ATTAAA polyadenylation site, and is capable of encoding a 284-amino-acid protein. This NIPSNAP1 protein has a strong sequence similarity limited to the central portion of a hypothetical protein (acc. P34492) from chromosome III of C. elegans, in which the other portions resemble a 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein. Thus, the NIPSNAP1 gene is a member of an evolutionarily well conserved, novel gene family with two members in human and mouse that have now been characterized, and one member in C. elegans. The second human gene, NIPSNAP2, is localized in the vicinity of marker D7S499 on chromosome 7. Although the function of the NIPSNAP protein family is unknown, clues about its role may reside in the co-expression of the C. elegans orthologue, within an operon encoding protein motifs known to be involved in vesicular transport.

Role of L-arginine, a substrate for nitric oxide-synthase, in gastroprotection and ulcer healing.

Nitric oxide (NO) synthesized from L-arginine interacts with prostaglandins (PG) and sensory neuropeptides in the regulation of mucosal integrity, but the role of L-arginine, a substrate for NO-synthase, in gastroprotection and healing of chronic gastric ulcers has been little studied. In this study we compared the effects of intragastric (i.g.) and systemic (i.v.) administration of L-arginine or D-arginine on gastric secretion and acute gastric lesions provoked in rats by i.g. application of 100% ethanol, acidified aspirin (ASA), or the exposure to 3.5h of water immersion and restraint stress (WRS). In addition, the effects of L-arginine on ulcer healing and the formation of new vessels (angiogenesis) were determined, using monoclonal antibody (MAb E-9). L-arginine (10-200 mg/kg i.g.) failed to significantly affect gastric secretion but dose-dependently reduced the gastric lesions induced by 100% ethanol. ASA, and WRS, the doses inhibiting 50% of these lesions being 65, 94, and 72 mg/kg, respectively. This protection was accompanied by a significant rise in the gastric blood flow (GBF), whereas L-arginine given i.v. failed to affect the ethanol-lesions and the GBF. D-arginine or the NO-related amino acids--L-glutamine, L-citrulline, or L-ornithine--failed to significantly influence these lesions. Suppression of the generation of mucosal PG by indomethacin or capsaicin-denervation attenuated the protection and hyperemia induced by L-arginine. The inhibition of constitutive NO synthase by L-NNA had no significant effect on the protection afforded by L-arginine, but reduced the gastric hyperemia accompanying this protection. L-arginine (150 mg/kg per day, i.g.) accelerated the ulcer healing and increased GBF at the ulcer margin, and angiogenesis, whereas treatment with L-NNA had an opposite effect. L-arginine added to NG-nitro-L-arginine (L-NNA) restored the ulcer healing, hyperemia, and angiogenesis. We conclude that: (1) the protective activity of L-arginine involves gastric hyperemia mediated by NO and a mild irritant effect due to enhanced generation of endogenous PG, and (2) the ulcer healing properties of L-arginine depend upon its hyperemic and angiogenic actions, possibly involving NO.

The use of computer-aided image analysis in the evaluation of epithelial nuclear area in the digestive tract of rats.

Semiautomatic computer-aided image analysis was used to measure nuclear area of epithelial cells (surface and glandular epithelium) in various parts of digestive tract in rats. In surface epithelium largest nuclei were observed in duodenum and jejunum. In glandular epithelium largest nuclei were found in duodenum. Nuclear area of epithelial cells within normal mucosa of digestive tract shows differences, that seems to be related to localization and resorption activity.

Computer-aided analysis of the nuclear area in proliferative lesions of the colonic and bronchial epithelium.

The aim of this study was to establish the diagnostical value of nuclear area in proliferative lesions of colonic and bronchial epithelium. The study included 11 adenomas and 9 adenocarcinomas of colon, 11 control samples of normal colonic epithelium as well as 5 cases of bronchial squamous cell metaplasia, 25 squamous cell carcinomas, 9 small cell carcinomas and 3 adenocarcinomas of bronchi and 13 control samples of normal bronchial epithelium. All tissue samples were formalin fixed, paraffin embedded and silver stained. Morphometrical analysis was performed using optical Axiophot microscope with immersion, B/W CCD camera connected to frame grabber card, computer PC AT 386 and morphometrical software Vist and Morpho. Nuclear area was measured semiautomatically. Within each case 100 nuclei were analyzed. Statistically significant differences between carcinoma and adenoma or normal epithelium were observed in colon. In bronchi nuclear area was significantly higher in squamous cell carcinoma and adenocarcinoma if compared with other lesions including small cell carcinoma or bronchial metaplasia and normal epithelium.

Morphometric measurements of the nuclear area in proliferative thyroid lesions.

The aim of this study was to establish the diagnostic value of nuclear area in proliferative lesions of thyroid gland. This study included 98 histological preparations of thyroid and 57 cytological preparations (touch imprints). Morphometric analysis was performed using light microscopy, B/W CCD camera connected to the frame grabber card, computer PC AT 386/3 and morphometric software Vist and Morpho. Nuclear area was measured semiautomatically. Our study indicated that the nuclear area was significantly higher in cytological preparations and that size of nuclei seems to depend on the type of neoplasm and to a lesser extent on whether a given tumor was malignant or benign.

Fine needle aspiration cytology combined with argyrophilic nucleolar organizer regions (AgNORs) in diagnosis of thyroid neoplasms.

NORs are the loops of DNA that contain the sites of ribosomal genes around which the nucleolus is formed during telophase of mitosis. In light microscopy they can be visualized by simple silver staining technique as small dark dots within the nucleus. It has been recognized that in many neoplasms, especially malignant AgNORs are more numerous and often atypical when compared with benign tumors and normal tissue. We have introduced this novel technique to the fine needle cytology of thyroid neoplasms (n = 56). We have analyzed the number, the area of AgNORs, the number of clustered AgNORs in the nucleus and the ratio of AgNOR area to the nuclear area and the area of single AgNOR by means of semiautomatic computerized image analysis. We have studied cytological samples consisting of 7 simple goiters, 7 hyperplasias, 15 follicular adenomas, 7 oxyphilic follicular adenomas, 6 follicular carcinomas, 8 oxyphilic follicular carcinomas, 6 papillary adenocarcinomas. In this study we have demonstrated that some differences in the AgNORs value are associated with the type of tumor rather than with malignancy. Location of the AgNORs seems to be very typical for some types of tumors. For example in oxyphilic neoplasms they form single clusters in the nucleus and in papillary adenocarcinomas they form at least two abundant clusters. In other proliferative lesions of the thyroid gland location of AgNORs is less typical.

The mouse ortholog of the human SMARCB1 gene encodes two splice forms.

The human SMARCB1 gene (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily b, member 1, previously named the INI1/hSNF5 gene) is a tumor suppressor gene located on chromosome 22q11.2 and is inactivated in malignant rhabdoid tumors. By using an EST-based approach, we cloned two splice forms of the Smarcb1 gene in mouse and a longer splice form of the human ortholog. Proteins corresponding to the longer (385 aa) and the shorter (376 aa) forms are 100% conserved between human and mouse. Meningiomas and schwannomas are tumors frequently deleting various regions on chromosome 22, including the SMARCB1 locus. We therefore directly sequenced seven SMARCB1 exons (90% of the open reading frame) in search for mutations in 41 meningiomas and 23 schwannomas. No inactivating mutations were observed, which suggests that the SMARCB1 gene is not involved in the pathogenesis of these tumors.

A sequence highly similar to PNG is located on chromosome 22q12 in intron 15 of the LIMK-2 gene.

In this report we describe a sequence (PNG22) highly similar to the Phospholipase C beta3 Neighboring Gene (PNG). We also report that PNG22 is located in the q12 region on chromosome 22 between markers D22S1144 and D22S280. This finding explains that PNG probes cross hybridize to sequences on chromosome 22. Fine mapping using our sequence data and the complete sequence of a PAC clone (DJ515N1), located in this region, determined that PNG22 is located in intron 15 of the LIMK-2 gene. PNG22 is 93% homologous to PNG, however it do not have the introns described for the PNG gene, instead matching the cDNA sequence. This leads us to suggest that PNG22 probably represents a PNG pseudogene. In this report we also list the exon intron borders and the genomic structure of LIMK-2 and place it on the Sanger Center chromosome 22 Physical map. It also explains the finding that PNG probes cross hybridize to sequences on chromosome 22.

Genomic structure, 5' flanking sequences, and precise localization in 1P31.1 of the human prostaglandin F receptor gene.

This paper describes the genomic structure of the human Prostaglandin F receptor gene (FP) with its exon-intron borders and 5' flanking sequences. Furthermore, the location of the gene has been localized to a very small region on 1p31.1 using FISH and radiation hybrids analysis. The PGF receptor (FP) is highly expressed in mouse tissues especially in the corpora lutea in ovaries and in the kidney. Recently, it has been shown that homozygous knockout-mice lacking the gene for this receptor are unable to deliver normal fetuses at term. It might be speculated that the lack of the FP gene has the same effect in human as in mouse. Mutation analysis in families with difficulties in parturition would therefore be of high interest. The results presented here provides data necessary for further investigations of the FP gene.

The germinal center kinase gene and a novel CDC25-like gene are located in the vicinity of the PYGM gene on 11q13.

Multiple endocrine neoplasia type 1 (MEN1) is tightly linked to the muscle-type glycogen phosphorylase (PYGM) gene in 11q13. This region of the human genome contains additional disease-related loci implicated in the development of insulin-dependent diabetes mellitus, familial paraganglioma type 2, spinocerebellar ataxia type 5, Bardet-Biedl syndrome and translocation t(11;17) described in B-cell non-Hodgkin's lymphoma. We approached cloning of candidate disease genes from 11q13 by large-scale genomic sequencing. We obtained > 106 kb of sequence around the PYGM gene and established a transcriptional map that includes: (i) two genes previously localized to 11q13, PYGM and a zinc-finger protein (ZFM1) gene; (ii) the germinal center kinase (GCK, human B-lymphocyte serine/threonine protein kinase) gene; (iii) a novel human CDC25-like (HCDC25L) gene; (iv) a dystrophia myotonica protein kinase-like (DMPKL) gene; and (v) a novel ubiquitously expressed gene of unknown function (germinal center kinase- neighboring gene, GCKNG).

Characterization of five novel human genes in the 11q13-q22 region.

The redundancy of sequences in dbEST has approached a level where contiguous cDNA sequences of genes can be assembled, without the need to physically handle the clones from which the ESTs are derived. This is termed EST based in silico gene cloning. With the availability of sequence chromatogram files for a subset of ESTs, the quality of EST sequences can be ascertained accurately and used in contig assembly. In this report, we performed a study using this approach and isolated five novel human genes, C11orf1-C11orf5, in the 11q13-q22 region. The full open reading frames of these genes were determined by comparison with their orthologs, of which four mouse orthologs were isolated (c11orf1, c11orf2, c11orf3 and c11orf5). These genes were then analyzed using several proteomics tools. Both C11orf1 and C11orf2 are nuclear proteins with no other distinguishing features. C11orf3 is a cytoplasmic protein containing an ATP/GTP binding site, a signal peptide located in the N-terminus and a similarity to the C. elegans protein "Probable ARP 2/3 complex 20kD subunit." C11orf4 is a peptide which displays four putative transmembrane domains and is predicted to have a cytoplasmic localization. It contains signal peptides at the N- and C-termini. C11orf5 is a putative nuclear protein displaying a central coiled coil domain. Here, we propose that this purely EST-based cloning approach can be used by modestly sized laboratories to rapidly and accurately characterize and map a significant number of human genes without the need of further sequencing.

Mutations in short stature homeobox containing gene (SHOX) in dyschondrosteosis but not in hypochondroplasia.

Dyschondrosteosis (DCO) and hypochondroplasia (HCH) are common skeletal dysplasias characterized by disproportionate short stature. The diagnosis of these conditions might be difficult to establish especially in early childhood. Point mutations and deletions of the short stature homeobox containing gene (SHOX) are detected in DCO and idiopathic short stature with some rhizomelic body disproportion, whereas mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are found in 40-70% of HCH cases. In this study, we performed mutational analysis of the coding region of the SHOX gene in five DCO and 18 HCH patients, all of whom tested negative for the known HCH-associated FGFR3 mutations. The polymorphic CA-repeat analysis, direct sequencing and Southern blotting were used for detection of deletions and point mutations. The auxological and radiological phenotype of these patients was carefully determined. Three novel mutations in DCO patients were found: (1) a deletion of one base (de1272G) (according to GenBank accession nos. Y11536, Y11535), resulting in a premature stop codon at position 75 of the amino acid sequence; (2) the transversion C485G resulting in the substitution Leu132Val; and (3) the transversion G549T causing an Arg153Leu substitution. These substitutions segregate with the DCO phenotype and affect evolutionarily conserved homeodomain residues, based on a comparison of homeobox containing proteins in 13 species. Moreover, these changes were not found in 80 unrelated, unaffected individuals. This strongly suggests that these mutations are pathogenic. The phenotype of our patients with DCO and HCH varied from mild to severe shortness and body disproportion. These results further support clinical and genetic heterogeneity of dyschondrosteosis and hypochondroplasia.

Characterization of a second human clathrin heavy chain polypeptide gene (CLH-22) from chromosome 22q11.

We report cloning and characterization of the second human clathrin heavy chain polypeptide gene (CLH-22) localized to chromosome 22q11. Hence H. sapiens is the first species for which two clathrin heavy chain genes have been reported. We provide 5470 bp cDNA sequence covering the entire open reading frame of the CLH-22 gene. The predicted polypeptide is composed of 1640 amino acids. Its 6 kb transcript is expressed in all of 16 tested human tissues, suggesting it is a housekeeping gene. Skeletal muscle, testis and heart show significantly higher expression levels. Compared to the previously characterized human clathrin heavy chain gene localized on chromosome 17 (CLH-17), CLH-22 shows different transcript size and expression profile in human tissues. Northern analysis of CLH-22 suggests that several alternatively spliced transcripts exist. A presumably single, 171 bp long alternatively spliced exon has been characterized. Amino acid sequence comparison between CLH-22 and CLH-17 shows an overall identify and similarity of 84.7 and 91.1%, respectively. At the nucleic acid level, identity between open reading frames of both genes is 74.3%. Sequence comparison with previously cloned genes in other species suggests that counterparts of the CLH-17 gene have been cloned in B. taurus and R. norvegicus, whereas presumptive mammalian homologues of the CLH-22 gene are yet to be characterized. Our Northern and Southern blot analyses of meningiomas clearly suggest the CLH-22 gene may be involved in the tumor development and can be considered as a candidate for a tumor suppressor.

Sequence and expression of the mouse homologue to human phospholipase C beta3 neighboring gene.

We describe the isolation and expression of a murine homologue of the Phospholipase C beta3 Neighboring Gene (PNG), located in the MEN1 region on chromosome 11q13. The PNG cDNA was isolated using a human PNG cDNA clone (SOM172). Human and mouse PNG do not have any marked similarity to other known genes on the DNA level, but the predicted protein display similarity to the C-terminal part of Phospholipase C beta2. Northern blots with mouse PNG probes revealed expression of a 1 kb message in multiple tissues, and an additional 2.3 kb band in testis. The predicted murine protein contains 203 amino acids. In situ hybridization histochemistry displayed png mRNA expression in several tissues of the midstage mouse embryo, including the central nervous system. In late stage embryos, png was highly expressed in skeletal muscle, retina and neocortex. In the adult animal, expression was restricted to testis and thymus.

The LZTFL1 gene is a part of a transcriptional map covering 250 kb within the common eliminated region 1 (C3CER1) in 3p21.3.

Deletions on 3p have been described in a large number of human tumors, suggesting the presence of a tumor suppressor gene(s). Using the elimination test, we previously defined a 1-Mb segment from human 3p21.3 (C3CER1). Genomic sequencing allowed us to construct a transcription map covering 250 kb containing five genes. We have characterized a human leucine zipper containing gene, leucine zipper transcription factor-like 1 (LZTFL1), and its mouse orthologue (Lztfl1), which was also mapped to mouse chromosome 9F. The LZTFL1 gene has two transcript isoforms displaying alternative polyadenylation. We have localized the human orthologue of the yeast SAC1 (suppressor of actin) gene as well as characterized and mapped the mouse Sac1 gene. Furthermore, the XT3 gene was characterized, encoding a member of the Na(+)/Cl(-) neurotransmitter superfamily. It has been shown that the XT3 gene had an alternatively spliced brain-specific isoform, predicted to remove 1 of 12 putative transmembrane domains. The transcription map also includes the CC chemokine receptor 9 gene (CCR9) and the LIM domain containing gene 1 (LIMD1). This work partially defines the gene content of C3CER1 that is a prerequisite for delineation of its role in tumorigenesis.

Structure of the promoter and genomic organization of the human beta'-adaptin gene (BAM22) from chromosome 22q12.

Adaptins are major structural components of heterotetrameric protein complexes called adaptors, which are essential in intracellular receptor transport via clathrin-coated vesicles. beta-adaptins constitute one of three known classes (alpha, beta, gamma) of adaptins, including beta and beta' subtypes. We previously cloned the human beta'-adaptin gene (BAM22) (GDB symbol, ADTB1) from chromosome 22q12 and proposed its involvement in the development of meningiomas. Here we describe the genomic organization of this gene, which consists of 22 exons spanning over approximately 100 kb. We also report results from point mutation screening of 7 randomly chosen exons analyzed in 110 sporadic meningiomas. As part of the genomic characterization of the BAM22 locus, we sequenced 40 kb covering exons 1-4 and 12 kb upstream from the start of gene transcription. Analysis of the sequence suggests that the BAM22 gene has a CG-rich promoter.

Duplications on human chromosome 22 reveal a novel Ret Finger Protein-like gene family with sense and endogenous antisense transcripts.

Analysis of 600 kb of sequence encompassing the beta-prime adaptin (BAM22) gene on human chromosome 22 revealed intrachromosomal duplications within 22q12-13 resulting in three active RFPL genes, two RFPL pseudogenes, and two pseudogenes of BAM22. The genomic sequence of BAM22vartheta1 shows a remarkable similarity to that of BAM22. The cDNA sequence comparison of RFPL1, RFPL2, and RFPL3 showed 95%-96% identity between the genes, which were most similar to the Ret Finger Protein gene from human chromosome 6. The sense RFPL transcripts encode proteins with the tripartite structure, composed of RING finger, coiled-coil, and B30-2 domains, which are characteristic of the RING-B30 family. Each of these domains are thought to mediate protein-protein interactions by promoting homo- or heterodimerization. The MID1 gene on Xp22 is also a member of the RING-B30 family and is mutated in Opitz syndrome (OS). The autosomal dominant form of OS shows linkage to 22q11-q12. We detected a polymorphic protein-truncating allele of RFPL1 in 8% of the population, which was not associated with the OS phenotype. We identified 6-kb and 1.2-kb noncoding antisense mRNAs of RFPL1S and RFPL3S antisense genes, respectively. The RFPL1S and RFPL3S genes cover substantial portions of their sense counterparts, which suggests that the function of RFPL1S and RFPL3S is a post-transcriptional regulation of the sense RFPL genes. We illustrate the role of intrachromosomal duplications in the generation of RFPL genes, which were created by a series of duplications and share an ancestor with the RING-B30 domain containing genes from the major histocompatibility complex region on human chromosome 6.