Identification of a carbonic anhydrase-like domain in the extracellular region of RPTP gamma defines a new subfamily of receptor tyrosine phosphatases.

The tyrosine phosphatase RPTP gamma is a candidate tumor suppressor gene since it is located on human chromosome 3p14.2-p21 in a region frequently deleted in certain types of renal and lung carcinomas. In order to evaluate its oncogenic potential and to explore its normal in vivo functions, we have isolated cDNAs and deduced the complete sequences of both human and murine RPTP gamma. The murine RPTP gamma gene has been localized to chromosome 14 to a region syntenic to the location of the human gene. Northern (RNA) blot analysis reveals the presence of two major transcripts of 5.5 and 8.5 kb in a variety of murine tissues. In situ hybridization analysis reveals that RPTP gamma mRNA is expressed in specific regions of the brain and that the localization of RPTP gamma changes during brain development. RPTP gamma is composed of a putative extracellular domain, a single transmembrane domain, and a cytoplasmic portion with two tandem catalytic tyrosine phosphatase domains. The extracellular domain contains a stretch of 266 amino acids with striking homology to the zinc-containing enzyme carbonic anhydrase (CAH), indicating that RPTP gamma and RPTP beta (HPTP zeta) represent a subfamily of receptor tyrosine phosphatases. We have constructed a model for the CAH-like domain of RPTP gamma based upon the crystal structure of CAH. It appears that 11 of the 19 residues that form the active site of CAH are conserved in RPTP gamma. Yet only one of the three His residues that ligate the zinc atom and are required for catalytic activity is conserved. On the basis of this model we propose that the CAH-like domain of RPTP gamma may have a function other than catalysis of hydration of metabolic CO2.

A homozygous deletion within the carbonic anhydrase-like domain of the Ptprg gene in murine L-cells.

Protein tyrosine phosphatases, on purely theoretical grounds, were suggested as possible tumor suppressor genes, and receptor protein tyrosine phosphatase gamma (PTPRG) has been proposed, on the basis of its location at human chromosome region 3p14.2, specifically as a tumor suppressor gene for renal cell carcinoma. We have isolated murine genomic and complementary DNA clones for analysis and mapping of the murine Ptprg locus; interspecific backcross analysis showed that the Ptprg locus maps to the centromeric region of mouse chromosome 14. We also observed a homozygous, intragenic deletion in the Ptprg gene in all clonal derivatives of the original L-cell strain, a methylcholanthrene-treated mouse connective tissue cell line which produces sarcomas in syngeneic mice. The deletion begins in the second intron of the carbonic anhydrase-like domain of the Ptprg gene and ends in the fourth intron of the carbonic anhydrase-like domain. At the genomic level, perhaps several hundred kilobases of DNA are deleted; at the complementary DNA level the 400 base pairs comprising exons 2, 3, and 4 of the carbonic anhydrase-like domain are deleted. By reverse transcription polymerase chain reaction, an amplified fragment is produced from L-cell mRNA which is 400 base pairs shorter than the wild type gene product, suggesting that the deleted gene is transcribed and may produce a protein product. Thus, mouse L-cells have lost one Ptprg allele and sustained an intragenic deletion in the other; such allele loss and mutation frequently occur at tumor suppressor gene loci.

Chromosome locations of the MYB related genes, AMYB and BMYB.

The MYB related loci, AMYB and BMYB, were localized to specific human chromosome regions by Southern blot analysis of their segregation patterns in a panel of rodent-human hybrid DNAs using radiolabeled AMYB and BMYB probes. The AMYB locus was present in hybrids retaining the chromosome region 8cen----8q22 and was absent in hybrids which had lost this chromosome region. The presence of the BMYB locus in rodent-human hybrids correlated with, and only with, chromosome region Xq13. Chromosomal in situ hybridization refined the localization of AMYB to region 8q22-23 and confirmed the localization of BMYB to region Xq13. Chromosome region 8q22 is involved in recurrent translocations in malignant lymphoma and in acute myeloid leukemia (AML-M2); therefore AMYB is a candidate for involvement in such translocations. A region on Xq13 is also involved in chromosomal abnormalities in acute myeloid leukemia and myelodysplasias.

Characterization of the receptor protein tyrosine phosphatase gene product PTP gamma: binding and activation by triphosphorylated nucleosides.

A full-length cDNA for a novel isoform of the human receptor tyrosine phosphatase gamma gene (PTPRG) was overexpressed in Sf9 insect cells, and the gene product, PTP gamma, was purified and characterized. The protein was expressed as a M(r) approximately 185,000 protein accompanied by a M(r) approximately 120,000 putative cleavage product on SDS-PAGE analysis. The protein undergoes N-linked glycosylation and constitutive phosphorylation of serine residues. When assayed for tyrosine-specific phosphatase activity, PTP gamma dephosphorylated myelin basic protein at a pH optimum of 7.5 and a Km of 12.6 microM; reduced carboxyamidomethylated and maleylated lysozyme (RCM-lysozyme) at a pH optimum of 6.0 and a Km of 12 microM; and p-nitrophenylphosphate with a pH optimum of 5.5 and a Km of 3.5 mM. Phosphatase activity was inhibited by ZnCl2 and sodium orthovanadate; Mg2+, Mn2+, and Ca2+ ions were ineffective. The partially purified form of the enzyme was allosterically activated by triphosphorylated nucleosides, with a preference for purines. This activation was prevented by Mg2+ addition and did not occur when a purified form of the enzyme was utilized, suggesting that its activation depends on specific activating factors or conformational constraints. Interestingly, PTP gamma protein was specifically bound by an ATP-agarose matrix through its intracellular domain, suggesting a link between binding of nucleotides and activation of the phosphatase.

Detailed genetic and physical map of the 3p chromosome region surrounding the familial renal cell carcinoma chromosome translocation, t(3;8)(p14.2;q24.1).

Extensive studies of loss of heterozygosity of 3p markers in renal cell carcinomas (RCCs) have established that there are at least three regions critical in kidney tumorigenesis, one most likely coincident with the von Hippel-Lindau gene at 3p25.3, one in 3p21 which may also be critical in small cell lung carcinomas, and one in 3p13-p14.2, a region which includes the 3p chromosome translocation break of familial RCC with the t(3;8)(p14.2;q24.1) translocation. A panel of rodent-human hybrids carrying portions of 3p, including a hybrid carrying the derivative 8 (der(8)(8pter-->8q24.1::3p14.2-->3pter)) from the RCC family, have been characterized using 3p anchor probes and cytogenetic methods. This 3p panel was then used to map a large number of genetically mapped probes into seven physical intervals between 3p12 and 3pter defined by the hybrid panel. Markers have been physically, and some genetically, placed relative to the t(3;8) break, such that positional cloning of the break is feasible.

Loss of heterozygosity at the familial RCC t(3;8) locus in most clear cell renal carcinomas.

Previously, we had observed that more than 80% of clear cell renal carcinomas (RCCs) exhibited loss of heterozygosity (LOH) between the microsatellite markers D3S1285 (in 3p14.1) and D3S1295 (in 3p21.1), a region which includes the protein tyrosine phosphatase gamma locus (PTPRG locus, PTP gamma gene) and the 3p14.2 break of the familial RCC-associated translocation, t(3;8)(p14.2;q24), which has been hypothesized to affect expression of an RCC suppressor gene or oncogene. Using seven microsatellite markers and four markers derived from a PTPRG YAC contig, we have further delineated the 3p14.2 region of LOH in RCCs. Eighty-nine % of clear cell RCCs (31 of 35) showed a common region of loss between the D3S1481 and D3S1312 loci which flank the 3p14.2 t(3;8) translocation breakpoint and the PTP gamma gene. The PTP gamma gene occupies approximately 780 kilobase pairs between markers D3S1480 and D3S1312, with its currently defined 5' end greater than 200 kilobase pairs centromeric to the 3p14.2 translocation break. Although most of the RCCs with LOH between D3S1481 and D3S1312 loci have lost at least a portion of one PTP gamma allele, we have tested all known exons of the remaining PTP gamma gene in a number of the kidney tumors and have not observed mutations. Thus, there may be another gene in the vicinity of the 3p14.2 break that is important not only in the familial RCCs in the t(3;8) family but in the majority of clear cell RCCs.

The human ryk cDNA sequence predicts a protein containing two putative transmembrane segments and a tyrosine kinase catalytic domain.

The human ryk tyrosine kinase cDNA was originally identified as a PCR-amplified cDNA fragment (JTK5) from K562 leukemia cells and found to represent a ubiquitously expressed gene (Partanen et al., 1990). The open reading frame of human ryk, reported here, encodes a novel type of putative tyrosine kinase of 607 amino acid residues, having two potential transmembrane domains and homology to receptor tyrosine kinases, such as met (HGF/SF-R) and IGF-1R, in its catalytic domain. The gene maps to human chromosome 3q11-25. Expression of the 3.4 kb ryk mRNA was found in all human adult tissues examined.

Papillon-Lefèvre syndrome: mutations and polymorphisms in the cathepsin C gene.

The Papillon-Lefèvre syndrome, inherited in an autosomal recessive pattern, manifests with palmoplantar keratoderma and early, destructive periodontitis. Recently, mutations in the gene encoding cathepsin C have been disclosed in a limited number of families with Papillon-Lefèvre syndrome. We have examined two multiplex families with Papillon-Lefèvre syndrome, and evaluated the gene encoding cathepsin C for mutations. The mutation detection strategy consisted of polymerase chain reaction amplification of all seven exons and flanking intronic sequences, followed by direct nucleotide sequencing. This strategy identified two missense mutations, W39S and G301S, affecting highly conserved amino acid residues within the cathepsin C polypeptide. The affected individuals were homozygotes whereas heterozygous carriers of the mutations were clinically unaffected, confirming the recessive nature of the mutations. Addition of these cathepsin C gene mutations into the expanding Papillon-Lefèvre syndrome mutation database allows further development of genotype/phenotype correlations towards understanding this severe genodermatosis.

Epidermolysis bullosa: novel and de novo premature termination codon and deletion mutations in the plectin gene predict late-onset muscular dystrophy.

Epidermolysis bullosa (EB) with late-onset muscular dystrophy (EB-MD) is a hemidesmosomal variant of EB due to mutations in the plectin gene (PLEC1). The age of onset of muscle involvement has been noted to vary from infancy to the fourth decade of life. Immunofluorescence of the patients' skin and muscle biopsies is usually negative for staining with antibodies recognizing plectin, a large cytoskeleton-associated anchorage protein. In this study we report novel plectin mutations in two families with EB. In both families, the proband was a newborn with neonatal blistering with no evidence for muscle weakness as yet. Peripheral blood DNA was isolated and examined by heteroduplex scanning strategy, protein truncation test (PTT), and/or direct sequencing of the plectin gene. One of the probands was compound heterozygote for nonsense mutations E2005X/K4460X, and the proband in the second family was compound heterozygote for deletion mutations 5083delG/2745-9del21, the latter mutation extending from -9 to +12 at the intron 22/exon 23 border. The mutations K4460X and 5083delG were not present in either one of the parents, thus being de novo events. In both cases, nonpaternity was excluded by microsatellite marker analysis. The stop codon mutations are predicted to result in the synthesis of a truncated protein lacking the carboxy-terminal globular domain of the protein and possibly causing nonsense-mediated decay of the corresponding mRNA. The 2745-9del21 deletion mutation abolishes the splice site at the intron 22/exon 23 junction, predicting abnormal splicing events. Because plectin deficiency is associated with muscular dystrophy, molecular diagnostics of the plectin gene provides prognostic value in evaluation of these patients who appear to be at risk to develop muscular dystrophy.

Cloning and chromosomal mapping of mouse ladinin, a novel basement membrane zone component.

Linear IgA disease is characterized by circulating IgA autoantibodies recognizing basement membrane zone components, including an anchoring filament protein, ladinin. In this study, we have cloned the mouse ladinin cDNA, elucidated the intron-exon organization of the corresponding gene (Lad1), and determined its chromosomal assignment. We have also characterized the promoter region of Lad1 and examined its tissue-specific expression. The mouse Lad1 gene consists of 10 exons spanning approximately 13.4 kb of the mouse genome on chromosome 1, and Southern analysis suggested that Lad1 is a single-copy gene. The coding region comprises 1584 nucleotides and encodes a 528-amino-acid polypeptide with a calculated molecular mass of 59 kDa. The deduced polypeptide contained two putative N-glycosylation and two O-glycosylation sites, and sequence analysis predicted a 15-amino-acid signal peptide. The 5' upstream region demonstrated the presence of consensus cis-elements for AP2 and SP1 and was GC rich, consistent with eukaryotic promoter. Northern analysis revealed expression in cultured keratinocytes, but not in fibroblasts, with the mRNA transcript being approximately 2.5 kb in size. A significant level of expression was also noted in the kidney and lung, and to a lesser degree in the liver, spleen, and brain. Ladinin is a novel component of the basement membranes and may function in contributing to the stability of the association of the epithelial layers with the underlying mesenchyme.

Pretibial epidermolysis bullosa: genetic linkage to COL7A1 and identification of a glycine-to-cysteine substitution in the triple-helical domain of type VII collagen.

Pretibial epidermolysis bullosa (PEB) is a rare variant of dominant dystrophic EB (DDEB) in which recurrent blistering with scarring predominantly involves the pretibial skin. Although blistering appears to be localized clinically, electron microscopy of the dermalepidermal junction in patients with PEB reveals anchoring fibril abnormalities that are not restricted to the predilection sites. Furthermore, PEB cannot be distinguished from the generalized (Cockayne-Touraine and Pasini) types of DDEB on the basis of anchoring fibril morphology alone. The generalized forms of DDEB have been linked to the type VII collagen gene (COL7A1) on chromosome 3p21. In this study, we sought to test the hypothesis that mutations underlying PEB also reside in COL7A1. We initiated mutational analysis in COL7A1 in a large five-generation PEB family of Taiwanese descent. We identified a G-to-T transversion at nt position 7867, which results in a glycine-to-cysteine substitution (G2623C) in exon 105. This mutation was confirmed in affected family members using the loss of a SmaI restriction site, and when used for linkage analysis, together with an intragenic PvuII polymorphism and several flanking markers, resulted in a LOD score of Z = 3.61 at theta = 0 in this family. This is the first demonstration of genetic linkage and mutation analysis in PEB, and illustrates that the Cockayne-Touraine, Pasini, and now the pretibial clinical variants of DDEB are allelic, resulting from different glycine substitution mutations in the type VII collagen gene.

Prenatal diagnosis for recessive dystrophic epidermolysis bullosa in 10 families by mutation and haplotype analysis in the type VII collagen gene (COL7A1).

BACKGROUND: Epidermolysis bullosa (EB) is a group of heritable diseases that manifest as blistering and erosions of the skin and mucous membranes. In the dystrophic forms of EB (DEB), the diagnostic hallmark is abnormalities in the anchoring fibrils, attachment structures beneath the cutaneous basement membrane zone. The major component of anchoring fibrils is type VII collagen, and DEB has been linked to the type VII collagen gene (COL7A1) at 3p21, with no evidence for locus heterogeneity. Due to life-threatening complications and significant long-term morbidity associated with the severe, mutilating form of recessive dystrophic EB (RDEB), there has been a demand for prenatal diagnosis from families with affected offspring. MATERIALS AND METHODS: Intragenic polymorphisms in COL7A1 and flanking microsatellite markers on chromosome 3p21, as well as detection of pathogenetic mutations in families, were used to perform PCR-based prenatal diagnosis from DNA obtained by chorionic villus sampling at 10-15 weeks or amniocentesis at 12-15 weeks gestation in 10 families at risk for recurrence of RDEB. RESULTS: In nine cases, the fetus was predicted to be normal or a clinically unaffected carrier of a mutation in one allele. These predictions have been validated in nine cases by the birth of a healthy child. In one case, an affected fetus was predicted, and the diagnosis was confirmed by fetal skin biopsy. CONCLUSIONS: DNA-based prenatal diagnosis of RDEB offers an early, expedient method of testing which will largely replace the previously available invasive fetal skin biopsy at 18-20 weeks gestation.

Structure of the human receptor tyrosine phosphatase gamma gene (PTPRG) and relation to the familial RCC t(3;8) chromosome translocation.

The receptor protein tyrosine phosphatase gamma gene, PTP gamma (locus name PTPRG), was previously mapped to chromosome region 3p14.2, within a 2- to 4-Mb region centromeric to the 3p14.2 breakpoint of the t(3;8) familial renal cell carcinoma (RCC)-associated constitutional chromosome translocation. Because of its chromosomal position, its enzymatic properties as a receptor phosphatase, which might oppose a growth activating kinase activity, its homozygous deletion in murine L cells, and its transcriptional activity in numerous normal tissues, including kidney, the PTP gamma gene was an attractive tumor suppressor gene candidate for renal cell carcinoma. To determine whether the PTP gamma gene was a target of loss of heterozygosity or mutation in RCCs and to determine its map position relative to the t(3;8) break at 3p14.2, we have isolated YAC and lambda genomic clones for the PTP gamma gene and other 3p14.2 markers and determined the relative positions of the t(3;8) break, a 3p14.2 de novo break possibly in a fragile site, and the 5' end of the PTP gamma gene. Additionally, the genomic structure, position of the proximal promotor, and intron-exon border sequences of the 30-exon 780-kb PTP gamma gene have been determined, which will facilitate analysis of the PTP gamma gene in tumors.

YAC-assisted cloning of transcribed sequences from the human chromosome 3p21 region.

The region surrounding the ZNF35 zinc finger protein gene on 3p21 is of particular interest, as this region of chromosome 3 is frequently involved in rearrangements and/or deletions associated with various human tumors including lung and renal carcinoma. We have analyzed yeast artificial chromosomes (YACs), identified by PCR screening, using oligonucleotides derived from the ZNF35 gene. PFGE and Southern blot/hybridization analysis revealed that the clones cover 750-kb including the ZNF35 gene. The use of specific somatic cell hybrids have allowed us to locate the YAC contig telomeric to the D3F15S2 locus, in a region which is frequently deleted in lung carcinomas. In addition, we have developed a novel cDNA hybridization protocol allowing the isolation of transcribed sequences present in the overlapping YAC clones. Using the cDNA hybridization selection, we have isolated and characterized one transcribed sequence (D3S1362E) from the 3p21 YAC contig and the corresponding cDNA has been isolated. DNA sequencing analysis indicated that the D3S1363E cDNA codes for a putative transcription factor. Northern blot analysis indicated that the D3S1362E sequence hybridized to multiple transcripts in skeletal muscle, and weakly hybridizing transcripts of similar sizes were detected in other tissues.

Chromosomal localization of four human zinc finger cDNAs.

cDNA clones encoding zinc finger motifs were isolated by screening human placenta and T-cell (Peer) cDNA libraries with zinc finger (ZNF) consensus sequences. Unique cDNA clones were mapped in the human genome by rodent-human somatic cell hybrid analysis and in some cases in situ chromosomal hybridization. ZNF80 mapped to 3p12-3qter, ZNF7 was previously mapped to 8q24 and is here shown by in situ hybridization and use of appropriate hybrids to map telomeric to the MYC locus. ZNF79 mapped to 9q34 centromeric to the ABL gene and between a constitutional chromosomal translocation on the centromeric side and the CML specific ABL translocation on the telomeric side. ZNF77 mapped to 19p while ZNF78L1 (pT3) mapped to 19q. Chromosome 19 carries many ZNF loci and other genes with zinc finger encoding motifs; the pT3 clone additionally detected a locus designated ZNF78L2, which mapped to chromosome region 1p, most likely in the region 1p32 where the MYCL and JUN loci map.

Assignment of genes encoding a unique cytokine (IL12) composed of two unrelated subunits to chromosomes 3 and 5.

IL12 (formerly NKSF or CLMF) is a unique cytokine composed of two unrelated disulfide-linked subunits. The larger 40-kDa subunit (p40) is a member of the cytokine receptor family, and the smaller 35-kDa subunit (p35) is related to IL6 and GCSF. The chromosomal localization of these two subunits was determined by PCR analysis of DNA from rodent-human hybrids. More refined mapping was obtained by PCR analysis of hybrids containing translocation chromosomes and for p40, by analysis of radiation hybrids. The subunits map to different chromosomes: p40 (IL12B) to 5q31-q33 and p35 (IL12A) to 3p12-3q13.2.

Mapping of 22 Notl linking clones on human chromosome 3 by polymerase chain reaction and somatic cell hybrid panels.

Twenty-two human chromosome 3 derived and partially sequenced Notl linking clones were mapped using two somatic cell hybrid panels. Somatic cell hybrid mapping was performed by Southern hybridization and/or by polymerase chain reaction (PCR), using 300-500 bp CpG-rich sequences surrounding Notl sites. Thus, 22 new Notl site-tagged (sequence tagged sites) STSs were created, distributed over the entire human chromosome 3. The majority of these linking clones tag known or unknown expressed sequences (genes). Together with other physical and genetic mapping methods, localization of Notl linking clones facilitates the construction of a long-range physical map and, at the same time, a transcriptional map of human chromosome 3.

Receptor protein-tyrosine phosphatase gamma is a candidate tumor suppressor gene at human chromosome region 3p21.

PTPG, the gene for protein-tyrosine phosphatase gamma (PTP gamma), maps to a region of human chromosome 3, 3p21, that is frequently deleted in renal cell carcinoma and lung carcinoma. One of the functions of protein-tyrosine phosphatases is to reverse the effect of protein-tyrosine kinases, many of which are oncogenes, suggesting that some protein-tyrosine phosphatase genes may act as tumor suppressor genes. A hallmark of tumor suppressor genes is that they are deleted in tumors in which their inactivation contributes to the malignant phenotype. In this study, one PTP gamma allele was lost in 3 of 5 renal carcinoma cell lines and 5 of 10 lung carcinoma tumor samples tested. Importantly, one PTP gamma allele was lost in three lung tumors that had not lost flanking loci. PTP gamma mRNA was expressed in kidney cell lines and lung cell lines but not expressed in several hematopoietic cell lines tested. Thus, the PTP gamma gene has characteristics that suggest it as a candidate tumor suppressor gene at 3p21.