[Show abstract][Hide abstract] ABSTRACT: Loss of heterozygosity (LOH) of chromosome 16q24.3 is a common genetic alteration observed in invasive ductal and lobular breast carcinomas. We constructed a physical map and generated genomic DNA sequence data spanning 2.4 Mb in this region. Detailed in silico and in vitro analyses of the genomic sequence data enabled the identification of 104 genes. It was hypothesized that tumor-suppressor genes would exhibit marked mRNA expression variability in a panel of breast cancer cell lines as a result of downregulation due to mutation or hypermethylation. We examined the mRNA expression profiles of the genes identified at 16q24.3 in normal breast, a normal breast epithelial cell line, and several breast cancer cell lines exhibiting 16q24.3 LOH. Three of the genes, CYBA, Hs.7970, and CBFA2T3, exhibited variability ten times higher than the baseline. The possible role of these genes as tumor suppressors is discussed.
[Show abstract][Hide abstract] ABSTRACT: Loss of heterozygosity (LOH) at the long arm of chromosome 16 occurs in at least half of all breast tumors and is considered to target one or more tumor suppressor genes. Despite extensive studies by us and by others, a clear consensus of the boundaries of the smallest region of overlap (SRO) could not be identified. To find more solid evidence for SROs, we tested a large series of 712 breast tumors for LOH at 16q using a dense map of polymorphic markers. Strict criteria for LOH and retention were applied, and results that did not meet these criteria were excluded from the analysis. We compared LOH results obtained from samples with different DNA isolation methods, ie., from microdissected tissue versus total tissue blocks. In the latter group, 16% of the cases were excluded because of noninterpretable LOH results. The selection of polymorphic markers is clearly influencing the LOH pattern because a chromosomal region seems more frequently involved in LOH when many markers from this region are used. The LOH detection method, i.e., radioactive versus fluorescence detection, has no marked effect on the results. Increasing the threshold window for retention of heterozygosity resulted in significantly more cases with complex LOH, i.e., several alternating regions of loss and retention, than seen in tumors with a small window for retention. Tumors with complex LOH do not provide evidence for clear-cut SROs that are repeatedly found in other samples. On disregarding these complex cases, we could identify three different SROs, two at band 16q24.3 and one at 16q22.1. In all three tumor series, we found cases with single LOH regions that designated the distal region at 16q24.3 and the region at 16q22.1. Comparing histological data on these tumors did not result in the identification of a particular subtype with LOH at 16q or a specific region involved in LOH. Only the rare mucinous tumors had no 16q LOH at all. Furthermore, a positive estrogen content is prevalent in tumors with 16q LOH, but not in tumors with LOH at 16q24.3 only.
Cancer Research 03/2001; 61(3):1171-7. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CD20 and the beta subunit of the high affinity receptor for IgE (FcepsilonRIbeta) are related four-transmembrane molecules that are expressed on the surface of hematopoietic cells and play crucial roles in signal transduction. Herein, we report the identification and characterization of a human gene, TETM4, that encodes a novel four-transmembrane protein related to CD20 and FcepsilonRIbeta. The predicted TETM4 protein is 200 amino acids and contains four putative transmembrane regions, N- and C-terminal cytoplasmic domains, and three inter-transmembrane loop regions. TETM4 shows 31.0 and 23.2% overall identity with CD20 and FcepsilonRIbeta respectively, with the highest identity in the transmembrane regions, whereas the N- and C-termini and inter-transmembrane loops are more divergent. Northern blot and RT-PCR analysis suggest that TETM4 mRNA has a highly restricted tissue distribution, being expressed selectively in the testis. Using fluorescence in situ hybridization and radiation hybrid analysis, the TETM4 gene has been localized to chromosome 11q12. The genes for CD20 and FcepsilonRIbeta have also been mapped to the same region of chromosome 11 (11q12-13.1), suggesting that these genes have evolved by duplication to form a family of four-transmembrane genes. TETM4 is the first nonhematopoietic member of the CD20/FcepsilonRIbeta family, and like its hematopoietic-specific relatives, it may be involved in signal transduction as a component of a multimeric receptor complex.
Biochemical and Biophysical Research Communications 02/2001; 280(1):374-9. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel gene product, GPR74, with homology to the seven transmembrane-domain receptor superfamily, has been cloned. GPR74 has been identified from the expressed sequence tags (EST) database. Subsequent PCR amplification of that sequence and screening of a human heart cDNA library led to the isolation of a 1.7-kb cDNA clone encoding a protein of 408 amino acids. GPR74 shows highest amino acid identity (33%) to the human neuropeptide Y-receptor subtype Y2. The human and mouse genes for GPR74 have been isolated and their exon-intron structures determined. In both species the gene consists of four exons spanning around 20 kb with the exon-intron borders being 100% conserved. Northern analysis of various human tissues reveals highest levels of mRNA expression in brain and heart. In situ hybridisation analysis of rat brain tissue confirms this result and identifies the hippocampus and amygdala nuclei as the brain areas with particular high expression of GPR74 mRNA. Fluorescence in situ hybridisation, PCR analysis on a radiation hybrid panel and interspecific mouse backcross mapping have localised the genes to human chromosome 4q21 and mouse chromosome 5. Expression of the human GPR74 cDNA as a GFP-fusion protein in various cell lines reveals the inability of the recombinant receptor protein to reach the cell surface. This is consistent with the lack of NPY specific binding in these cells and suggests that unknown factors are required for a full functional receptor complex.
Molecular Brain Research 06/2000; 77(2):199-208. · 2.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two novel G-protein-coupled receptors, one from human, GPR72, and one from mouse, GPR73 have been isolated, sequenced and their genomic organisation determined. Non-isotopic in situ hybridisation and radiation hybrid mapping have identified GPR72 to be localised on human chromosome 11q21.1, and GPR73 on human chromosome 2p14. Interspecific mouse backcross mapping has localised the genes to mouse chromosomes 9 and 6, respectively. Northern analysis reveals GPR72 mRNA expression only in brain tissue. However, GPR73 mRNA can be found in heart, skeletal muscle and pancreas. Both receptors are closely related with 36 and 33% overall amino acid identity, respectively, to the Y-receptor family. However, although successful cell surface expression in a heterologous expression system can be achieved no specific binding to this ligand family can be detected, indicating that perhaps additional factors are required for binding.
Biochimica et Biophysica Acta 05/2000; 1491(1-3):369-75. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In a search for candidate tumor suppressor genes within a 650-kb common region of loss of heterozygosity (LOH) at 16q24.3 in breast cancer tissues, a 2.6-kb cDNA, named copine VII (CPNE7), was characterized. The gene is 2654 bp and codes for a 633-residue protein with high homology to the other members of the copine family, such as copine I, copine III, and N-copine. The predicted amino acid sequence contains two copies of a C2 domain in the N-terminus. Since these domains have been found in several membrane-binding proteins involved in different intracellular processes, copine VII was viewed as a potential tumor suppressor gene. Mutation analysis was carried out by single-strand conformation polymorphism analysis of 18 breast tumor tissue samples with ascertained LOH on chromosome 16q24.3. Since only two polymorphisms were identified, no evidence was found to indicate that copine VII is the tumor suppressor gene at 16q24.3 involved in breast cancer.
[Show abstract][Hide abstract] ABSTRACT: SPG7 is a newly identified gene involved in an autosomal recessive form of hereditary spastic paraplegia (HSP), a genetically heterogeneous group of neurodegenerative disorders. This gene encodes a protein characterized as a nuclear-encoded mitochondrial metalloprotease. The present report describes the genomic structure of the SPG7 gene. It is organized into 17 exons ranging from 78 to 242 bp and spans approximately 52 kb within three overlapping cosmids. The exon/intron boundaries and all splice junctions are consistent with the published consensus sequences for donor and acceptor sites. The provided genomic structure of SPG7 should facilitate the screening for mutations in this gene in patients with HSP and other related mitochondrial disease syndromes. SPG7 has been mapped to chromosome 16q24.3, a region of frequent loss of heterozygosity (LOH) seen in sporadic breast and prostate cancer. We have performed single-strand conformation polymorphism analysis of ten exons of this gene in a number of sporadic breast cancer samples showing LOH at 16q24.3. No mutations were detected; only single nucleotide polymorphisms were observed in exon 11, intron 7, intron 10 and intron 12. An expression analysis study has revealed the differential expression of SPG7 mRNA in various tissues and at different developmental stages.
Human Genetics 07/1999; 105(1-2):139-44. · 4.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Differential display polymerase chain reaction has been used to isolate genes regulated in vascular endothelial cells by the angiogenic factor vascular endothelial cell growth factor (VEGF). Analysis of one of the bands consistently up-regulated by VEGF led us to the identification of a cDNA from a human umbilical vein endothelial cell library that is 77% identical to the human K+-Cl- cotransporter1 (KCC1). We have referred to the predicted protein as K+-Cl- cotransporter 3 (KCC3). Hydrophobicity analysis of the KCC3 amino acid sequence showed an almost identical pattern to KCC1, suggesting 12 membrane-spanning segments, a large extracellular loop with potential N-glycosylation sites, and cytoplasmic N- and C-terminal regions. The KCC3 mRNA was highly expressed in brain, heart, skeletal muscle, and kidney, showing a distinct pattern and size from KCC1 and KCC2. The KCC3 mRNA level in endothelial cells increased on treatment with VEGF and decreased with the proinflammatory cytokine tumor necrosis factor alpha, whereas KCC1 mRNA levels remained unchanged. Stable overexpression of KCC3 cDNA in HEK293 cells produced a glycoprotein of approximately 150 kDa, which was reduced to 120 kDa by glycosidase digestion. An increased initial uptake rate of 86Rb was seen in clones with high KCC3 expression, which was dependent on extracellular Cl- but not Na+ and was inhibitable by the loop diuretic agent furosemide. The KCC3 genomic localization was shown to be 15q13 by fluorescence in situ hybridization. Radiation hybrid analysis placed KCC3 within an area associated with juvenile myoclonic epilepsy. These results suggest KCC3 is a new member of the KCC family that is under distinct regulation from KCC1.
Journal of Biological Chemistry 05/1999; 274(15):10661-7. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The recently identified Fanconi anaemia A (FAA) gene is located on chromosomal band 16q24.3 within a region that has been frequently reported to show loss of heterozygosity (LOH) in breast cancer. FAA mutation analysis of 19 breast tumours with specific LOH at 16q24.3 was performed. Single-stranded conformational polymorphism (SSCP) analysis on cDNA and genomic DNA, and Southern blotting failed to identify any tumour-specific mutations. Five polymorphisms were identified, but frequencies of occurrence did not deviate from those in a normal control population. Therefore, the FAA gene is not the gene targeted by LOH at 16q24.3 in breast cancer. Another tumour suppressor gene in this chromosomal region remains to be identified.
British Journal of Cancer 04/1999; 79(7-8):1049-52. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel gene product, GPR56, with homology to the seven transmembrane-domain receptor superfamily, has been cloned by PCR amplification using degenerate oligonucleotide primers and subsequent screening of a human heart cDNA library. The isolated 2.8-kb cDNA clone encodes a protein of 693 amino acids that shows highest identity (32%) to HE6, a member of a subclass of the class B secretin-like G-protein-coupled receptors. Northern analysis of various human tissues revealed a wide distribution of the transcript with highest levels found in thyroid gland, brain, and heart. In situ hybridization analysis of human thyroid gland as well as rat heart and brain tissue confirms these results and identifies the hippocampus and hypothalamic nuclei as brain areas with particularly high expression of GPR56 mRNA. The high level of mRNA expression, its wide distribution, and the mucin-like extracellular domain of the receptor protein suggest a possible role for this receptor in cell-cell interaction processes. The human gene for GPR56 has been isolated and its exon-intron structure determined. The total length of the human GPR56 gene is approximately 15 kb, and it consists of 13 exons. Fluorescence in situ hybridization, PCR analysis of somatic cell hybrids, and interspecific mouse backcross mapping have localized the genes to human chromosome 16q13 and mouse chromosome 8.
[Show abstract][Hide abstract] ABSTRACT: In sporadic breast cancer, loss of heterozygosity (LOH) frequently occurs in three discrete regions of the long arm of chromosome 16q, the most telomeric of which is located at 16q24.3. Among the genes mapped to this region, PISSLRE is a plausible candidate tumor suppressor gene. It codes for a putative cyclin-dependent kinase that, as with other members of this family, is likely to be involved in regulating the cell cycle and therefore may have a role in oncogenesis. We characterized the genomic structure of PISSLRE and found that the splicing of this gene is complex. A variety of different transcripts were identified, including those due to cryptic splice sites, exon skipping, insertion of intronic sequences, and exon scrambling. The last phenomenon was observed in a rare PISSLRE transcript in which exons are joined at a nonconsensus splice site in an order different from that predicted by the genomic sequence. To screen the PISSLRE gene in breast tumors with ascertained LOH at 16q24.3, we have analyzed each exon by single-strand conformational polymorphism. No variation was found in the coding sequence, leading us to conclude that another tumor suppressor must be targeted by LOH in sporadic breast cancer.
[Show abstract][Hide abstract] ABSTRACT: Loss of heterozygosity involving the long arm of chromosome 16 is a frequent event seen in a number of human carcinomas, including breast, prostate, hepatocellular, and ovarian cancers. A region found to be commonly deleted in breast and prostate carcinomas is located at 16q24.3, which suggests the presence of a tumor suppressor gene that may be altered in these two malignancies. A detailed physical and transcription map of this region that includes the loci defining the smallest region of deletion has been constructed. This report describes the characterization of a transcript located in this region, the growth arrest-specific 11 (GAS11) gene, which was viewed as a potential tumor suppressor gene due to the expression of its mouse homolog specifically during growth arrest. The gene consists of 11 exons spanning approximately 25 kb. Northern blot analysis identified two ubiquitously expressed mRNAs of 3.4 and 1.8 kb produced by the use of alternative polyadenylation sites. Another gene, C16orf3 (chromosome 16 open reading frame 3), was found to lie within intron 2 of GAS11. This gene appears intronless, is transcribed in the orientation opposite to that of GAS11, and is expressed at low levels. These genes were examined for mutations in breast tumor DNA, and both were excluded as tumor suppressor genes involved in breast cancer.
[Show abstract][Hide abstract] ABSTRACT: A number of localizations for the putative susceptibility gene(s) have been identified for both Crohn's disease and ulcerative colitis. In a genome wide scan, Hugot et al. (1996) identified a region on chromosome 16 which appeared to be responsible for the inheritance of inflammatory bowel disease in a small proportion of families. Subsequent work has suggested that this localization is important for susceptibility to Crohn's disease rather than ulcerative colitis (Ohmen et al. 1996; Parkes et al. 1996). We investigated the contribution of this localization to the inheritance of inflammatory bowel disease in 54 multiplex Australian families, and confirmed its importance in a significant proportion of Crohn's disease families; we further refined the localization to a region near to D16S409, obtaining a maximum LOD score of 6.3 between D16S409 and D16S753.
Annals of Human Genetics 08/1998; 62(Pt 4):291-8. · 2.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A breast cancer tumor suppressor gene has been localized to chromosome 16q24.3 by loss of heterozygosity (LOH) studies of breast tumor DNA. To identify candidate genes for this suppressor function, we have constructed a detailed physical map extending approximately 940 kb from the telomere of the long arm of chromosome 16 that encompasses the minimum LOH interval. This contig consists of a minimum overlapping set of 35 cosmids and a single PAC clone that were aligned by restriction enzyme site mapping. Cosmids were initially identified by screening filters with markers localized to the region by physical mapping using mouse/human somatic cell hybrids, and subsequently cosmid ends were used to complete the contig. A total of seven known genes, including PRSM1, PISSLRE, and the recently cloned Fanconi anemia A (FAA) gene, and potential transcripts from exon-trapping experiments have been located to this contig. A minimum of 14 new transcripts have been identified based on homology of trapped exons with database sequences. This contig and expressed sequence map will form the basis for the identification of the breast cancer tumor suppressor gene in this region.
[Show abstract][Hide abstract] ABSTRACT: This report presents the chromosomal localization of cadherin genes. Cadherins are cellular adhesion molecules. Since disturbance of intracellular adhesion is important for invasion and metastasis of tumor cells, cadherins are considered prime candidates for tumor suppressor genes. A variety of solid tumors show loss of heterozygosity of the long arm of chromosome 16, which is indicative of the potential location of tumor suppressor genes. Refined and new localizations of six cadherin genes (CDH3, 5, 8, 11, 13, and 15) to the long arm of chromosome 16 are shown. CDH15 was localized to 16q24.3, in a region that exhibits loss of heterozygosity in a number of sporadic breast cancer tumors. Previous localization of CDH13 (H-cadherin) to 16q24 suggested this gene as a tumor suppressor candidate in the 16q24.3 loss of heterozygosity region; however, refined mapping presented in this report localizes CDH13 proximal to this region. A human EST homologous to the chicken cadherin-7 was partially sequenced and found to represent a new human cadherin. This cadherin mapped to chromosome 18q22-q23, a region that exhibits loss of heterozygosity in head and neck squamous cell carcinomas. CDH16 was localized to 8q22.1, a region exhibiting loss of heterozygosity in adult acute myeloid leukemia.
[Show abstract][Hide abstract] ABSTRACT: To assess the relative contributions of trans-acting factors (replication and repair functions) and cis-acting elements (repeat and flanking DNA composition) to the mechanism of trinucleotide repeat sequence mutation we have analysed the distribution of copy number polymorphisms at 12 loci associated with dynamic mutations in 15 populations of different ethnic origins. Genome wide instability of repeats in a particular population would be evidence of trans-acting factor instigation of the mutation process, whereas instability at a particular locus (perhaps even in several populations) would be evidence that the composition of the particular locus was the most significant factor contributing to mutation. The FRA16A locus is highly polymorphic in only the European population. Some other loci exhibit distinct distributions of alleles between different populations. Therefore sequences in the vicinity of the repeat -- the cis component of a particular locus -- appear(s) to be more important in the mutation mechanism than sporadic genome-wide instability induced by trans-acting factors such as the DNA mismatch repair enzymes.
Annals of Human Genetics 10/1996; 60(Pt 5):391-400. · 2.22 Impact Factor