Structure and Methylation-Based Silencing of a Gene (DBCCR1) within a Candidate Bladder Cancer Tumor Suppressor Region at 9q32–q33

Loss of heterozygosity (LOH) on chromosome 9q is the most frequent genetic alteration in transitional cell carcinoma (TCC) of the bladder, indicating the presence of one or more relevant tumor suppressor genes. We previously mapped one of these putative tumor suppressor loci to 9q32–q33 and localized the candidate region within a single YAC 840 kb in size. This locus has been designatedDBC1(for deleted in bladder cancer gene 1). We have identified a novel gene,DBCCR1,in this candidate region by searching for expressed sequence tags (ESTs) that map to YACs spanning the region. Database searching using the entireDBCCR1cDNA sequence identified several human ESTs and a few homologous mouse ESTs. However, the predicted 761-amino-acid sequence had no significant homology to known protein sequences. Mutation analysis of the coding region and Southern blot analysis detected neither somatic mutations nor gross genetic alterations in primary TCCs. AlthoughDBCCR1was expressed in multiple normal human tissues including urothelium, mRNA expression was absent in 5 of 10 (50%) bladder cancer cell lines. Methylation analysis of the CpG island at the 5′ region of the gene and the induction ofde novoexpression by a demethylating agent indicated that this island might be a frequent target for hypermethylation and that hypermethylation-based silencing of the gene occurs in TCC. These findings makeDBCCR1a good candidate forDBC1.     

Assignment of 48 ESTs to Chromosome 13 Band q14.3 and Expression Pattern for ESTs Located in the Core Region Deleted in B-CLL

An expression map containing 48 ESTs was constructed to identify a tumor-suppressor gene involved in B-cell chronic lymphocytic leukemia (B-CLL), which was previously assigned to chromosome band 13q14.3 close to genetic markers D13S25 and D13S319. Thirty-nine of these 48 ESTs, together with 11 additional ones listed in databases, were initially assigned to chromosome 13q14 between markers D13S168 and D13S176. Nine others have recently been located in the D13S319 region. Our results indicate that 48 of the 59 ESTs analyzed belong to a YAC contig of chromosome 13 band q14, and 22 are contained on YAC 933e9, which encompasses the B-CLL critical region. Ten of these 22 ESTs were accurately assigned on a PAC, BAC, and cosmid contig encompassing the smallest minimal deletion area described so far in B-CLL, and 20 were tested for their expression on 27 normal or tumor tissues. One EST appears to be a likely candidate for the tumor-suppressor gene involved in B-CLL.     

Juxta-centromeric region of human chromosome 21 is enriched for pseudogenes and gene fragments

A physical map including four pseudogenes and 10 gene fragments and spanning 500 kb in the juxta-centromeric region of the long arm of human chromosome 21 is presented. cDNA fragments isolated from a selected cDNA library were characterized and mapped to the 831B6 YAC and to two BAC contigs that cover 250 kb of the region. An 85 kb genomic sequence located in the proximal region of the map was analyzed for putative exons. Four pseudogenes were found, including psiIGSF3, psiEIF3, psiGCT-rel whose functional copies map to chromosome 1p13, chromosome 2 and chromosome 22q11, respectively. The TTLL1 pseudogene corresponds to a new gene whose functional copy maps to chromosome 22q13. Ten gene fragments represent novel sequences that have related sequences on different human chromosomes and show 97-100% nucleotide identity to chromosome 21. These may correspond to pseudogenes on chromosome 21 and to functional genes in other chromosomes. The 85 kb genomic sequence was analyzed also for GC content, CpG islands, and repetitive sequence distribution. A GC-poor L isochore spanning 40 kb from satellite 1 was observed in the most centromeric region, next to a GC-rich H isochore that is a candidate region for the presence of functional genes. The pericentric duplication of a 7.8 kb region that is derived from the 22q13 chromosome band is described. We showed that the juxta-centromeric region of human chromosome 21 is enriched for retrotransposed pseudogenes and gene fragments transferred by interchromosome duplications, but we do not rule out the possibility that the region harbors functional genes also.     

High-resolution oligonucleotide array-CGH pinpoints genes involved in cryptic losses in chronic lymphocytic leukemia

Although recurrent chromosomal alterations occur in chronic lymphocytic leukemia (CLL), relatively few affected tumor suppressors and oncogenes have been implicated. To improve genetic characterization of CLL, we performed high-resolution gene copy number analysis of 20 CLL patients using oligonucleotide array comparative genomic hybridization (aCGH). The most recurrent losses were observed in 13q and 11q with variable sizes. The 11q losses varied between 7.44 Mb and 41.72 Mb in size and targeted ATM among others. Lost regions in 13q were generally smaller, spanning from 0.79 Mb to 29.33 Mb. The minimal common region (158 kb) in 13q14.3, which was also homozygously deleted in some cases, harbored five genes: TRIM13, KCNRG, DLEU2, DLEU1, and FAM10A4. Additionally, two micro-RNA genes (MIRN15A and MIRN16-1) locate to the region. New cryptic losses were detected in 1q23.2-->q23.3, 3p21.31, 16pter-->p13.3, 17p13.3-->p13.2, 17q25.3-->qter, and 22q11.22. In conclusion, our oligonucleotide aCGH study revealed novel aberrations and provided detailed genomic profiles of the altered regions.     

Four novel mutant alleles of the arylsulfatase B gene in two patients with intermediate form of mucopolysaccharidosis VI (Maroteaux-Lamy syndrome)

We have used pulsed-field gel electrophoresis to construct a long range restriction map of the myxoid liposarcoma (MLS) breakpoint region in 12q13-14. The CHOP/GADD153 gene, consistently translocated in myxoid liposarcomas, is located less than 55 kb from the putative oncogene GLI. We have used fluorescent in situ hybridization to orient the map with respect to the chromosome, and to show that GLI (and thus A2MR) is located proximal to the MLS breakpoint.     

Molecular analysis of the human chromosome 5q13.3 region in patients with hairy cell leukemia and identification of tumor suppressor gene candidates.

The pathogenesis of hairy cell leukemia (HCL) remains largely unknown since no specific genetic lesion has been identified in this disease. Previous cytogenetic analysis from our group has shown that chromosome abnormalities involving the 5q13 band are common in HCL, occurring in approximately 1/3 of patients. The data suggest that the 5q13.3 band is likely to harbor a gene involved in the transformational events of this disease. We have recently found two cosmids flanking the 5q13.3 breakpoint in patients with HCL, and the distance between them is approximately 35 kb, as analyzed by fiber-FISH. The two cosmids have been located between the markers SGC34998 and WI-15505/WI-6897 by radiation hybrid mapping. Five of 11 patients with HCL had a hemizygous deletion of the two cosmids, indicating that the function of a tumor suppressor gene may be lost. With the aim of delineating the critical region of 5q13.3 loss in patients with HCL, we have constructed an integrated contig of YAC, BAC, PAC, P1, and cosmid clones that covers the region. Within this area, three expressed sequences were identified as candidates for the putative 5q13.3 tumor suppressor gene involved in the pathogenesis of HCL.     

A sequence-ready BAC clone contig of human chromosome 10p15 spanning the loss of heterozygosity region in glioma

Deletion of chromosome 10 is one of the most common chromosomal alterations in glioma. At 10p15, the telomeric region of the short arm of chromosome 10, loss of heterozygosity (LOH) has been frequently observed by microsatellite analysis, suggesting the presence of a tumor suppressor gene. We examined LOH in 34 gliomas on chromosome 10, and frequent LOH on 10p was detected on 10p15, in agreement with deletion mapping studies on chromosome 10. We then constructed a bacterial artificial chromosome (BAC) clone contig covering the critical region, which spanned the interval between D10S249 and D10S533 on 10p15. The map contained 68 BAC clones connected by 74 sequenced tag sites (STSs) and covered approximately 2.7 Mb, with one gap. A total of 74 STSs, including 6 microsatellite markers, 29 expressed sequenced tags (ESTs), and 39 BAC end STSs, were physically arranged. Twenty-eight ESTs were mapped in the interval between D10S249 and D10S559 (approximately 1200 kb), and another EST was mapped in the interval between D10S559 and D10S533 (approximately 1300 kb). This sequence-ready BAC clone contig map will be a basic resource for high-quality sequencing and positional cloning of the putative tumor suppressor gene at 10p15 in glioma.     

A 1.5-Mb physical map of the hidrotic ectodermal dysplasia (Clouston syndrome) gene region on human chromosome 13q11

The HED (hidrotic ectodermal dysplasia) or Clouston syndrome gene (named ED2) has been mapped to the pericentromeric region of chromosome 13 (13q11) to a 2.4-cM interval flanked by markers D13S1828 and D13S1830. We have developed a BAC/PAC-based contig map of this region. This contig, comprising 23 clones and spanning 1.5 Mb, was established by mapping of 27 BAC/PAC end-derived STSs, 11 known polymorphic markers, 2 previously mapped genes, and 14 ESTs. The genomic clone overlaps were confirmed by restriction fragment fingerprint analysis. This contig provides the basis for genomic sequencing and gene identification in the ED2 critical region. Of the 14 ESTs mapped to the contig, 6 show homology to human genes and 8 appear to be novel. Expression patterns of the genes/ESTs were tested by Northern blot and RT-PCR. Full characterization of some of these genes, as well as the novel ESTs, will be useful in assessing their involvement in the HED/Clouston syndrome.     

A 2-Mb sequence-ready contig map and a novel immunoglobulin superfamily gene IGSF4 in the LOH region of chromosome 11q23.2

Human chromosome 11q23.2 has been proposed to contain a tumor suppressor gene(s) whose deletion has been associated with cancer of the lung and breast and with neuroblastoma. To analyze the genomic structure and to isolate a candidate tumor suppressor gene from this region, we constructed a 2-Mb sequence-ready contig map using bacteriophage P1 (P1), bacterial artificial chromosome (BAC), and P1-derived artificial chromosome (PAC). The map comprises a contig of 24 overlapping P1, BAC, and PAC clones. To isolate gene fragments from the region, we performed direct cDNA library screening, exon trapping, EST mapping, and genomic sequencing using the P1, BAC, and PAC clones. Sequence analysis of 5 clones, which spans 23% (458,738 bp) of the region, and extensive gene scanning along the entire region revealed that the region is extraordinarily scarce in genes, but we identified one ubiquitously expressed novel gene and one testis-specific gene fragment. The novel gene, which we call IGSF4 (immunoglobulin superfamily 4), is transcribed into a 1.6- or 4.4-kb RNA encoding a 442-amino-acid protein. It shares strong homology with mouse IGSF-B12 and cell adhesion molecules NCAM1 and NCAM2 within their Ig-like C2-type domains. The IGSF4 gene, a novel gene that is shown to be located in the common loss of heterozygosity region, possesses a number of interesting features and may be good candidate for a tumor suppressor gene.     

A common deletion at chromosomal region 17q21 in sporadic prostate tumors distal to BRCA1

Prostate cancer is the most common cancer in males in the United States, yet the etiology of this disease is still poorly understood. In previous work from our laboratory, one or more deleted regions were found in prostate tumors distal to the breast and ovarian cancer susceptibility gene (BRCA1) on chromosome 17. This suggested that genes at 17q21 may play a pivotal role in prostate cancer progression, and there may be new tumor suppressor genes at this locus. We now present a physical map built with P1, P1 artificial chromosome, and bacterial artificial chromosome clones encompassing a DNA sequence anchored by multiple STS markers. The analysis of prostate tumors indicated an 85-kb novel commonly deleted interval flanked by D17S1184-D17S183-D17S1203-D17S1860, which is at least 470 kb distal to the BRCA1 gene. Fifty-four of 126 prostrate cancer cases (43%) showed a deletion by a direct FISH technique using P1 probes in this region. Searching with clone end sequences in the sequence database BLAST, the deleted clone covered genomic DNA sequence that contained upstream binding factor (UBF), EPB3 genes, SHCL1, ASB-4-like sequence, and acidic protein-like sequence. PCR for the ESTs confirmed that these genes or ESTs are within the deletion region. Our results will be helpful for finding candidate tumor suppressor genes in prostate cancer.     

多发性骨髓瘤患者13q14.3区域一个候选抑瘤基因MYETS1的克隆与序列分析

为克隆位于多发性骨髓瘤(multiple myeloma,MM)患者染色体13q14.2～13q21.1区域候选抑瘤基因,通过生物信息学分析获取疾病基因定位区域内代表新基因的ESTs,并运用半定量RT-PCR检测它们在正常人与MM患者骨髓组织中的表达水平,发现一条在MM患者骨髓组织中明显表达下调的EST(GenBank收录号:H86826).Northern印迹杂交显示H86826在骨髓组织中转录本大小为1.5kb.通过购买商品化克隆IM-AGE223589测序获得了H86826所代表的基因的1491 bp全长cDNA序列(GenBank收录号:AY368652),人类基因组命名委员会将其命名为MYETS1(myeloma tumor suppressor 1).生物信息学分析其为一个编码分子质量为15.1 kD、等电点为6.13的135个氨基酸的新基因.该基因的功能正在进一步的研究之中.     

Allelotype of human thyroid tumors: loss of chromosome 11q13 sequences in follicular neoplasms.

Two classes of genes are the targets of mutations involved in human tumorigenesis: oncogenes, the activation of which leads to growth stimulation, and tumor suppressor genes, which become tumorigenic through loss of function, often through allelic deletion. To obtain evidence for a role for tumor suppressor genes in thyroid tumorigenesis, we examined DNA from 80 thyroid neoplasms for loss of heterozygosity in multiple chromosomal loci using 19 polymorphic genomic probes. None of the informative thyroid tumors studied had allelic loss detected with probes for chromosome 2q (D2S44), 3p (D3F15S2, D3S32), 3q (D3S46), 4p (D4S125), 6p (D6S40), 8q (D8S39), 9q (D9S7), 12p (D12S14), 13q (D13S52), 17p (D17S30), or 18q (D18S10). One of eight of the follicular adenomas had a 10q deletion detected with marker D10S15, and one of 26 had a 10q deletion detected with D10S25. One of two of the follicular carcinomas had an 11p deletion in the H-ras locus. The most significant findings were on chromosome 11q13, the site containing the putative gene predisposing to multiple endocrine neoplasia type I. Four of 27 follicular adenomas had loss of heterozygosity for probes in this region. Allelic deletions were detected with the following probes: D11S149, PYGM, D11S146, and INT2. None of 13 informative papillary carcinomas and none of two follicular carcinomas had loss of heterozygosity detectable with these 11q13 markers. Allelic loss is a relatively infrequent event in human thyroid tumors. Deletions of chromosome 11q13 are present in about 14% of follicular, but not papillary, neoplasms.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-resolution mapping of the gamma-aminobutyric acid receptor subunit beta 3 and alpha 5 gene cluster on chromosome 15q11-q13, and localization of breakpoints in two Angelman syndrome patients.

The gamma-aminobutyric acid (GABAA) receptors are a family of ligand-gated chloride channels constituting the major inhibitory neurotransmitter receptors in the nervous system. In order to determine the genomic organization of the GABAA receptor beta 3 subunit gene (GABRB3) and alpha 5 subunit gene (GABRA5) in chromosome 15q11-q13, we have constructed a high-resolution physical map using the combined techniques of field-inversion gel electrophoresis and phage genomic library screening. This map, which covers nearly 1.0 Mb, shows that GABRB3 and GABRA5 are separated by less than 100 kb and are arranged in a head-to-head configuration. GABRB3 encompasses approximately 250 kb, while GABRA5 is contained within 70 kb. This difference in size is due in large part to an intron of 150 kb within GABRB3. We have also identified seven putative CpG islands within a 600-kb interval. Chromosomal rearrangement breakpoints--in one Angelman syndrome (AS) patient with an unbalanced translocation and in another patient with a submicroscopic deletion--are located within the large GABRB3 intron. These findings will facilitate chromosomal walking strategies for cloning the regions disrupted by the DNA rearrangements in these AS patients and will be valuable for mapping new genes to the AS chromosomal region.