Human chromosome 3P regions of putative tumor-suppressor genes in renal, breast, and ovarian carcinomas

Allelic imbalances (AI) of polymorphic markers at the short arm of chromosome 3 (3p) were mapped using DNA samples of renal cell carcinoma (RCC, 80 cases), breast carcinoma (BC, 95 cases), and epithelial ovarian cancer (EOC, 50 cases) at the same dense panel of markers (up to 24 loci). Six regions with the increased AI frequency (versus the average values determined for all the analyzed 3p markers) at RCC, BC or EOC were found in 3p chromosome. Four 3p regions presumably contain suppressor genes of tumor growth (TSG) observed in the epithelial tumors of various types. Region between D3S2409 and D3S3667 markers in the 3q21.31 region was identified in this study for the first time. The AI peak in D3S2409-D3S3667 region was statistically significant (P < 0.001, according to Fisher) when representative sample of 95 BC patients was analyzed. The data on increased frequency of polymorphic marker allele amplification suggest that the D3S2409-D3S3667 region contains both putative TSG and protooncogenes.     

From identification of genomic polymorphism to diagnostic and prognostic markers of human epithelial tumors

The review considers the results obtained by several groups in the fields of identification of polymorphic loci in the human genome, localization and analysis of genes associated with epithelial tumors of various origins, and generation of molecular markers of socially important oncological diseases. In the first two cases, work was initiated and supported by the Russian program Human Genome. To find new polymorphic loci in the human genome, di-, tri-, and tetranucleotide repeats were searched for in an ordered cosmid library of chromosome 13, NotI and cosmid clones of chromosome 3, and in brain EST. In total, nine polymorphisms and almost 200 STS were identified. Markers of NotI clones of chromosome 3 were associated with particular genes. Polymorphic loci NL1-024, NL2-007, and EST04896 were employed in analysis of deletions from chromosome 3p in tumor DNA. Deletion mapping of 3p in epithelial tumors of five types revealed six critical regions containing potential tumor suppressor genes. Of these, two were in the distal region of chromosome 3p and four, in region 3p21.3. A significant correlation was observed for the frequency of allelic deletions and the stage and the grade of tumors (P < 0.05). On the strength of these findings, genes of region 3p were associated with both tumor development and progression, and proposed as prognostic markers. Regions LUCA and AP20 (3p21.3) showed a high (90%) frequency of aberrations, including homozygous deletions in almost 20% cases. The peak of allelic deletions from region D3S2409-D3S3667 (600 kb) was statistically valid (P = 10(-3)). Regions AP20 and D3S2409-D3S3667 (3p21.3) were for the first time associated with tumorigenesis. Clusters of tumor suppressor genes were identified in regions LUCA, AP20, and D3S2409-D3S3667. Methylation of RASSF1A and RARbeta2 (3p) was associated with early carcinogenesis, and that of SEMA3B, with tumor progression. These findings are useful for early diagnostics and post-surgery prognosis of tumors.     

From Identification of Genomic Polymorphisms to Diagnostic and Prognostic Markers of Human Epithelial Tumors

The review considers the results obtained by several groups in the fields of identification of polymorphic loci in the human genome, localization and analysis of genes associated with epithelial tumors of various origins, and generation of molecular markers of socially important oncological diseases. In the first two cases, work was initiated and supported by the Russian program Human Genome. To find new polymorphic loci in the human genome, di-, tri-, and tetranucleotide repeats were searched for in an ordered cosmid library of chromosome 13, NotI and cosmid clones of chromosome 3, and in brain EST. In total, nine polymorphisms and almost 200 STS were identified. Markers of NotI clones of chromosome 3 were associated with particular genes. Polymorphic loci NL1-024, NL2-007, and EST04896 were employed in analysis of deletions from chromosome 3p in tumor DNA. Deletion mapping of 3p in epithelial tumors of five types revealed six critical regions containing potential tumor suppressor genes. Of these, two were in the distal region of chromosome 3p and four, in region 3p21.3. A significant correlation was observed for the frequency of allelic deletions and the stage and the grade of tumors (P < 0.05). On the strength of these findings, genes of region 3p were associated with both tumor development and progression, and proposed as prognostic markers. Regions LUCA and AP20 (3p21.3) showed a high (90%) frequency of aberrations, including homozygous deletions in almost 20% cases. The peak of allelic deletions from region D3S2409–D3S3667 (600 kb) was statistically valid (P = 10^–3). Regions AP20 and D3S2409–D3S3667 (3p21.3) were for the first time associated with tumorigenesis. Clusters of tumor suppressor genes were identified in regions LUCA, AP20, and D3S2409–D3S3667. Methylation of RASSF1A and RAR-beta2 (3p) was associated with early carcinogenesis, and that of SEMA3B, with tumor progression. These findings are useful for early diagnostics and post-surgery prognosis of tumors.     

Allele Deletion Mapping of the Short Arm of Human Chromosome 3 in Renal Carcinoma

Allelic deletions along the short arm of human chromosome 3 were mapped in 57 pairs of DNA samples from tumor and normal tissue of renal carcinoma patients in order to locate potential tumor suppressor genes. Twenty highly polymorphic microsatellite markers were used for deletion mapping. Allelic deletions were found in most of the samples (91%). Extended terminal deletions (56%) prevailed over shorter internal and multiple deletions and dominated (65%) in the most aggressive histopathological kidney cancer subtype, clear-cell carcinoma. Frequency analysis of loss of heterozygosity allowed detection of the human chromosome 3 regions most essential for renal carcinomas: the region adjacent to the gene VHL(3p26–p25), the region of homozygous deletions AP20 (3p22–p21.33), and a new region between markers D3S2420 and D3S2409 (3p21.31, 2.2 Mbp).     

Mapping allelic deletions on the short arm of human chromosome 3 in kidney neoplasms]

Allelic deletions along the short arm of human chromosome 3 were mapped in 57 pairs of DNA samples from tumor and normal tissue of renal carcinoma patients in order to locate potential tumor suppressor genes. Twenty highly polymorphic microsatellite markers were used for deletion mapping. Allelic deletions were found in most of the samples (91%). Extended terminal deletions (56%) prevailed over shorter internal and multiple deletions and dominated (65%) in the most aggressive histopathological kidney cancer subtype, clear-cell carcinoma. Frequency analysis of loss of heterozygosity allowed detection of the human chromosome 3 regions most essential for renal carcinomas: the region adjacent to the gene VHL (3p26-p25), the region of homozygous deletions AP20 (3p22-p21.33), and a new region between markers D3S2420 and D3S2409 (3p21.31, 2.2 Mbp).     

Methylation of the <Emphasis Type="Italic">RASSF1A</Emphasis> promoter region and the allelic imbalance frequencies in chromosome 3 critical regions correlate with progression of clear cell renal carcinoma

The short arm of chromosome 3 (3p) contains several critical regions that have increased frequencies of allelic deletions and harbor a set of tumor suppressor genes. In particular, the range of functions performed by RASSF1A (LUCA region, 3p21.31) includes those potentially associated with carcinogenesis. Among 3p genes, RASSF1A has the highest methylation frequency in epithelial tumors of various locations. For the first time, two different methods (methylation-specific PCR and methylation-sensitive restriction analysis) independently showed that the methylation level of the CpG island in the RASSF1A promoter region significantly correlated with grade and clinical stage of clear cell renal cell carcinoma (RCC). An analysis of 23 3p polymorphic markers in a representative set of 80 RCC cases characterized clinically and histologically revealed that RCC progression significantly correlated with the frequency of allelic imbalances in some critical regions of 3p (LUCA and AP20), but not in 3p as a whole. These data suggest that RCC progression is associated with the methylation of the RASSF1A promoter and, possibly, with structural and functional alterations in other 3p genes. In addition, significant correlation between RASSF1A methylation and allelic losses at the nearby polymorphic marker locus suggests the “two hit” model for the inactivation of this tumor suppressor gene in RCC.     

Genetic and physical map of the interferon region on chromosome 9p.

A region of chromosome 9, surrounding the interferon-beta (IFNB1) locus and the interferon-alpha (IFNA) gene cluster on 9p13-p22, has been shown to be frequently deleted or rearranged in a number of human cancers, including leukemia, glioma, non-small-cell lung carcinoma, and melanoma. To assist in better defining the precise region(s) of 9p implicated in each of these malignancies, a combined genetic and physical map of this region was generated using the available 9p markers IFNB1, IFNA, D9S3, and D9S19, along with a newly described locus, D9S126. The relative order and distances between these loci were determined by multipoint linkage analysis of CEPH (Centre d'Etude du Polymorphisme Humain) pedigree DNAs, pulsed-field gel electrophoresis, and fluorescence in situ hybridization. All three mapping approaches gave concordant results and, in the case of multipoint linkage analysis, the following gene order was supported for these and other closely linked chromosome 9 markers present in the CEPH database: pter-D9S33-IFNB1/IFNA-D9S126-D9S3-D9S19 -D9S9/D9S15-ASSP3-qter. This map serves to extend preexisting chromosome 9 maps (which focus primarily on 9q) and also reassigns D9S3 and D9S19 to more proximal locations on 9p.     

Physical mapping of chromosome 3p25-p26 by flourescence in situ hybridisation (FISH)

As part of our effort to isolate and characterise the von Hippel-Lindau (VHL) disease gene, we constructed a physical map of chromosome 3p25-26 by fluorescence in situ hybridisation (FISH) studies on a panel of cytogenetic rearrangements involving this region. Biotinylated cosmid and lambda probes were hybridised to metaphase chromosome spreads and positioned with respect to each cytogenetic breakpoint. These studies unequivocally established the order of five loci linked to the VHL disease gene: cen-(RAF1,312)-D3S732-D3S1250-D3S601-D3S18-pter and determined the position of three other probes within this map. These results ordered RAF1 and D3S732 for the first time, confirmed the localisation of D3S1250 between RAF1 and D3S601 and determined the position of D3S651 with respect to other chromosome 3p25-p26 loci. The establishment of an ordered set of cytogenetic aberrations will enable the rapid assignment of polymorphic and nonpolymorphic cloned sequences within the chromosome region 3p25-p26.     

Five new microsatellite polymorphisms at the q21 region of human chromosome 21

Five clones, containing polymorphic CA-repeat sequences, have been isolated from a specific human chromosome 21 phage library and have been localised to band q21 of chromosome 21 using a somatic cell hybrid panel. These highly repetitive sequences (D21S1263, D21S1264, D21S1415, D21S1417 and D21S1420) have been characterised in the CEPH reference parents and have heterozygosities ranging from 0.30 to 0.81 and an average polymorphism information content (PIC) of 0.62. The relative order of these markers, based on the somatic cell hybrid panel, is cen-D21S1417, D21S1420-D21S1263, D21S1415-D21S1264-tel. The most polymorphic marker (D21S1264) has been included in the chromosome 21 genetic map. They have also been localised in the CEPH/ Généthon YAC panel, providing a refined localisation of these polymorphic sequences. These five CA-repeat markers should provide a better characterisation of the q21 region of chromosome 21.     

Loss of heterozygosity on 17p in human breast carcinomas: defining the smallest common region of deletion

The marker D17S5, mapping to the short arm of chromosome 17, was recently reported by us and others to undergo frequent heterozygous deletion in human primary breast carcinomas, implicating the presence of a tumor suppressor gene in this region. To narrow down the location of this gene more precisely, we have performed a deletion-mapping study in an extended series of 78 breast carcinomas, using nine polymorphic markers for the short arm and two polymorphic markers for the long arm of chromosome 17. Partial allele losses on 17p were observed in nine cases, which, taken together, suggest that the target gene for the deletions maps to the region extending between the markers D17S5 (17p13.3) and D17S67 (17p12).     

Physical Mapping of a Commonly Deleted Region, the Site of a Candidate Tumor Suppressor Gene, at 12q22 in Human Male Germ Cell Tumors

A candidate tumor suppressor gene (TSG) site at 12q22 characterized by a high frequency of loss of heterozygosity (LOH) and a homozygous deletion has previously been reported in human male germ cell tumors (GCTs). In a detailed deletion mapping analysis of 67 normal-tumor DNAs utilizing 20 polymorphic markers mapped to 12q22–q24, we identified the limits of the minimal region of deletion at 12q22 between D12S377 (proximal) and D12S296 (distal). We have constructed a YAC contig map of a 3-cM region of this band between the proximal marker D12S101 and the distal marker D12S346, which contained the minimal region of deletion in GCTs. The map is composed of 53 overlapping YACs and 3 cosmids onto which 25 polymorphic and nonpolymorphic sequence-tagged sites (STSs) were placed in a unique order. The size of the minimal region of deletion was approximately 2 Mb from overlapping, nonchimeric YACs that spanned the region. We also developed a radiation hybrid (RH) map of the region between D12S101 and D12S346 containing 17 loci. The consensus order developed by RH mapping is in good agreement with the YAC STS-content map order. The RH map estimated the distance between D12S101 and D12S346 to be 246 cR₈₀₀₀and the minimal region of deletion to be 141 cR₈₀₀₀. In addition, four genes that were previously mapped to 12q22 have been excluded as candidate genes. The leads gained from the deletion mapping and physical maps should expedite the isolation and characterization of the TSG at 12q22.     

Increased recombination adjacent to the Huntington disease-linked D4S10 marker.

Huntington disease (HD) is caused by a genetic defect distal to the anonymous DNA marker D4S10 in the terminal cytogenetic subband of the short arm of chromosome 4 (4p16.3). The effort to identify new markers linked to HD has concentrated on the use of somatic cell hybrid panels that split 4p16.3 into proximal and distal portions. Here we report two new polymorphic markers in the proximal portion of 4p16.3, distal to D4S10. Both loci, D4S126 and D4S127, are defined by cosmids isolated from a library enriched for sequences in the 4pter-4p15.1 region. Physical mapping by pulsed-field gel electrophoresis places D4S126 200 kb telomeric to D4S10, while D4S127 is located near the more distal marker D4S95. Typing of a reference pedigree for D4S126 and D4S127 and for the recently described VNTR marker D4S125 has firmly placed these loci on the existing linkage map of 4p16.3. This genetic analysis has revealed that the region immediately distal to D4S10 shows a dramatically higher rate of recombination than would be expected based on its physical size. D4S10-D4S126-D4S125 span 3.5 cM, but only 300-400 kb of DNA. Consequently, this small region accounts for most of the reported genetic distance between D4S10 and HD. By contrast, it was not possible to connect D4S127 to D4S125 by physical mapping, although they are only 0.3 cM apart. A more detailed analysis of recombination sites within the immediate vicinity of D4S10 could potentially reveal the molecular basis for this phenomenon; however, it is clear that the rate of recombination is not continuously increased with progress toward the telomere of 4p.     

Integrated High-Resolution BAC, P1, and Transcript Map of the CHH Region in Chromosome 9p13

A bacterial artificial chromosome (BAC) and P1 contig of the proximal part of chromosome 9p centromeric of markers D9S165 and D9S304 is described. This 1.1- to 1.7-Mb portion of chromosome 9p13 was previously not physically mapped. It contains 24 genes or expressed sequence tags, five polymorphic AC repeats, and three new polymorphic single-strand conformation polymorphism variants. Several of the genes thus mapped are excellent candidates for disease-causing genes whose loci have previously been assigned to proximal 9p. Our primary interest is in the cartilage-hair hypoplasia gene (CHH) that resides within the contig between markers D9S163 and D9S1791 based on linkage evidence.     

Polymorphic microsatellites and Wilson disease (WD)

Wilson disease (WD), an autosomal recessive disorder of copper metabolism, has been previously mapped to chromosome 13q. Highly informative PCR-based polymorphic microsatellites closely linked to the WD locus (WND) at 13q14.3, as well as sequence-tagged sites for closely linked loci, are described. Two polymorphic microsatellite markers at D13S118 and D13S119 lie within 3 cM of WND. Two others (D13S227 and D13S228) were derived from a yeast artificial chromosome containing D13S31. These were placed on a genetic linkage map of chromosome 13 and were typed in 74 multiplex WD families from a variety of geographic origins (166 affected members). Multipoint analysis provides very high odds that the location of WND is between D13S31/D13S227/D13S228 and D13S59. Previous odds with RFLP-based markers were only 7:1 more likely than any other location. Current odds are 5,000:1. Preclinical testing of three cases of WD by using the highly informative polymorphic microsatellite markers is described. The markers described here ensure that 95% of predictive tests using DNA from both parents and from at least one affected sib will have an accuracy >99%.     

Linkage map of human chromosome 9 microsatellite polymorphisms.

Ten microsatellite markers composed of polymorphic (CA)n or (AAAT)n repeats were mapped to chromosome 9. PIC values for these markers ranged from 0.46 to 0.82. The marker at the D9S54 locus was localized to 9pter-p22 by means of a somatic cell hybrid; another marker at D9S103 was similarly localized to 9q34-qter. Two-point lod scores and individual meiotic recombination events were used to position the 10 markers relative to each other. The best order resulting from these analyses was D9S54-D9S104-[D9S52-D9S43-D9S50]-D9S53+ ++- [D9S106-D9S105]-D9S51-D9S103, with order of the loci within brackets uncertain. Two-point linkage analysis was also used to approximate the positions of the microsatellite markers relative to those of 33 markers contained in the public CEPH database (v.3) and to one other available microsatellite marker at the D9S15 locus.     

A Huntington disease-like neurodegenerative disorder maps to chromosome 20p.

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor disturbance, cognitive loss, and psychiatric manifestations. The disease is associated with a CAG trinucleotide-repeat expansion in the Huntington gene (IT15) on chromosome 4p16.3. One family with a history of HD was referred to us initially for predictive testing using linkage analysis. However, the chromosome 4p region was completely excluded by polymorphic markers, and later no CAG-repeat expansion in the HD gene was detected. To map the disease trait segregating in this family, whole-genome screening with highly polymorphic dinucleotide-, trinucleotide-, and tetranucleotide-repeat DNA markers was performed. A positive LOD score of 3.01 was obtained for the marker D20S482 on chromosome 20p, by two-point LOD-score analysis with the MLINK program. Haplotype analysis indicated that the gene responsible for the disease is likely located in a 2.7-cM region between the markers D20S193 and D20S895. Candidate genes from the mapping region were screened for mutations.     

Dominant hereditary inclusion-body myopathy gene (IBM3) maps to chromosome region 17p13.1

We recently described an autosomal dominant inclusion-body myopathy characterized by congenital joint contractures, external ophthalmoplegia, and predominantly proximal muscle weakness. A whole-genome scan, performed with 161 polymorphic markers and with DNA from 40 members of one family, indicated strong linkage for markers on chromosome 17p. After analyses with additional markers in the region and with DNA from eight additional family members, a maximum LOD score (Zmax) was detected for marker D17S1303 (Zmax=7.38; recombination fraction (theta)=0). Haplotype analyses showed that the locus (Genome Database locus name: IBM3) is flanked distally by marker D17S945 and proximally by marker D17S969. The positions of cytogenetically localized flanking markers suggest that the location of the IBM3 gene is in chromosome region 17p13.1. Radiation hybrid mapping showed that IBM3 is located in a 2-Mb chromosomal region and that the myosin heavy-chain (MHC) gene cluster, consisting of at least six genes, co-localizes to the same region. This localization raises the possibility that one of the MHC genes clustered in this region may be involved in this disorder.     

Molecular genetic investigations of the mechanism of tumourigenesis in von Hippel-Lindau disease: analysis of allele loss in VHL tumours

Von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome characterised by the development of retinal and central nervous system haemangioblastomas, renal cell carcinoma (RCC), phaeochromocytoma and pancreatic tumours. The VHL disease gene maps to chromosome 3p25-p26. To investigate the mechanism of tumourigenesis in VHL disease, we analysed 24 paired blood/tumour DNA samples from 20 VHL patients for allele loss on chromosome 3p and in the region of tumour suppressor genes on chromosomes 5, 11, 13, 17 and 22. Nine out of 24 tumours showed loss of heterozygosity (LOH) at at least one locus on chromosome 3p and in each case the LOH included the region to which the VHL gene has been mapped. Chromosome 3p allele loss was found in four tumour types (RCC, haemangioblastoma, phaeochromocytoma and pancreatic tumour) suggesting a common mechanism of tumourigenesis in all types of tumour in VHL disease. The smallest region of overlap was between D3S1038 and D3S18, a region that corresponds to the target region for the VHL gene from genetic linkage studies. The parental origin of the chromosome 3p25-p26 allele loss could be determined in seven tumours from seven familial cases; in each tumour, the allele lost had been inherited from the unaffected parent. Our results suggest that the VHL disease gene functions as a recessive tumour suppressor gene and that inactivation of both alleles of the VHL gene is the critical event in the pathogenesis of VHL neoplasms. Four VHL tumours showed LOH on other chromosomes (5q21, 13q, 17q) indicating that homozygous VHL gene mutations may be required but may not be sufficient for tumourigenesis in VHL disease.     

Ordering of human chromosome 3p markers by radiation hybrid mapping.

To construct a panel of radiation hybrids (RHs) for human chromosome 3p mapping, mouse microcell hybrid cells, A9(neo3/t)-5, containing a single copy of human chromosome 3p with pSV2neo plasmid DNA integrated at 3p21-p22 were irradiated and fused to mouse A9 cells. A panel of 96 RHs that retain several sizes and portions of human chromosome 3p segments was used to map 25 DNA markers for chromosome 3p. Eight of them, H28, H29, H32, H33, H35, H38, H48, and H64, were cloned from Alu-primed PCR products using A9(neo3/t)-5 cell DNA as a template. The most likely order of the 24 markers, except for H28, based on the statistical ordering method proposed by Falk, was cen-D3S4-D3S3-D3S30-H29-D3S13-D3S2-+ ++H48-D3F15S2-D3S32-D3S23-CCK-H35-H33- D3S11-D3S12-RARB-THRB(ERBA2-pBH302)- H64-H38-RAF1-D3S18-H32-D3S22-pter. The order and location of these markers were in good agreement with those previously determined by other mapping methods, suggesting that a panel of these 96 RHs is a valuable source for a rapid mapping of human chromosome 3p markers.     

Replication of linkage of familial hypobetalipoproteinemia to chromosome 3p in six kindreds

Familial hypobetalipoproteinemia (FHBL) is a genetically heterogeneous condition characterized by very low apolipoprotein B (apoB) concentrations in plasma and/or low levels of LDL-cholesterol (LDL-C) with a propensity to developing fatty liver. In a minority of cases, truncation-specifying mutations of the apoB gene (APOB) are etiologic, but the genetic basis of most cases is unknown. We previously reported linkage of FHBL to a 10 cM region on 3p21.1-22 in one kindred. The objectives of the current study were to identify other FHBL families with linkage to 3p and to narrow the FHBL susceptibility region on 3p. Six additional FHBL kindreds unlinked to the APOB region on chromosome 2 were genotyped with polymorphic markers spanning a region of approximately 13 cM on chromosome 3. Quantitative linkage analyses indicated that the FHBL in these families was linked to 3p21.1-22. Haplotype analysis identified several meiotic crossover events, allowing us to narrow the critical region from 10 cM to 2.0 cM, between markers D3S2407 and D3S1767.