A comparison of genomic structures and expression patterns of two closely related flanking genes in a critical lung cancer region at 3p21.3.

In the search for a tumour suppressor gene in the 3p21.3 region we isolated two genes, RBM5 and RBM6. Gene RBM5 maps to the region which is homozygously deleted in the small cell lung cancer cell line GLC20; RBM6 crosses the telomeric breakpoint of this deletion. Sequence comparison revealed that at the amino acid level both genes show 30% identity. They contain two zinc finger motifs, a bipartite nuclear signal and two RNA binding motifs, suggesting that the proteins for which RBM5 and RBM6 are coding have a DNA/RNA binding function and are located in the nucleus. Northern and Southern analysis did not reveal any abnormalities. By SSCP analysis of 16 lung cancer cell lines we found only in RBM5 a single presumably neutral mutation. By RT-PCR we demonstrated the existence of two alternative splice variants of RBM6, one including and one excluding exon 5, in both normal lung tissue and lung cancer cell lines. Exclusion of exon 5 results in a frameshift which would cause a truncated protein of 520 amino acids instead of 1123 amino acids. In normal lung tissue, the relative amount of the shorter transcript was much greater than that in the lung tumour cell lines, which raises the question whether some tumour suppressor function may be attributed to the derived shorter protein.

Analysis of multiple renal cell adenomas and carcinomas suggests allelic loss at 3p21 to be a prerequisite for malignant development.

Multiple renal cell tumours from three unrelated patients have been analysed for loss of heterozygosity of 3p, mutation of VHL, and chromosome 7 and 17 imbalances. Loss of 3p alleles is characteristic for clear cell type tumours and the combination of +7, +17 for chromophilic cell type tumours. Thus, we could classify adenomas and carcinomas of the three patients according to the genomic patterns of the tumours. Adenomas appeared to be mostly of the chromophilic cell type. In some adenomas, however, allelic losses of chromosome 3 were detected, pointing to a clear cell phenotype. Irrespective of showing loss or retention of the 3p25 region, none of the adenomas had a VHL mutation. Therefore, inactivation of VHL does not seem to be an early event in the development of renal cell tumours. Results of an analysis of regions of loss and retention of alleles of 3p markers in multiple tumours of the individual patients suggest that losses at either 3p25 or 3p12-p14 are associated with adenomas. Additional loss at 3p21 is most likely required to lead to development of a more malignant clear cell carcinoma.

Ordering of polymorphic markers in the chromosome region 3p21.

Starting from five markers, with a well-defined order from distal to proximal 3p21, nine other markers could be inserted in this 3p21 map. Five were precisely mapped genetically. The other markers were ordered by FISH and/or deletion hybrid mapping. The overall 3p21 order from distal to proximal is as follows: D3S1298-D3S1260-(D3S966, D3S1448 (= D3S1449)-D3S1029-D3S32-D3S643-D3F15S2 -D3S2968-D3S1235-D3S1289-D3S1447-D3S1295.

Defining the position of the breakpoint of the constitutional t(3;6) occurring in a family with renal cell carcinoma.

In a family with a constitutional translocation t(3;6), the oldest member carrying the translocation had developed multiple nonpapillary renal cell carcinomas (RCCs). The translocation breakpoint was positioned between 3p13 and 3p14.1. This is close to the region in which a t(3;8) breakpoint has been reported in a family with hereditary RCC. We defined the location of the t(3;6) and t(3;8) breakpoints by fluorescence in situ hybridization (FISH) analysis with yeast artificial chromosomes (YACs) from the 3p14-13 region. Both interphase nuclei and metaphase cells from translocation-carrying members of both families have been used, allowing the definition of flanking YACs for each breakpoint. We could thereby clearly confirm that the breakpoints are different, the t(3;8) breakpoint being most distal. In addition, we have shown that both translocation breakpoints are located distal to the homozygously deleted region in the U2020 lung cancer cell line.

Construction of a pulsed-field map around D3S3 using partially demethylated DNA.

Heavy methylation of restriction sites in the relevant chromosomal region can be a major problem in the construction of a long-range restriction map. In the region around the locus D3S3 there appeared to be few accessible restriction sites. Therefore, we cultured cells in the presence of 5-azacytidine and used the partially demethylated DNA to construct a relatively detailed restriction map spanning approximately 1 Mb. Using partially demethylated DNA is a recommendable approach when a genome region appears inaccessible for pulsed-field analysis because excessively long restriction fragments are obtained.

Somatic mutations of the von Hippel-Lindau disease tumour suppressor gene in non-familial clear cell renal carcinoma.

Loss of heterozygosity (LOH) studies have suggested that somatic mutations of a tumour suppressor gene or genes on chromosome 3p are a critical event in the pathogenesis of non-familial renal cell carcinoma (RCC). Germline mutations of the von Hippel-Lindau (VHL) disease gene predispose to early onset and multifocal clear cell renal cell carcinoma, and the mechanism of tumorigenesis in VHL disease is consistent with a one-hit mutation model. To investigate the role of somatic VHL gene mutations in non-familial RCC, we analysed 99 primary RCC for VHL gene mutations by SSCP and heteroduplex analysis. Somatic VHL gene mutations were identified in 30 of 65 (46%) sporadic RCC with chromosome 3p allele loss and one of 34 (3%) tumours with no LOH for chromosome 3p. The VHL gene mutations were heterogeneous (17 frameshift deletions, eight missense mutations, four frameshift insertions, one nonsense and one splice site mutation), but no mutations were detected in the first 120 codons of cloned coding sequence. Most RCCs with somatic VHL mutations (23 of 27 (85%) informative cases) had chromosome 3p25 allele loss in the region of the VHL gene so that both alleles of the VHL gene had been inactivated as expected from a two-hit model of tumorigenesis. Detailed histopathology was available for 59 of the tumours investigated: 18 of 43 (42%) RCC with a clear cell appearance had a somatic VHL gene mutation but none of 16 non-clear cell RCC (eight chromophilic, three chromophobe and five oncocytoma) (chi2 = 7.77, P < 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)

A homozygous deletion in a small cell lung cancer cell line involving a 3p21 region with a marked instability in yeast artificial chromosomes.

All types of lung carcinoma are characterized by a high frequency of loss of sequences from the short arm of chromosome 3, the smallest region of overlap containing D3F15S2 in band p21. Here we characterize a 440-kilobase segment from this region, which we found homozygously deleted in one of our small cell lung cancer-derived cell lines. The homozygous deletion maps between UBE1L and ZnF16, just centromeric to D3F15S2. Yeast artificial chromosomes with inserts originating from the deleted region are very unstable and readily lose parts of their insert.