Dysregulation of cyclin dependent kinase 6 expression in splenic marginal zone lymphoma through chromosome 7q translocations.

The increased or inappropriate expression of genes with oncogenic properties through specific chromosome translocations is an important event in the pathogenesis of B-cell lymphoproliferative diseases. Recent studies have found deletions or translocations of chromosome 7q to be the most common cytogenetic abnormality observed in SLVL, a leukemic variant of SMZL, with the q21-q22 region being most frequently affected. In three patients with translocations between chromosomes 2 and 7, the cloning of the breakpoints at 7q21 revealed that each was located within a small region of DNA 3.6 kb upstream of the transcription start site of cyclin dependent kinase 6 (CDK6). In each case the translocation event was consistent with aberrant VJ recombination between the immunoglobulin light chain region (Ig kappa) on chromosome 2p12 and DNA sequences at 7q21, resembling the heptamer recombination site. The t(7;21) breakpoint in an additional patient with splenic marginal zone lymphoma (SMZL), resided 66 kb telomeric to the t(2;7) breakpoints juxtaposing CDK6 to an uncharacterized transcript. In two of the SLVL patient samples, the CDK6 protein was found to be markedly over expressed. These results suggest that dysregulation of CDK6 gene expression contributes to the pathogenesis of SLVL and SMZL.

Detailed molecular delineation of 13q14.3 loss in B-cell chronic lymphocytic leukemia.

A region of chromosome 13q14.3, telomeric to the Retinoblastoma gene RB-1 is frequently deleted in patients with B-cell chronic lymphocytic leukemia (B-CLL). A cosmid and P1-derived artificial chromosome (PAC) contig spanning over 600 kb has been constructed, which encompasses this locus. The contig clones have been used to order a number of markers along the minimally deleted region and to localize a series of CpG islands corresponding to possible candidate genes. A novel polymorphic dinucleotide repeat, 6E3.2, present in one of the ordered cosmid clones has been isolated for use in deletion mapping studies of patient DNA. Leukemic samples from 229 CLL patients have been screened for loss of heterozygosity using microsatellite markers and analyzed for hemizygous and homozygous deletions by Southern blot techniques using genomic probes selected from cosmids across the region. Hemizygous deletions were found in 31% of cases with an additional 10% showing homozygous loss. The use of these probes has defined the commonly deleted area to less than 130 kb, centromeric to the locus D13S272.

Cytogenetic, fluorescence in situ hybridisation, and clinical evaluation of translocations with concomitant deletion at 13q14 in chronic lymphocytic leukaemia.

Deletions and translocations of 13q14 are the most frequent structural chromosome abnormalities found in chronic lymphocytic leukaemia (CLL). We have identified 13q14 translocations in the blood of 30 of 450 (6.6%) CLL patients by conventional cytogenetics, using tetradecanoyl phorbol 12-myristate 13-acetate (TPA) as a mitogen. The translocations are characterised by multiple partner chromosomes and a high incidence, 6 of 30 cases, of complex rearrangements. Seven cases were also studied by fluorescence in situ hybridisation (FISH) using four previously ordered YACs, to define the breakpoints further. Deletions with varying proximal and distal breakpoints were found in six cases. Two of the cases had deletions of the cytogenetically normal chromosome 13 at q14, and in one case the 13q14 translocation was a secondary genetic event. No difference in the clinical features between the patients with 13q14 translocation and 54 patients with 13q14 deletions or four patients with both a translocation and a deletion was observed. These data suggest that the genetic consequence of 13q14 translocations in CLL is the loss of a tumour suppressor gene.

PCR analysis of polymorphisms at the D13S25 locus.

Evidence that the D13S25 locus lies close to a potential tumour suppressor gene implicated in the pathogenesis of B-CLL has been based on detection of LOH and bi-allelic loss using the pH2-42 probe. The SspI polymorphism detected by this probe has been identified by sequencing adjacent clones and a polymorphic (TA)n repeat has been found. Amplification of the region encompassing both polymorphic markers by PCR increases the informativity to 80%.

Frequent homozygous deletions of the D13S25 locus in chromosome region 13q14 defines the location of a gene critical in leukaemogenesis in chronic B-cell lymphocytic leukaemia.

Cytogenetic studies of B-cell chronic lymphocytic leukaemia show structural abnormalities involving the 13q14 chromosome region as the only karyotypic change in a significant proportion of tumours. This observation suggests the location of a gene important in leukaemogenesis. A series of 68 BCLL tumours have been analysed for allele loss using a series of probes from 13q14. Using intragenic polymorphic markers from the retinoblastoma predisposition gene LOH was observed in 25% of tumours including 3/6 showing cytogenetically obvious deletions of the 13q14 region and 3/6 showing translocations involving 13q14. However, three deletions with proximal breakpoints in 13q14 did not show allele loss, demonstrating that the breakpoint lay distal to RB1. Using the D13S25 locus, which lies 1.6 cM distal to RB1, allele loss was seen in 90% of tumours with structural rearrangements of 13q14 and 75% of tumours with an apparently normal karyotype. 50% of these tumours showed homozygous loss of D13S25, suggesting that a 'tumour suppressor gene' lies in this region. The more distal D13S31 locus, 1 cM distal to D13S25, was infrequently involved in allele loss demonstrating that the minimum region of overlap for homozygous deletions is approximately 1 Mbp around the D13S25 locus.