An apparent interlocus gene conversion-like event at a putative tumor suppressor gene locus on human chromosome 6q27 in a Burkitt's lymphoma cell line.

A region of minimal deletion in B-cell non-Hodgkin's lymphoma (B-NHL) has recently been defined between D6S186 and D6S227 spanning 5-9 Mb at 6q26-q27, predicting the presence of at least one tumor suppressor gene (TSG) at this locus. During the construction of a deletion map in the B-NHL tumor panel, we report the identification of a Burkitt's lymphoma cell line, BL74, having an apparent homozygous deletion at the D6S347 locus, internal to the critical region. Since this case may facilitate the localization of the target TSG, a detailed structural molecular characterization and search for candidate genes were undertaken at this locus. While BL74 underwent a loss of heterozygosity at 6q26-q27, D6S347 was also likely subjected to a somatic interlocus gene conversion-like event between two homologous but distinct loci, resulting in the homozygous replacement of a 1860- to 2067-bp segment of one locus with the corresponding segment copied from the other locus. Two genes at this locus were identified, but their lack of expression in B-cell lineages tentatively excludes them as candidate TSGs. Another still unidentified gene at this locus may be disrupted by the gene conversion-like event, which would represent a novel mechanism of TSG inactivation.

Cloning and mapping of human chromosome 6q26-q27 deleted in B-cell non-Hodgkin lymphoma and multiple tumor types.

Frequent deletions of the distal region on the long arm of chromosome 6 have been reported in multiple human tumors including B-cell non-Hodgkin lymphoma (B-NHL), suggesting the presence of one or more tumor suppressor genes (TSGs) at this locus. Previously, we identified a region of minimal molecular deletion at 6q25-q27 (RMD-1) in B-NHL cases. To facilitate positional cloning efforts to identify the RMD-1 TSG(s), a yeast artificial chromosome (YAC) contig consisting of 110 clones was constructed across 6q26-q27 by sequence-tagged site/probe content mapping. The contig integrates 79 ordered markers including restriction fragment length polymorphisms, minisatellites, microsatellites, YAC-insert termini, expressed sequence tags, and known genes. It spans 34 cM and has a minimal tiling path of approximately 12 clones, covering an estimated 9-14 Mb with nearly every marker on the map showing at least double linkage to its adjacent markers. Dual-color fluorescence in situ hybridization of selected marker pairs on normal pachytene chromosome 6 further confirmed the YAC-based mappings. Utilizing a loss of constitutional heterozygosity assay in the B-NHL tumor panel, 24 additional 6q26-q27 polymorphic markers (21 mapping to the contig) further defined RMD-1 between markers D6S186 proximally and D6S227 distally. The minimal tiling path of the B-NHL RMD-1 consists of approximately 8 YAC clones, providing a size estimate of 5-9 Mb. This interval contains, in their entirety, several smaller candidate TSG critical regions previously delimited in other tumor systems. The AF-6 gene, mapping within RMD-1, revealed no mutations in a small subset of B-NHL. The deletion and physical maps presented herein provide a framework for the identification of the gene(s) involved in B-NHL as well as other malignancies and diseases mapped to this region and provide the initial reagents for large-scale genomic sequencing.

Analysis of a 69-kb contiguous genomic sequence at a putative tumor suppressor gene locus on human chromosome 6q27.

Multiple neoplasias including B-cell non-Hodgkin's lymphoma, breast carcinoma, and ovarian carcinoma, have been associated with frequent deletions of the distal region on the long arm of human chromosome 6, suggesting the presence of one or more tumor suppressor gene(s) at this locus. Loss of heterozygosity analysis of breast and ovarian tumors has further restricted the minimal region of loss within 6q27. To further characterize this genomic region for gene content including putative tumor suppressor genes as well as other elements that may contribute to tumorigenesis, a 68940-bp contiguous sequence, encompassing markers D6S193 and D6S297, was generated by random shotgun sequencing of a cosmid, P1, and PAC contig. In addition, exon trapping was performed utilizing a subset of these clones. Sixteen trapped exons, ranging in size from 44 to 399 bp, span this approximately 69-kb region. Many other putative exons have been identified computationally. Further analysis has identified 13 potential promoters and 13 putative polyadenylation sites in the region. Northern analysis identified a transcript mapping within this interval that is expressed in ovarian, breast, and lymphoid-derived tumor cell lines. Consideration of these data, together with the demonstration of several regions of high CpG content, suggests the possibility of several genes at this locus.

Cytogenetic and molecular analysis of 6q deletions in Burkitt's lymphoma cell lines.

Although abnormalities of chromosome 6 have frequently been observed in Burkitt's lymphoma (BL), they have so far not been defined by modern cytogenetic and molecular methods. By a combination of high-resolution chromosome banding, fluorescence in situ hybridization (FISH), and loss of heterozygosity (LOH) analysis, we have examined the nature of aberrations affecting chromosome 6 in 7 previously established BL cell lines. All cell lines exhibited the characteristic translocations associated with BL; 5 had t(8;14)(q24;q32) and 2 had t(8;22)(q24;q11). Three cell lines had deletions of 6q; 3 others had rearrangements affecting 6q, whereas one cell line had apparently normal chromosomes 6. FISH analysis of the three deletions established that they were interstitial. LOH analysis with probes mapped to the 6q26-27 region confirmed the sub-telomeric interstitial deletion in cell line BL-108, which had a del(6)(q23q27). All informative loci mapped to 6q26-27 (5/7) were deleted in BL-74, which had no apparent cytogenetic abnormality in chromosome 6, thus documenting a sub-microscopic deletion. These data define the cytogenetic and molecular limits of 6q deletions in BL and are consistent with our previous demonstration of LOH analysis of the site of a candidate tumor suppressor gene in the 6q25-27 region.

Deletions involving two distinct regions of 6q in B-cell non-Hodgkin lymphoma.

The recurrent loss of genetic material from a specific chromosomal region in a given tumor type suggests the presence of a tumor-suppressor gene, the loss or inactivation of which may be relevant for tumorigenesis. In this study, we provide molecular evidence for the recurrent association between deletions on the long arm of chromosome 6 and B-cell non-Hodgkin lymphoma (B-NHL). Normal and tumor DNAs from 71 cases of B-NHL were studied for loss of constitutional heterozygosity (LOH) at 19 loci on chromosome 6 using a panel of restriction fragment length polymorphism (RFLP) probes. LOH, indicating deletion of all or part of 6q, was detected in 16 of 71 cases (22.5%), ranging from low-grade to high-grade B-NHL. The isolated loss of 6p or the loss of other chromosomes (8, 17, 22) tested as controls for specificity was not observed in any case. Comparison of the extent of the deletions among different cases allowed the identification of two distinct regions of minimal deletion (RMD) at 6q25 to 6q27 (RMD-1) and at 6q21 to 6q23 (RMD-2), respectively, suggesting the existence of two tumor-suppressor genes. These data support a role for 6q deletions in B-NHL pathogenesis and provide a basis for identifying the corresponding tumor-suppressor genes.