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Translocation of the BCL2 gene from chromosome 18 to chromosome 14 results in constitutive expression of the gene. We have recently demonstrated that the major breakpoint region (mbr) of BCL2, which is implicated in 70% of t(14;18) translocations present in human follicular lymphoma, is a matrix attachment region. Since these regions are implicated in control of both transcription and replication, we wished to determine whether BCL2 translocation was also accompanied by changes in replication timing of the translocated allele. Using both fluorescence in situ hybridization and allele-specific PCR, we have demonstrated that the translocated allele replicates at the G1/S boundary, while the wildtype allele continues to replicate as usual in mid-S phase. These differences are accompanied by allele-specific changes in BCL2 expression. Since the net structural effect of t(14;18) translocations within the mbr is to disrupt the BCL2 MAR and replace it with the IGH MARs located just downstream of each breakpoint, we conclude that MAR exchange is a significant, selectable outcome of these translocations. We propose that subsequent changes of replication and transcriptional patterns for the translocated BCL2 allele result from this exchange and represent important early steps in lymphomagenesis., (Copyright 2001 Academic Press.)

The t(14,18) chromosomal translocation that occurs in human follicular lymphoma constitutively activates the BCL2 gene and disrupts control of apoptosis. Interestingly, 70% of the t(14,18) translocations are confined to three 15-bp clusters positioned within a 150-bp region (major breakpoint region or [MBR]) in the untranslated portion of terminal exon 3. We analyzed DNA-protein interactions in the MBR, as these may play some role in targeting the translocation to this region. An 87-bp segment (87MBR) immediately 3' to breakpoint cluster 3 was essential for DNA-protein interaction monitored with mobility shift assays. We further delineated a core binding region within 87MBR: a 33-bp, very AT-rich sequence highly conserved between the human and mouse BCL2 gene (37MBR). We have purified and identified one of the core factors as the matrix attachment region (MAR) binding protein, SATB1, which is known to bind to AT-rich sequences with a high propensity to unwind. Additional factors in nuclear extracts, which we have not yet characterized further, increased SATB1 affinity for the 37MBR target four- to fivefold. Specific binding activity within 37MBR displayed cell cycle regulation in Jurkat T cells, while levels of SATB1 remained constant throughout the cell cycle. Finally, we demonstrated in vivo binding of SATB1 to the MBR, strongly suggesting the BCL2 major breakpoint region is a MAR. We discuss the potential consequences of our observations for both MBR fragility and regulatory function.

Approximately 10% of ovarian cancers are due to mutations in highly penetrant inherited cancer susceptibility genes. The highly polymorphic HRAS1 minisatellite locus, located just downstream from the proto-oncogene H-ras-1 on chromosome 11p, consists of four common progenitor alleles and several dozen rare alleles, which apparently derive from mutations of the progenitors. Mutant alleles of this locus represent a major risk factor for cancers of the breast, colorectum, and bladder, and it was found that BRCAI mutation carriers with at least one rare HRAS1 allele have a greater risk of ovarian cancer than BRCA1 carriers with only common HRAS1 alleles. There are no conclusive studies of HRAS1 alleles in sporadic epithelial ovarian cancer. A case-control study of HRAS1 alleles was performed on DNA from 136 Caucasian patients with ovarian cancer and 108 cancer-free controls using conventional (Southern blot) and PCR-based methods to determine the frequency of rare HRAS1 alleles. Odds ratios (ORs) were estimated using unconditional logistic regression methods. A single degree of freedom test was used to assess the significance of linear trend across categories of increasing exposure. A statistically significant association between rare HRAS1 alleles and risk of ovarian cancer was observed [OR, 1.70; 95% confidence interval (CI), 1.03-2.80; P = 0.04]. Having only one rare allele was associated with a relative risk of 1.66 (95% CI, 0.91-3.01), whereas having two rare alleles increased the relative risk to 2.86 (95% CI, 0.75-10.94; trend P = 0.03). Analysis of HRAS1 allele types by the age of the case at diagnosis revealed that younger cases (<45 years) had a borderline statistically significant increased association with rare HRAS1 alleles compared to older cases (> or = 0 years; OR, 1.89; 95% CI, 0.90-3.98; P = 0.09). Rare HRAS1 alleles contribute to ovarian cancer predisposition in the general population. Thus, the HRAS1-variable number of tandem repeats locus may function as a modifier of ovarian cancer risk in both sporadic and hereditary ovarian cancer.

Inherited mutations in the cystatin B gene ( CSTB ) are responsible for progressive myoclonus epilepsy type 1 (EPM1; MIM 254800). This autosomal recessive disease is characterized by variable progression to mental retardation, dementia and ataxia. The majority of EPM1 alleles identified to date contain expansions of a dodecamer repeat located upstream of the transcription start site of the CSTB gene. Normal alleles contain two or three copies of the repeat, whereas pathogenic alleles contain >40 repeats. We examined the meiotic stability of pathogenic, expanded EPM1 alleles from 17 EPM1 families by employing a fluorescence-based PCR-based genotyping assay capable of detecting single dodecamer repeat unit differences on an automated DNA sequencer. We followed 74 expanded allele transmissions to 30 affected individuals and 22 carriers. Thirty-five of 74 expanded allele transmissions demonstrated either contraction or expansion of the minisatellite, typically by a single repeat unit. Thus expanded alleles of the EPM1 minisatellite demonstrate a mutation rate of 47%, the highest yet observed for pathogenetic alleles of a human minisatellite.

DNA sequence analysis of 130 alleles of the HRAS1 minisatellite has demonstrated that breast cancer-associated variants arise as a consequence of both replication errors and gene conversions. Unlike mutations at other variable number of tandem repeats (VNTRs), high-risk variants of the HRAS1 minisatellite do not demonstrate positional polarity. Instead, most mutations occur at three hotspots, with replication errors confined to one hotspot, gene conversions to a second and a mixed pattern of mutation at the third. DNA sequence analysis of 66 low-risk a1 alleles revealed no evidence for hypermutation. Therefore, while the HRAS1 minisatellite may serve as a reporter for a broad-based group of mutational mechanisms, these results are consistent with a direct pathogenetic contribution by high-risk alleles as the biological basis underlying cancer association of this VNTR.

We have tested a simple procedure, disease association by locus stratification, for identifying breast cancer patients with pathogenetic allelic variants at several candidate loci. The strategy was based on the assumption of epistatic interactions of the candidates. We analyzed 66 independent cases from sib pairs affected with breast cancer that had previously been collected during an investigation of pathogenetic-allele-sharing at the HRAS1 mini-satellite locus. An exon 24 polymorphism of ATM, substituting arginine for proline was associated with breast cancer in these cases with an overall odds ratio of 4.5 (95% confidence interval, 1.2-20.5, nominal p = 0.02, 2-tail Fisher exact test). In the presence of a rare HRAS1 allele, the odds ratio increased to 6.9 (95% CI, 1.2-38.3, p = 0.03). Thus, our procedure identified at least one allelic variant of ATM associated with breast cancer, and indicated that the ATM locus may interact with HRAS1.

Background: The role of mutations in protooncogenes and their regulatory sequences in the pathogenesis of cancer is under close scrutiny. Minisatellites are unstable repetitive sequences of DNA that are present throughout the human genome. The highly polymorphic HRAS1 minisatellite locus just downstream from the protooncogene H-ras-1 consists of four common progenitor alleles and several dozen rare alleles, which apparently derive from mutations of the progenitors. We previously observed an association of the rare mutant alleles with many forms of cancer, and we undertook the present study to pursue this observation further., Methods: We conducted a case-control study, typing 736 HRAS1 alleles from patients with cancer and 652 from controls by Southern blotting of leukocyte DNA. We also carried out a meta-analysis of this study and 22 other published studies, estimating the relative risk of cancer (such as bladder, breast, or colorectal cancer) when one of the rare HRAS1 alleles was present., Results: Both the present case-control study (odds ratio, 1.83; 95 percent confidence interval, 1.28 to 2.67; P = 0.002) and the present study combined with our previous study (odds ratio, 2.07; 95 percent confidence interval, 1.47 to 2.92; P < 0.001), as well as the meta-analysis of all 23 studies (odds ratio, 1.93; 95 percent confidence interval, 1.63 to 2.30; chi-square = 57.58; P < 0.001), replicated our original finding and demonstrated a significant association of rare HRAS1 alleles with cancer. We found significant associations for four types of cancer: carcinomas of the breast, colorectum, and urinary bladder and acute leukemia. We also identified suggestive but not statistically significant associations for cancers of the lung and prostate and for non-Hodgkin's lymphoma., Conclusions: Mutant alleles of the HRAS1 minisatellite locus represent a major risk factor for common types of cancer. Although the relative risk associated with the presence of one rare allele is moderate, the aggregate prevalence of one rare allele is moderate, the aggregate prevalence of this class of mutant alleles implies an extremely important attributable risk: 1 in 11 cancers of the breast, colorectum, and bladder.

We have recently shown that constitutively expressed members of the rel/NF-kappa B family of transcription factors bind the HRAS1 minisatellite, VTRHRAS1. We now report that, like other NF-kappa B binding sites, VTRHRAS1 displays pleiotropic transcriptional regulatory activity that is promoter- and cell-type-specific. Both enhancement and suppression are restricted to the human bladder carcinoma cell line EJ, in which we have previously defined a unique form of NF-kappa B p50. We also observe allelic variation in functional activity: the rare a2.1 allele, one member of a class of HRAS1 alleles overrepresented in the genomes of cancer patients, possesses twofold greater enhancer activity than the low-risk alleles, a0.1, a1, and a2. Finally, VTRHRAS1 enhancer activity is upregulated by the adenovirus E1A 13S gene product, demonstrating the potential of the minisatellite for influencing gene expression through several distinct interactions with the transcriptional apparatus.

The 50bp repeat unit of the minisatellite within the DH-JH interval of the human immunoglobulin heavy chain locus binds a nuclear factor present in a wide variety of cell types. The binding site contains the myc/HLH motif, CACGTG, and represents a 15 of 17 base match for the USF/MLTF binding site adjacent to the adenovirus major late promoter (MLP). Unlike the USF/MLTF site, the IGH minisatellite possesses no enhancer activity. However, it can significantly suppress, in cis and in trans, USF-site-mediated transcriptional activation of the MLP. In murine myeloma cells, the IGH minisatellite can suppress, in trans, MLP activation by the murine heavy chain gene enhancer, E mu. These activities potentially represent a DNA-based form of squelching.

We have cloned and characterized a 55-kb region of DNA surrounding HRAS1. It contains a cluster of two, and possibly three, genes associated with CpG islands within the 32 kb immediately upstream of HRAS1. We have sequenced cDNAs representing one of these genes, provisionally designated HRC1. The locus, which is located 29 kb upstream of HRAS1, is divergently transcribed. HRC1 cDNA probe recognizes fragments on Southern blots of DNA from other vertebrate species. In human DNA, multiple homologous fragments are detected in addition to the predicted ones containing HRC1. Therefore, this locus may represent a member of an evolutionarily conserved gene family. HRC1 expression is upregulated with HRAS1 in the EJ bladder carcinoma cell line, suggesting the possibility of coordinate regulation. The deduced translational product of the longest open reading frame (1119 nucleotides, 373 amino acids) predicts a protein with regions rich in glutamine and proline and a region similar to the helix-loop-helix motif adjacent to a carboxy-terminal leucine zipper dimerization motif with four heptad repeats. Alternate splicing of terminal exons occurs, resulting in the truncation of one proline-rich domain and preservation of the leucine zipper. Thus, a biologically important region of chromosome 11p consists of a gene cluster. At least one of these genes, in addition to HRAS1, may be involved in regulation of cell growth or differentiation.

The 28 base pair repeat unit of a minisatellite 1000 bp downstream from the human HRAS1 gene (VTRHRAS1) bound four proteins (p45, p50, p72 and p85) in nuclear extracts from a variety of human cell lines which were indistinguishable from several members of the rel/NF-kappa B family of transcriptional regulatory factors. VTRHRAS1 bound the constitutively expressed, but not the inducible, forms of these proteins. Analysis of partially purified binding factors from different cell lines demonstrated qualitative differences in the p50 subunit; phosphocellulose fractionation also revealed considerable heterogeneity in the p72 and p85 subunits. These results suggest the possibility that the HRAS1 minisatellite, in serving as a tandem array of rel/NF-kappa B binding sites, may function in the transcriptional regulation of HRAS1 and nearby genes.

Restriction fragment length polymorphism analyses of the Ha-ras-1 proto-oncogene were undertaken in white and black populations residing in the Baltimore-Washington metropolitan area to address whether specific rare alleles of the Ha-ras-1 proto-oncogene locus vary in their distribution among different racial groups. High-molecular-weight genomic DNA samples from the lungs of 80 lung cancer patients and 92 accident victims were digested with appropriate restriction enzymes and subjected to Southern analysis using the 6.6-kb BamHI human Ha-ras-1 recombinant fragment from the plasmid pEC. Thirty allelomorphs of different sizes were detected among the 172 study subjects. An association was observed between race and specific alleles. Rare alleles were more frequent in black cancer patients and trauma victims than in whites. Within each racial category, lung cancer patients had an excess of rare alleles. These data indicate the importance of controlling for racial variation when designing studies to determine human cancer risk factors.

Three genetic markers within the promoter-exon 1 region of the HRAS1 locus have been employed to investigate lineage relationships among alleles of the highly polymorphic variable tandem repeat (VTR) immediately downstream of the HRAS1 gene. These markers were in absolute linkage disequilibrium with the HRAS1 VTR, allowing the assignment of unique upstream haplotypes to each of the four common VTR alleles. Analysis of 17 rare alleles revealed a stratification of allele fragment size and upstream haplotype in which each rare VTR allele possessed the markers characteristic of the common allele nearest in size. Therefore, hyperallelism emanated from the four common alleles in a defined fashion, the size of a rare allele specifying its origin. As discussed below, this result implies that unequal crossing-over between homologues is unlikely to be the predominant mechanism for generating new VTR alleles at this minisatellite locus.

The hypothesis that rare variable nucleotide tandem repeat alleles of the Ha-ras-1 polymorphism are an inherited predisposing factor in human lung carcinogenesis has been evaluated in an age, race, and smoking matched case-control study. Twenty-three different alleles were identified by their restriction fragment length in DNA isolated from peripheral blood lymphocytes and were categorized into three groups: common; intermediate; and rare. The frequencies of rare alleles in blacks with either squamous cell carcinoma, large cell carcinoma, or small cell carcinoma were found to be significantly higher than those among groups of control subjects that were comprised of chronic obstructive pulmonary disease patients and patients with cancer at sites other than the lung. A similar trend which did not reach statistical significance was observed in whites. These data are consistent with the hypothesis that inheritance of Ha-ras-1 rare restriction fragment length alleles represents a genetic risk factor for some human lung cancers. The biological basis of this observation remains to be clarified, and it is possible that ethnic variations in rare allele frequencies are responsible for the differences noted. However, the data suggest that further evaluation of the Ha-ras-1 polymorphism as a marker of individual lung cancer susceptibility is warranted.

A reexamination of human minisatellite (hypervariable) regions following the cloning and sequencing of the new minisatellite, VTR1.1, revealed that many of these structures possessed a strongly conserved copy of the chi-like octamer, GC[A/T]GG[A/T]GG. In oncogene translocations apparently created by aberrant VDJ recombinase activity, this VTR octamer was often found within a few bases of the breakpoint (p less than 10(-10)). Three bcl2 rearrangements which occurred within 2 bp of one another were located precisely adjacent to this consensus; it defined the 5' border of that oncogene's major breakpoint cluster. Several c-myc translocations also occurred within 2 bp of this sequence. While the appearance of a chi-like element in polymorphic minisatellite sequences is consistent with a role promoting either recombination or replication slippage, the existence of such elements at sites of somatic translocations suggests chi function in site-specific recombination, perhaps as a subsidiary recognition signal in immunoglobulin gene rearrangement. We discuss the implications of these observations for mechanisms by which oncogene translocations and minisatellite sequences are generated.

A highly polymorphic locus associated with the variable tandem repetition of a 35 bp consensus sequence was mapped to chromosome 10, band q26. Examination of leukocyte DNA from a cancer patient revealed the twenty-fold amplification of one allelic fragment of this locus, while the other allelic fragment demonstrated a normal copy number. In another patient, Southern blotting of leukocyte DNA detected the deletion of the 3'-flanking region from one tandem repeat allele. These results indicate that variable tandem repeats may mark highly unstable regions of DNA in the human genome which can be altered by changes more extensive than simple tandem repeat variation.

A rapid and exciting accumulation of data about cellular oncogenes in human tumors has resulted from convergent research on DNA-mediated gene transfer, retroviruses, and tumor cytogenetics. Such work promises to increase our understanding of the genetic events that predispose to, and result in, malignant disease. This knowledge may quickly find clinical application in tumor classification and prediction of risk. Ultimately, therapeutic benefits may be achieved as we begin to explore the mechanisms by which transforming gene products act to defeat the normal regulatory processes of cells.

Many of the genes that are likely participants in the pathogenesis of human neoplasia have been identified. The major classes of events that activate these genes in tumors have also been described. We expect that continuing research on the function of oncogenes will greatly inform our understanding of fundamental growth control processes and, eventually, influence our approaches to treating cancer patients.

White blood cell DNA from cancer patients and DNA from tumours and tumour-derived cell lines frequently demonstrated allelic restriction fragments of the Harvey ras oncogene locus not found in the unaffected population. The presence of such unusual alleles may be linked to susceptibility to cancer.

Although monoclonal B-cell populations can be identified both by surface-marker analysis and by immunoglobulin-gene rearrangements, this has not been possible with T cells. We have employed cDNA probes that are specific for the entire beta chain of the T-cell receptor, and for its constant and variable regions, to investigate gene rearrangements in T-cell chronic lymphocytic leukemia and related disorders. In three malignant proliferations of helper (T4-positive) T cells, rearrangements of the beta-chain constant-region gene were readily demonstrated. A patient from the Caribbean who had adult T-cell lymphoma and antibody to human T-cell lymphotrophic virus Type I (HTLV-I), a patient with virus-negative chronic lymphocytic leukemia, and a patient with cutaneous T-cell lymphoma (Sézary variant) made up the T4-positive group. Three additional patients with chronic T8 (cytotoxic-suppressor) lymphocytosis and neutropenia were studied; in two; rearrangements were found. In all five patients with constant-region rearrangements, deletions of variable-region restriction fragments were observed as well. The presence of rearrangements of a T-cell receptor gene provides presumptive evidence for the clonal nature of T-cell proliferation and for its neoplastic character.

The polymorphic restriction fragments of the human Ha-ras locus, produced by the variable tandem repetition (VTR) of a short consensus sequence, fall into three classes based on allelic frequencies. Alleles of the "rare" class (individual frequencies less than 0.5%) have been detected only in white blood cell and tumor DNA of cancer patients. This phenomenon is independent of ethnic origin. No significant association of rare alleles with cancer patients has been demonstrated at an independent tandem repeat locus, VTR4.1. The results suggest that the Ha-ras restriction fragment length polymorphism is useful in cancer risk assessment.

We have detected a 6-bp deletion in the untranslated first exon of a unique HRAS1 gene cloned from lymphocyte DNA of a familial melanoma patient. The deletion is without apparent functional consequence. Using an RNase protection assay, we have demonstrated the deletion in leukocyte DNAs of individuals unrelated to the patient. In these cases, the deletion marker is specifically associated with one class of common HRAS1 allele, thereby establishing the origin of the unique allele. We discuss the means by which DNA sequence heterogeneity at other loci may be rapidly analyzed.

We have prospectively examined 66 consecutive initial diagnostic lymph node biopsies from unselected patients suspected of having malignant lymphoma for clonal immunophenotypic and immunogenotypic markers. By morphological and cell surface phenotypic criteria 52 had non-Hodgkin's lymphoma derived from the B-cell lineage and in these we compared surface immunoglobulin criteria for clonality with immunoglobulin gene rearrangement as detected by JH, C kappa, and C lambda gene probes. We found that the addition of BglII and HindIII double digests to the standard BamHI and EcoRI restriction enzymes made it possible to detect rearrangement in the vast majority of lymphomas and that rearrangement of both JH alleles is the rule. A rearranged heavy and/or light chain gene was detected in 47 of 52 (91%) tumors and the JH probe alone detected rearrangements in 87% of tumors when multiple restriction enzymes were used. In contrast, surface immunoglobulin, the standard clonal marker for monoclonal B-cell malignancy, was either undetectable or did not exhibit light chain restriction in 29 of 52 tumors as detected by flow cytometric analysis. Further, in 24 of these 29 tumor DNAs we could detect an Ig gene rearrangement. In follicular (nodular) lymphoma which often gives ambiguous immunophenotypic results by cell suspension techniques, monoclonal gene rearrangements were detected in 16 of 18 tumor DNAs. Monoclonal surface immunoglobulin was detected in only 8 of 18 of this subset of cases. The 52 tumors were also analyzed for potential oligoclonality. We found that the use of BglII, a restriction enzyme that closely spanned the JH region, increased the sensitivity of detecting rearrangements and facilitated the identification of isotype switch variants. In only a single (1 of 52) tumor DNA were more than two rearranged bands seen with JH, C kappa, and C lambda probes, suggesting a multiclonal origin. Additional cases thought to potentially represent oligoclonality by immunophenotypic criteria proved to be isotype switch variants. We conclude that Ig gene rearrangement is an extremely sensitive method for defining monoclonality in lymphoma cell populations, particularly if multiple restriction enzymes are used, and that the vast majority of the clonal diversity seen in initial diagnostic biopsies is the result of isotypic switch within a given clone rather than true oligoclonality.

Host restriction of oncogenesis of RNA tumor viruses in vivo is associated with several gene loci. One of these genes, the Fv-1 locus in mice, is expressed in vitro and may be studied in mouse-embryo cultures that are restrictive or permissive for replication of Friend leukemia virus. Two strains of Friend leukemia virus, N-or B-tropic, show reciprocal ability to replicate successfully in either NIH Swiss (N-type) or BALB/c (B-type) cells that differ at the Fv-1 locus. These two strains of virus and two cell lines form a system to measure host restriction in vitro. Measurement of adsorption of Friend leukemia virus to permissive or restrictive cells reveals no difference in rate or total amount of virus bound. Furthermore, studies with virions of vesicular stomatitis virus phenotypically mixed within an envelope containing Friend leukemia virus protein show no differences in penetration or replication of vesicular stomatitis virus. These results strongly suggest that host restriction of Friend leukemia virus is due to an intracellular event in the viral replication cycle.