Academic literature on the topic 'Translocation breakpoint regions'

Abstract Translocations involving the IgH locus are one of the most common genetic abnormalities observed in multiple myeloma (MM). Unlike several hematological malignancies, MM IgH translocations involve multiple partner chromosomes. Although IgH translocations are not unique to MM, the molecular anatomy of the translocations appears to be different from that observed in most B-cell malignancies. In general, the breakpoints occur within the switch regions of the IgH locus and the translocations appear to result from illegitimate class switch recombination (CSR) events. Previous analysis of the breakpoint junctions from t(4;14) samples suggested that the majority of these translocations result from illegitimate CSR events. These events were characterized by der(4) breakpoints containing Smu-chromosome 4 junctions and der(14) breakpoints with chromosome 4-downstream switch region junctions. However, not all t(4;14) breakpoints fit this “classical” model, as some derivative chromosomes were observed with hybrid switch regions. Unfortunately, the mechanism that generates these hybrid switch regions has been unclear. In general, only a single derivative was cloned from each patient or cell line. In the one case reported elsewhere in which both derivatives were cloned, the mechanism did not appear to be linked to the CSR process and thus represented a “non-classical” translocation. The poor prognostic impact of t(4;14) myeloma has been well established by several groups, including our own. In an attempt to identify recurrent breakpoint sites and to identify the potential mechanism(s) leading to t(4;14) translocations, we cloned the breakpoint junctions of both derivative chromosomes from 4 cell lines and 5 patients with MB4-2 and MB4-3 breakpoints. Furthermore, we cloned der(4) breakpoints from 4 additional patients, three of which are FGFR3 non-expressers for which we could not detect a der(14) breakpoint using our PCR based strategy. We defined the t(4;14) breakpoint region as encompassing 64.5 kb of chromosome 4, flanked by LETM1 exon 3 and MMSET exon 5, based on combining the previously published breakpoints with our newly cloned and sequenced breakpoints. Current dogma suggests that t(4;14) translocation events are randomly distributed throughout the defined breakpoint region, but this idea is not supported by our sequencing data. We identified two hotspots, which contain breakpoints from 9 of the 27 patients or cell lines with at least one cloned derivative. Interestingly, these regions only represent 1 kb of the entire breakpoint region. Therefore 33% of the cloned breakpoints exist within only 1.5% of the total breakpoint region. Moreover, for the 13 MM samples for which both derivatives are cloned, although 6/13 (46%) fit the classical model of CSR mediated switch translocations, surprisingly, 7/13 (54%) appear to be non-classical translocations. The non-classical translocations are defined by little to no loss of sequence from the involved switch region and the presence of a hybrid switch region on one of the two derivative chromosomes. Importantly, the non-classical translocations may not involve B-cell specific mechanisms and could potentially occur before or after a successful CSR event. Therefore, the classical illegitimate CSR event model can explain only half of the t (4; 14) breakpoints cloned to date.

Abstract Abstract 3490 IGH loci translocations in multiple myeloma are primary events in the aetiology of the disease. There are 5 main translocation partner chromosomes which result in the over-expression of key oncogenes. These translocations are t(4;14), t(6;14), t(11;14), t(14;16) and t(14;20) and result in the over-expression of MMSET and FGFR3, CCND3, CCND1, MAF and MAFB, respectively. The translocations have a major impact on response and survival with the t(4;14), t(14;16) and t(14;20) resulting in poor prognosis. It is therefore imperative that these chromosomal abnormalities be identified. Translocations have traditionally been identified by fluorescence in situ hybridisation (FISH). Using targeted capture techniques, similar to exome capture technology, followed by massively parallel sequencing it should be possible to identify the translocations and the specific breakpoints. We have developed a targeted capture using the SureSelect (Agilent) system by tiling RNA baits across the IGH locus. Baits covered the V, D and J segments as well as being tiled across the entire constant region, including the switch regions. DNA from samples (n=120) were assayed using 150 ng of DNA and a modified capture protocol. The translocation partner had previously been identified by FISH in 36 samples which comprised 11 t(4;14), 3 t(6;14), 11 t(11;14), 9 t(14;16), 2 t(14;20). The remaining 84 samples were assayed by RQ-PCR for over-expression of the partner oncogenes to determine the translocation. Several identified translocations were verified by PCR. In 90% of samples which had FISH performed the correct IGH translocation was detected using the capture technique. The number of paired reads detecting the translocation varied from 2 to 102. Breakpoints could be determined for all of these samples and were mapped for each translocation group. In the t(4;14) group the breakpoints were clustered around exons 1, 4 and 5, corresponding to the MB4-1, MB4-2 and MB4-3 IgH-MMSET hybrid transcripts. Of the 11 t(4;14) with FISH only 2 did not express FGFR3 and had deletion of der(14). In these samples the breakpoint was located between LETM1 and MMSET, confirming that loss of FGFR3 expression is due to deletion of der(14) and not due to the location of the breakpoint. The sample with the breakpoint furthest from MMSET was located 67 kbp upstream of the start of translation within LETM1, in a position similar to that found in the KMS-11 cell line. In the t(11;14) samples the breakpoints varied dramatically on chromosome 11 but were always centromeric to CCND1. Breakpoints varied from 1.1 kbp centromeric to the start of CCND1 transcription to 1.1 Mbp centromeric, within the PPP6R3 gene. However, most breakpoints (70%) were in the intergenic region between MYEOV and CCND1. The distance from the breakpoint to CCND1 did not inversely correlate with CCND1 expression, in fact the sample with the breakpoint furthest from CCND1, within PPP6R3, had the highest expression of CCND1 as determined by gene expression array. No samples had breakpoints within the mantle cell lymphoma major translocation cluster. However, 2 samples had their breakpoint within 100 bp of one another, indicating a possible common breakpoint. Of the t(6;14) samples 2 had breakpoints in the first intron of CCND3, upstream of the start of translation. The remaining sample had its breakpoint 550 kbp upstream of the transcription start site within UBR2. The t(14;16) samples all had their breakpoints within the last intron of WWOX, 0.48–1.03 Mbp centromeric of MAF and in the location of the common fragile site FRA16D. The breakpoints cluster into 2 groups on either side of the fragile site. The t(14;20) breakpoints were located in the 1.5 Mbp intergenic region centromeric of MAF. The breakpoint furthest from MAF was 1.2 Mbp centromeric of the gene. In conclusion, we have developed and validated a targeted capture and sequencing approach for identifying translocations into the IGH locus in myeloma. This approach is important because of its capacity for high throughput low cost testing strategies that can identify these important prognostic events making a myeloma specific diagnostic platform and personalised medicine a reality for patients with myeloma. Importantly sequence analysis of the peri-breakpoint regions gives insight into molecular mechanisms acting early in the process of myelomagenesis. Disclosures: No relevant conflicts of interest to declare.

Our results indicate that there are two major breakpoint cluster regions in chromosome 18 DNA for t(14;18) translocations in follicular lymphomas. The absence of a pFL-1 homologous transcript in a cell line containing a pFL-2-detectable translocation suggests that there may be two different pathogenetic consequences of t(14;18) translocations. One possibility is that, despite the distances between them (greater than 20 kb), breakpoints in the two cluster regions in some way affect transcription of the same gene product, which has not yet been identified. Alternatively, two separate transcriptional units may be involved. The availability of DNA probes for each of the two t(14;18) breakpoint cluster regions will allow further studies regarding the biologic significance of these two genetically distinct classes of t(14;18) translocations.

Abstract Array based comparative genomic hybridization (CGH) has revolutionized the study of chromosomal imbalances but generally is incapable of detecting balanced genomic rearrangements like reciprocal translocations, which play central roles in the pathogenesis and diagnosis of lymphomas, leukemias and other tumors. The precise identification of immunoglobulin heavy chain (IgH) translocation partners, for example, is essential for the classification of B cell lymphomas and for predicting prognosis in plasma cell neoplasms like multiple myeloma. Using IgH translocations as a model for balanced genomic rearrangements, we have developed a simple modification of array CGH that we call translocation-CGH (transCGH) and that enables the rapid identification of IgH translocation partners and precise mapping of translocation-associated breakpoints to unprecedented resolution. To render IgH translocations detectable on CGH arrays, genomic DNA from test and reference samples is modified prior to array hybridization in an enzymatic linear amplification reaction that employs a single IgH joining (JH) or switch (Sμ/Sα/Sε) region primer, resulting in specific amplification of any fusion partner sequences that may be inserted (via translocation or other rearrangement) downstream of the IgH primer. Using a single tiling-density oligonucleotide array representing such common IgH partner loci as MYC, BCL2 and CCND1 (cyclin D1), transCGH successfully identified and mapped to ∼100bp resolution an assortment of known IgH fusion breakpoints in various cell lines and primary lymphomas, including JH-CCND1 breakpoints in MO2058 and Granta 519 cell lines (mantle cell lymphoma), a cytogenetically cryptic Sα-CCND1 fusion in U266 (myeloma), JH-MYC and Sμ-MYC breakpoints in MC116 and Raji (Burkitt lymphoma), and JH-BCL2 breakpoints in DHL16 (large cell lymphoma; minor cluster region) and in an archival case of follicular lymphoma (major breakpoint region). We then used transCGH to analyze 4 archival cases of mantle cell lymphoma and one t(11;14)-positive case of B cell prolymphocytic leukemia, all of which lacked PCR-detectable translocation breakpoints at the CCND1 major translocation cluster (MTC). Five novel CCND1 translocation breakpoints were identified and mapped to ∼100bp resolution, allowing the rapid design of patient-specific PCR primers for amplification, sequencing, and confirmation of the predicted breakpoints. One breakpoint mapped to within 500bp of the MTC, whereas the other 4 were scattered across a ∼150kb region flanking the MTC. To our knowledge, this represents the largest series of non-MTC mantle cell lymphoma breakpoint sequences reported to date. It also illustrates how transCGH can facilitate the rapid cloning of previously unidentified IgH translocation breakpoints dispersed over very large genomic regions. Because transCGH requires only genomic DNA and can simultaneously detect both balanced IgH translocations and genomic imbalances at ultra-high resolution on the same array, it may become a useful alternative to molecular cytogenetic methods (e.g. FISH) for clinical testing of B cell and plasma cell neoplasms. transCGH also will facilitate the development of highly sensitive breakpoint-specific PCR assays for detecting minimal residual disease. Finally, because the primer used in the linear amplification reaction is fully customizable, transCGH can readily be adapted to identify and map other balanced translocations (or more complex genomic fusions) that involve non-IgH loci, provided that one of the fusion partners is known.

Abstract Negative interference describes a situation where two genetic regions have more double crossovers than would be expected considering the crossover rate of each region. We detected negative crossover interference while attempting to genetically map translocation breakpoints in maize. In an attempt to find precedent examples we determined there was negative interference among previously published translocation breakpoint mapping data in maize. It appears that negative interference was greater when the combined map length of the adjacent regions was smaller. Even positive interference appears to have been reduced when the combined lengths of adjacent regions were below 40 cM. Both phenomena can be explained by a reduction in crossovers near the breakpoints or, more specifically, by a failure of regions near breakpoints to become competent for crossovers. A mathematical explanation is provided.

Abstract The Philadelphia chromosome (Ph1) of chronic myelogenous leukemia (CML) contains sequences from chromosome 9, including the ABL protooncogene, that have been translocated to the breakpoint cluster region (bcr) of chromosome 22, giving rise to a bcr-ABL fusion gene, whose product has been implicated in the genesis of CML. Although chromosome 22 translocation breakpoints in CML virtually always occur within the 5.8- kilobase (kb) bcr, chromosome 9 breakpoints have been identified within the known limits of ABL in only a few instances. For a better understanding of the variability of the breakpoints on chromosome 9, we studied the CML cell line BV173. Using pulsed-field gel electrophoresis (PFGE), large-scale maps of the t(9;22) junctions were constructed. The chromosome 9 breakpoint was shown to have occurred within an ABL intron, 160 kb upstream of the v-abl homologous sequences, but still 35 kb downstream of the 5′-most ABL exon. bcr-ABL and ABL-bcr fusion genes were demonstrated on the Ph1 and the 9q+ chromosomes, respectively; both of these genes are expressed. These results suggest that the 9;22 translocation breakpoints in CML consistently occur within the limits of the large ABL gene. RNA splicing, sometimes of very large regions, appears to compensate for the variability in breakpoint location. These studies show that PFGE is a powerful new tool for the analysis of chromosomal translocations in human malignancies.

The Philadelphia chromosome (Ph1) of chronic myelogenous leukemia (CML) contains sequences from chromosome 9, including the ABL protooncogene, that have been translocated to the breakpoint cluster region (bcr) of chromosome 22, giving rise to a bcr-ABL fusion gene, whose product has been implicated in the genesis of CML. Although chromosome 22 translocation breakpoints in CML virtually always occur within the 5.8- kilobase (kb) bcr, chromosome 9 breakpoints have been identified within the known limits of ABL in only a few instances. For a better understanding of the variability of the breakpoints on chromosome 9, we studied the CML cell line BV173. Using pulsed-field gel electrophoresis (PFGE), large-scale maps of the t(9;22) junctions were constructed. The chromosome 9 breakpoint was shown to have occurred within an ABL intron, 160 kb upstream of the v-abl homologous sequences, but still 35 kb downstream of the 5′-most ABL exon. bcr-ABL and ABL-bcr fusion genes were demonstrated on the Ph1 and the 9q+ chromosomes, respectively; both of these genes are expressed. These results suggest that the 9;22 translocation breakpoints in CML consistently occur within the limits of the large ABL gene. RNA splicing, sometimes of very large regions, appears to compensate for the variability in breakpoint location. These studies show that PFGE is a powerful new tool for the analysis of chromosomal translocations in human malignancies.

Abstract Follicular lymphomas (FL) are associated with the chromosomal translocation t(14;18)(q32;q21). Most breakpoints of chromosome 18 (60%) occur in the major breakpoint region (MBR) of the BCL-2 gene. Further breakpoints have been detected in the minor cluster region (mcr), less frequent breakpoints are found in regions called 3′-MBR, 5′-mcr and icr. On chromosome 14 most breakpoints are located within one of the six JH-genes. Therefore, BCL-2 translocations with breakpoints within the MBR and mcr are generally detected by PCR using combinations of different BCL-2 primers with one JH-consensus primer. We have developed a multiplex quantitative real-time PCR strategy that that can be used to detect t(14;18) translocations with breakpoints located within all regions mentioned above. To minimize the costs for expensive probes we used the JH-consensus sequence as a target for one “consensus probe” (fluorescent labelled minor groove binder probe) for all assays in combination with 6 different JH intron primers. To reduce the size of amplified PCR fragments 12 BCL-2 primers were chosen in combination with 6 JH intron primers for the detection of all 5 breakpoint regions. It is very important to choose short DNA target sequences for amplification: (a) to establish a real-time PCR with a high amplification efficacy; (b) to be able to amplify target sequences also from partially degraded DNA isolated from formaldehyde-fixed paraffin-embedded tissue sections; (c) to achieve a high sensitivity to detect 1–3 copies per assay. Peripheral bood mononuclear cells (PBMNC) and formalin fixed, paraffin embedded lymph node tissue obtained from 139 FL patients at the time of diagnosis (LN and PBMNC, n = 54; LN only, n = 3; PBMNC only, n = 82) were tested by multiplex quantitative real-time PCR. 80 breakpoints were identified within the MBR (61%) region. For comparison, 78/80 breakpoints were also detected by our standard real-time PCR assay with one BCL-2-MBR- primer and one JH consensus primer in combination with a fluorescent probe located within the BCL-2 sequence [Doelken et al., BioTechniques, 1998]. Two additional translocations with breakpoints located 5′ of the target sequence of the standard PCR were found by using two additional MBR primers. In addition, five mcr breakpoints (5%), one breakpoint in the 3′MBR region and one breakpoint in the icr region were found. Based on these results the prevalence of breakpoints in various regions of the BCL-2 gene in FL patients is: MBR = 61% (80/139); mcr = 5% (5/139); 3′MBR = 1% (1/139); icr = 1% (1/139); 5′mcr = 0%). Furthermore, based on quantitative PCR results the t(14;18) translocations detected in this study were undoubtedly lymphoma associated and did not belong to t(14;18)-positive non-lymphoma B cell clones found in healthy persons. By applying this multiplex quantitative real-time PCR strategy t(14;18) translocations with breakpoints in five different breakpoint clusters can be detected in about 70% of patients with follicular lymphoma. The assays can be used for a fast and reliable quantitative detection of t(14;18) translocations on DNA isolated from fresh lymph nodes or pathological specimens as well as blood samples at the time of diagnosis. In almost all cases quantitative results will allow a distinction whether the translocation found is lymphoma associated or not, which will in turn allow a quantitative MRD analysis on follow-up samples during and after treatment.

Context.— B-cell lymphomas exhibit balanced translocations that involve immunoglobulin loci and result from aberrant V(D)J recombination, class switch recombination, or somatic hypermutation. Although most of the breakpoints in the immunoglobulin loci occur in defined regions, those in the partner genes vary; therefore, it is unlikely that 2 independent clones would share identical breakpoints in both partners. Establishing whether a new lesion in a patient with history of lymphoma represents recurrence or a new process can be relevant. Polymerase chain reaction (PCR)–based clonality assays used in this setting rely only on evaluating the length of a given rearrangement. In contrast, next-generation sequencing (NGS) provides the exact translocation breakpoint at single-base resolution. Objective.— To determine if translocation breakpoint coordinates can serve as a molecular fingerprint unique to a distinct clonal population. Design.— Thirty-eight follicular lymphoma/diffuse large B-cell lymphoma samples collected from different anatomic sites and/or at different time points from 18 patients were analyzed by NGS. For comparison, PCR-based B-cell clonality and fluorescence in situ hybridization studies were performed on a subset of cases. Results.— IGH-BCL2 rearrangements were detected in all samples. The breakpoint coordinates on derivative chromosome(s) were identical in all samples from a given patient, but distinct between samples derived from different patients. Additionally, 5 patients carried a second rearrangement also with conserved breakpoint coordinates in the follow-up sample(s). Conclusions.— Breakpoint coordinates in the immunoglobulin and partner genes can be used to establish clonal relatedness of anatomically/temporally distinct lesions. Additionally, an NGS-based approach has the potential to detect secondary translocations that may have prognostic and therapeutic significance.

Abstract The t(11;14)(q13;q32) chromosomal translocation, which is the hallmark of mantle cell lymphoma (MCL), is found in approximately 30% of multiple myeloma (MM) tumors with a 14q32 translocation. Although the overexpression of cyclin D1 has been found to be correlated with MM cell lines carrying the t(11;14), rearrangements of theBCL-1/cyclin D1 regions frequently involved in MCL rarely occur in MM cell lines or primary tumors. To test whether specific 11q13 breakpoint clusters may occur in MM, we investigated a representative panel of primary tumors by means of Southern blot analysis using probes derived from MM-associated 11q13 breakpoints. To this end, we first cloned the breakpoints and respective germ-line regions from a primary tumor and the U266 cell line, as well as the germ-line region from the KMS-12 cell line. DNA from 50 primary tumors was tested using a large panel of probes, but a rearrangement was detected in only one case using the KMS-12 breakpoint probe. Our results confirm previous findings that the 11q13 breakpoints in MM are scattered throughout the 11q13 region encompassing the cyclinD1 gene, thus suggesting the absence of 11q13 breakpoint clusters in MM.

Schizophrenia is the most severe of all the psychoses, affecting approximately 1% of the world's population. Evidence accumulated from numerous family, twin and adoption studies has firmly established a significant genetic component. The densely spaced markers now available throughout much of the human genome have facilitated whole genome positional cloning efforts. However the presence of a cytogenetic rearrangement associated with the disorder, such as the balanced t(1;11) translocation described here, provides an extremely valuable research tool. Generation and mapping of markers in the region of this translocation led to the construction of a 3Mb yeast artificial chromosome (YAC) contig across the chromosome 11 breakpoint. This thesis begins with the isolation of YAC end clones by 'splinkerette PCR', an improved alternative to vectorette PCR, which facilitated the contig construction. A search for foetal brain-expressed genes from this YAC was then undertaken, using two complementary methods of cDNA selection, ('hybrid fishing' and 'end ligation' coincidence sequence cloning). A thorough analysis of the product library and confirmatory hybridisation to the YAC and cDNA led to the identification of an additional member of the α-tubulin gene family, and several novel gene fragments, comprising at least two genes. Extensive sequence and expression analysis of the α-tubulin gene suggest that it is a processed psuedogene, although its potential as a candidate gene for psychiatric illness in this family cannot be discounted. The novel gene fragments have been mapped relative to each other and to rare cutter restriction sites in cloned genomic DNA, allowing one group of four fragments to be tentatively assigned to a single CpG island-associated gene. These can now be tested as genetic susceptibility factors in schizophrenia.

Haematological cancers like leukemia and lymphoma are characterized by genetic abnormalities, specifically chromosomal translocations. Analyses of the translocation breakpoint regions in patients have shown that some loci in the genome are more susceptible to breakage than others. However, very little is known about the mechanism of generation of many such chromosomal translocations. In the present study, we have attempted to understand the mechanism of fragility of three regions, which are prone to breaks during translocations in follicular lymphoma (FL) and T-cell leukemia. The t(14;18) translocation in FL is one of the most common chromosomal translocations. Most breaks on chromosome 18 are located at the 3’ UTR of the BCL2 gene and are broadly classified into three clusters, namely major breakpoint region (mbr), minor breakpoint cluster region (mcr) and the intermediate cluster region (icr). The RAG complex has been shown to cleave BCL2 mbr by recognizing an altered DNA structure. In the present study, by using a gel based assay, nature of the non-B DNA structure at BCL2 mbr was identified as parallel intramolecular G-quadruplex. Various studies including circular dichroism (CD), mutagenesis, DMS modification assay and 1H NMR showed the presence of three guanine tetrads in the structure. Further, evidence was also found for the formation of such a G-quadruplex structure within mammalian cells. In an effort to characterize the mechanism of fragility of mcr, a unique pattern of RAG cleavage was observed in a sequence dependent manner. Three independent nicks of equal efficiency were generated by RAGs at the cryptic sequence, “CCACCTCT”, at mcr and at a cytosine upstream of it, unlike a single specific nick at the 5’ of heptamer during V(D)J rearrangement. Interestingly, RAG nicking at mcr occured in the presence of both Mg2+ and Mn2+. Using recombination assay, followed by sequencing of the junctions, we find that mcr can recombine with standard RSS in vivo, albeit at a very low frequency. Mutations to this novel motif abolish recombination at the mcr within the cells. In order to determine the prevalence of t(14;18) translocation in the healthy Indian population, nested PCR approach followed by Southern hybridization was used. Results showed 34% prevalence of t(14;18) translocation in the Indian population. Although, no gender based difference was observed, an age dependent increase was found in adults. Further, presence of the t(14;18) transcripts was also detected. The mechanism underlying the fragility of the t(10;14) translocation involving HOX11 gene in T-cell leukemia is not known. Using primer extension assays on a plasmid DNA containing HOX11 breakpoint region, presence of consistent pause sites corresponding to two G-quadruplex forming regions, flanking the patient breakpoints, were detected. These replication blocks were dependent on K+ ions. Native gel shift assays, mutation analysis, S1 nuclease and CD, further revealed formation of intermolecular G-quadruplexes, unlike the BCL2 mbr. Further, sodium bisulfite modification assay indicated the presence of such structures in the genomic DNA within cells. Hence, we propose that two independent G-quadruplex structures formed in the HOX11 gene could interact with each other, thereby resulting in fragility of the intervening sequences, where majority of the patient breakpoints are mapped. Overall, this study has attempted to understand the role of both sequence and structure of DNA, in generating chromosomal fragility during t(14;18) translocation in FL and t(10;14) translocation in T-cell leukemia. These results may facilitate future studies in unraveling the mechanism leading to genomic instability in other lymphoid cancers.

Haematological cancers like leukemia and lymphoma are characterized by genetic abnormalities, specifically chromosomal translocations. Analyses of the translocation breakpoint regions in patients have shown that some loci in the genome are more susceptible to breakage than others. However, very little is known about the mechanism of generation of many such chromosomal translocations. In the present study, we have attempted to understand the mechanism of fragility of three regions, which are prone to breaks during translocations in follicular lymphoma (FL) and T-cell leukemia. The t(14;18) translocation in FL is one of the most common chromosomal translocations. Most breaks on chromosome 18 are located at the 3’ UTR of the BCL2 gene and are broadly classified into three clusters, namely major breakpoint region (mbr), minor breakpoint cluster region (mcr) and the intermediate cluster region (icr). The RAG complex has been shown to cleave BCL2 mbr by recognizing an altered DNA structure. In the present study, by using a gel based assay, nature of the non-B DNA structure at BCL2 mbr was identified as parallel intramolecular G-quadruplex. Various studies including circular dichroism (CD), mutagenesis, DMS modification assay and 1H NMR showed the presence of three guanine tetrads in the structure. Further, evidence was also found for the formation of such a G-quadruplex structure within mammalian cells. In an effort to characterize the mechanism of fragility of mcr, a unique pattern of RAG cleavage was observed in a sequence dependent manner. Three independent nicks of equal efficiency were generated by RAGs at the cryptic sequence, “CCACCTCT”, at mcr and at a cytosine upstream of it, unlike a single specific nick at the 5’ of heptamer during V(D)J rearrangement. Interestingly, RAG nicking at mcr occured in the presence of both Mg2+ and Mn2+. Using recombination assay, followed by sequencing of the junctions, we find that mcr can recombine with standard RSS in vivo, albeit at a very low frequency. Mutations to this novel motif abolish recombination at the mcr within the cells. In order to determine the prevalence of t(14;18) translocation in the healthy Indian population, nested PCR approach followed by Southern hybridization was used. Results showed 34% prevalence of t(14;18) translocation in the Indian population. Although, no gender based difference was observed, an age dependent increase was found in adults. Further, presence of the t(14;18) transcripts was also detected. The mechanism underlying the fragility of the t(10;14) translocation involving HOX11 gene in T-cell leukemia is not known. Using primer extension assays on a plasmid DNA containing HOX11 breakpoint region, presence of consistent pause sites corresponding to two G-quadruplex forming regions, flanking the patient breakpoints, were detected. These replication blocks were dependent on K+ ions. Native gel shift assays, mutation analysis, S1 nuclease and CD, further revealed formation of intermolecular G-quadruplexes, unlike the BCL2 mbr. Further, sodium bisulfite modification assay indicated the presence of such structures in the genomic DNA within cells. Hence, we propose that two independent G-quadruplex structures formed in the HOX11 gene could interact with each other, thereby resulting in fragility of the intervening sequences, where majority of the patient breakpoints are mapped. Overall, this study has attempted to understand the role of both sequence and structure of DNA, in generating chromosomal fragility during t(14;18) translocation in FL and t(10;14) translocation in T-cell leukemia. These results may facilitate future studies in unraveling the mechanism leading to genomic instability in other lymphoid cancers.

Genome integrity is essential for normal cellular functions. Erroneous repair of DNA double-strand breaks (DSBs) could lead to chromosomal rearrangements, including chromosomal translocations that may result in altered protein expression or chimeric proteins. Cell-type specific translocations, as observed in human lymphomas, are important for oncogenic progression. One such instance is of t(14;18) translocation involving BCL2 (chromosome 18) and IgH (chromosome 14) loci, that causes follicular lymphoma (FL). In another illustrious example, diverse translocations involving a common partner, BCL6 (chromosome 3), have been reported in diffuse large B cell lymphoma (DLBL). In the present study, impact of deviations in DNA structure in facilitating chromosomal fragility was investigated. In silico, in vitro and ex vivo approaches were employed to study the translocation breakpoint regions. Ex vivo assays were conducted to evaluate the effect of non-B DNA structures on physiological process such as transcription. Patient breakpoint analyses for BCL2 and BCL6 genes revealed breakpoint clusters in both the translocation cases. Bioinformatic studies suggest formation of G-quadruplex at BCL6 cluster III, while the nature of structure was unclear in case of BCL2 MBR. Gel shift assays showed faster mobility in case of both the translocation breakpoint sequences under study when compared to corresponding control sequences. Circular dichroism studies showed a characteristic G-quadruplex spectrum for BCL6. However, for BCL2, an uncharacteristic spectrum with a positive plateau pattern was observed, which was previously reported in literature for cruciform-like structures. Sodium bisulfite modification assay revealed single-strandedness at the fragile region when plasmid with BCL6 breakpoint was probed. These sequences had not been previously predicted by bioinformatic analysis; subsequent study of these revealed GNG motifs that aided in G4-structure formation. Additionally, gel shift assays, circular dichroism studies, and dimethyl sulphate probing supported the occurrence of G4-structure in these BCL6 sequences. However, no such protection at guanines was observed at BCL2, ruling out formation of G-quadruplex or triplex at this region. Further, it was observed that BCL2 peak III and BCL6 breakpoint region can block replication, unlike respective mutants, which allowed replication to proceed unhindered. Extrachromosomal episomal assay in pre-B cells, Nalm6 and Reh showed that BCL2 sequence can halt transcription. Similarly, BCL6 sequence could also halt transcription in the extrachromosomal episomal assay. Thus, our results suggest formation of G-quadruplex structures at BCL6 breakpoint region and a potential cruciform DNA structure at BCL2 MBR, with the capacity to affect replication and transcription. Furthermore, it is speculated that formation of such structures could impart fragility to breakpoint regions and eventually, lead to translocations, which needs to be investigated further. Taking into account the impact of formation of non-B DNA structures on cellular processes, several techniques have been developed to help study non-B DNA structures in vitro. However, detecting their intracellular presence has been challenging. Potential solutions include developing small molecule probes and antibodies against these forms and along these lines; Recently, an antibody, BG4 was designed to study G4 structures in cells. In present study, BG4 antibody was further characterized by biochemical and biophysical methods. BG4 showed specific binding towards G-rich DNA derived from multiple genes, which formed G4-DNA, unlike its complementary C-rich sequence or oligomer containing random sequence. Competition assays further supported the antibody specificity. Further, BG4 bound the inter- and intra-molecular G4-DNA, of parallel orientation, in single-stranded DNA. Interestingly, gel shift assays for multiple sequences indicated that mere presence of G4-DNA motif on duplex DNA was insufficient for antibody binding. BioLayer Interferometry (BLI) revealed high affinity (Kd 17.4 nM) with a known G4 substrate. Dimethyl sulphate probing of BG4-bound G4 substrate indicated higher protection of guanine residues that were specifically involved in G4-formation. Importantly, BG4 bound the G4-DNA in telomere region within supercoiled DNA context, as demonstrated by mobility shift of supercoiled plasmid, unlike in control plasmid bearing random sequence. BG4 binding within cells indicated efficient foci formation in four cell lines tested, thus demonstrating presence of G4-DNA in cellular context. Importantly, number of BG4 foci, indicative of G4 structures, were shown to be modulated upon shRNA mediated knockdown of a G4-resolvase, WRN, and upon inducing nutrient deprivation in cells by serum starvation. It would be of interest to investigate how this antibody and others could be utilized to study influence of different cellular conditions on extent of formation of non-B DNA structures. Taken together, our studies of the translocation breakpoint regions in BCL6 and BCL2 suggest a general mechanism associated with many common translocations seen in lymphoid cancers. Herein, formation of non-B DNA structures would generate partially single-stranded regions in the genome, which could render them susceptible to single-strand specific DNA nucleases or enzymes, thus promoting breakage and eventually, chromosomal translocations. In the other part of the study, further characterization has been conducted for an antibody known to detect one of such non-B DNA structures, G4-DNA, in in vitro and under two modified cellular conditions. Antibodies of such nature could be further utilized for determining G4 existence in certain genomic areas of interest, as probable diagnostic tools, and more ambitiously, as therapeutic agents.