The chromosomal translocation t(8;14) is the hallmark of Burkitt's-lymphoma (BL) and fuses the proto-oncogene c-MYC to the IGH locus. We analyzed the genomic structure of MYC/IGH fusions derived from a large series of 78 patients with t(8;14) and asked (i) whether distinct breakpoint clusters exist within the MYC gene and (ii) whether any pairwise association between particular IGH and MYC breakpoints exist. Identification of such associations will help elucidate the etiology of the breaks on the MYC locus. Scan statistic analyses revealed two distinct, but large clusters within c-MYC containing 60/78 (77%) of the breakpoints. Clusters 1 and 2 were 560 and 779 bp in length within a 4555 bp breakpoint cluster region. Breaks within IGH switch μ and joining region did not differ with respect to their corresponding MYC breakpoints. However, there was a highly significant correlation between breakpoints 5′ of MYC cluster 1 and fusions to IGH switch γ region and breakpoints downstream of MYC cluster 2 and fusions to IGH switch α region (χ2-test: P<0.005). Chromatin changes governing choice of IGH-Fc region recombination may parallel changes in the MYC gene 5′ region chromatin leading to some degree of coordinated ontological specificity in breakpoint location.
B-cell development takes place in distinct stages as directed in part via signals transmitted through, and structural modifications of, the B-cell receptor when directed by an immune response. Early stages of B-cell development occur in the bone marrow including the recombination of the immunoglobulin (IG) genes via the recombination-activating gene (RAG 1/2) endonuclease complex. B-cells that express a functional B-cell receptor differentiate into mature naive B-cells and leave the bone marrow to undergo clonal expansion in the germinal centers (GC). The IG genes are further modified by both somatic hypermutation (SHM) and class-switch recombination (CSR) resulting in antigen-activated B cells.1 The molecular processes that remodel Ig genes involve distinct mechanisms of DNA double-strand breaks and DNA repair. For V(D)J recombination RAGs recognize and bind to site-specific highly defined recombination sequence signals (RSSs).2, 3 In contrast, the DNA cleavage during CSR and SHM is not sequence-specific and is initiated over large target regions via the activation-induced deaminase (AID) endonuclease. Mistakes during these DNA cleavage processes may lead to chromosomal translocations involving the Ig loci and a proto-oncogene. The proto-oncogene comes under the control of the active Ig locus creating a deregulated, constitutive expression of the oncogene and plays an important role in the pathogenesis of B-cell malignancies.4, 5, 6
Three different models have emerged to explain how RAG proteins mediate chromosomal translocations: substrate-selection errors (also called the RAG misrecognition model), the end-donation model and the transposition model (reviewed in detail in Roth D B7). With respect to the RAG-misrecognition model, the presence of RSS-like sequences (pseudo-RSS) near chromosomal breakpoints leaves little doubt that the DNA break within the antigen-receptor containing chromosome is mediated via RAG, but the cause of the DNA break within the proto-oncogene has been unclear.8 In most of the cloned chromosomal breakpoints the identified pseudo-RSS are not located proximal to the breakpoint and deviate so far from the consensus-RSS that the involvement of RAG seems unlikely.4, 7, 9, 10 Thus, until recently it remained a largely unresolved question of whether the pseudo-RSS occur merely coincidental or really indicate the involvement of the V(D)J recombinase.11, 12
In follicular lymphoma (FL) the BCL-2/IGH fusion is the molecular equivalent of the translocation t(14;18) and demonstrates clear involvement of RAG in the IGH-JH breakpoints. Recently, work of the Lieber laboratory shed new light on the question how the RAG complex cleaves the BCL-2 proto-oncogene at its well-defined major breakpoint cluster region (Mbr) located in the untranslated portion of its third exon.13, 14, 15, 16 BCL-2 breakpoints are dependent upon the formation of stable non-B DNA secondary structure, which is cleaved by RAG activity in the absence of RSS. Therefore, the t(14;18)-associated BCL-2/IGH fusion is presumably mediated by the RAG complex only. In summary, the RAG complex is capable of acting as a nuclease based on sequence or structural specificity. Notably, the occurrence of other non-B form DNA structures (loops, cruciforms, left-handed Z-helices, triplexes or tetraplexes) in the human genome coincide with genomic rearrangements, for example, gross deletions or constitutional translocations.17 Whether these genomic instabilities are generated by RAG or perhaps other nucleases that are attracted by the unusual DNA structures is unknown.
In contrast to the recently characterized RAG-mediated DNA cleavage errors, mistakes during AID-mediated CSR are less well understood, but tight regulatory control of AID is also necessary to prevent generalized genomic mutations and genomic instability.1, 18, 19
Sporadic Burkitts lymphoma (sBL) carry the translocation t(8;14).20, 21 Breakpoints on chromosome 14 occur either within the IGH switch regions (Sα, Sγ and Sμ) or in the joining region (JH). Thus, in contrast to FL, in sBL the cleavage sites of IGH suggest involvement of either RAG (JH breakpoints) or AID (switch region breakpoints), depending on the individual tumor.2, 18 Whether AID or RAG also generate the DSB in MYC is unknown. If these nucleases induce the DSB in MYC, one might speculate that various breakpoint clusters within the MYC gene may also exist depending on the action of either RAG or AID. To investigate this, we analyzed a large series of sBL with MYC/IGH fusion and asked two questions: (1) Do one or more breakpoint clusters exist within the MYC gene?, (2) Do the putative MYC breakpoint cluster(s) associate with particular switch or the JH breakpoint regions? Such a putative association between the different IGH regions and the MYC breakpoint clusters may give correlative evidence for the distinct activities of either AID or RAG for generation of the breakpoints within MYC.
Materials and methods
Informed consent from the guardians for each patient was obtained. Tumor biopsy material, bone marrow, lymph nodes or ascites from 76 pediatric patients suffering from sporadic Burkitt's lymphoma/B-cell leukemia with translocation t(8;14)(q24;q32) were collected in the framework of the Berlin-Frankfurt-Muenster (BFM) study group. We analyzed two additional patients with translocation t(8;14)(q24;q32) who were not enrolled in our BFM study. From October 1996 until February 2003 1261 newly diagnosed Non-Hodgkin-Lymphomas were enrolled. Among them, 528 children suffered from Burkitts-Lymphoma (BL) or B-ALL, respectively. The selection of cases was solely based on the availability of high-molecular weight DNA. Whenever viable tumor cells were available, standard karyotyping was performed. Patients with endemic BL were excluded from this study. The procedure for isolation and storage of the tumor cells and mononuclear cells from bone marrow we described recently.22
DNA preparation, detection of der(14) MYC/IGH and der(8) IGH/MYC breakpoints
High-molecular-weight genomic DNA was prepared from frozen tumor cells and mononuclear cells by washing them twice with PBS before using the QIAamp DNA or DNA Blood Mini Kit (Qiagen, Hilden, Germany) following the instructions provided by the manufacturer.
The breakpoint region involving the MYC gene on chromosome 8q24 and the IGH locus on chromosome 14q32 were determined by long-distance (LD)-PCR using the Expand Long Template PCR System (Roche, Mannheim, Germany).23 To detect the rearrangement involving the c-myc gene on chromosome 8q24 and the IgH locus on chromosome 14q32, one primer for the c-myc gene (c-myc/M6 at position 4885 in exon 2) and four primers for the IgH locus were combined: three primers for the constant region (Cμ03, Cγ02, Cα01) and one for the JH. Primers for both genes represent the antisense strands in reverse direction, due to the head-to-head orientation of c-myc and IgH genes. Primer sequences for isolation of the der(14) MYC/IGH fusions are indicated in Supplementary Table 1.
The quality of the genomic DNA and adequacy of sample for the amplification of long DNA fragments were tested in each sample by using c-myc/M6 and c-myc/M9 primers together with an upstream primer c-myc up (at position 1), which yielded a PCR product of 4.9 and 8.2 kbp, respectively.
Each reaction mixture (50 μl) contained 250 ng of genomic DNA, 300 nM of downstream and upstream primer, 500 μ M of each dNTP, buffer III with 22.5 nM MgCl2 and 2.6 units of a polymerase mix as indicated in the Expand Long Template PCR System Kit (Roche, Mannheim, Germany). Reaction conditions were as follows: denaturation at 94°C for 2 min followed by 10 cycles of denaturation at 94°C for 30 s, annealing at 68°C for 30 s, extension at 68°C for 4 min, followed by 20 identical cycles with gradual increment of extension time (15 s/cycle) and a final extension for 10 min at 68°C. PCR were performed in a Thermal Cycler 9600 (Applied Biosystems, Darmstadt, Germany). For sequence analysis the LD-PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Sequence analysis of the PCR products was performed with MYC-specific primers (c-myc1 to c-myc16, Supplementary Table 2). The sequences thus obtained were analyzed by BLAST homology search (www.ncbi.nlm.nih.gov/BLAST/) and the junctional nucleotide sequences from each specific MYC/IGH fusion were deposited into the EMBL Nucleotide Sequence Databank (www.ebi.ac.uk/submission/webin.html). The assigned accession numbers are listed in Table 1 according to the MYC/IGH breakpoint. Similarly, the reciprocal der(8) IGH/MYC fusions were amplified and sequenced with the help of PCR/sequencing primers shown in Supplementary Table 3.
Statistical significance and the number of clusters of translocation breakpoints within the MYC gene (bp 1–4555) was evaluated by scan statistics and gap statistics as described in Segal G H24 with 999 runs of permutations using the software-package SaTScan V.3. 025 and custom programs implemented in the free statistical environment R (www.r-project.org). The IGH-loci-specific distributions of MYC-breakpoints were compared using the Kruskal–Wallis test or Mann–Whitney U-test (pairwise comparison). Associations of MYC-clusters with IGH-regions were investigated via contingency table analysis using χ2-tests.
Screening for human repetitive elements
A direct search for several known signals including the repetitive elements (SINEs, LINEs, LTRs, transposon fossils), V(D)J-RSS motifs, topoisomerase II binding site, eukaryotes replication origin sequences, translin binding site, χ-like sequences, scaffold/matrix attachment region, pyrimidine trait (Y12), putative triple helices and the purl-binding site was performed to analyze the sequence surrounding the identified MYC clusters. Matches to V(D)J RSS and Topoisomerase II binding site were scored using position-specific scoring matrices calculated based on the log odds ratio of known motif nucleotide frequency distribution to the uniform background. For the other motifs with limited number of known exemplar sequences, regular expression matches were done. Human repetitive elements were scanned by RepeatMasker (Smit AFA and Green P, 1996, unpublished results. RepeatMasker at http://ftp.genome.washington.edu/RM/RepeatMasker.html). In addition, an indirect search was performed using de novo statistical motif finders Gibbs Motif Sampler (GMS)26 and MEME27 to look for enriched motif in the proximity, (−100, +100 bp), of breakpoints. Found motifs from 100 runs GMS were then aggregated into a consensus motif.
Pattern of the 78 MYC/IGH breakpoints
Using the LD-PCR we determined the MYC/IGH rearrangement from 78 pediatric sBL patients enrolled in the trial NHL-BFM 95 (Table 1). The pattern of the breakpoint locations within the MYC gene and the IGH regions is shown in Figure 1. The breakpoint locations within the MYC gene were evenly distributed, 5′ of exon 1 (37%), in exon 1 (31%) and in intron 1 (32%). The breakpoint locations within the IGH regions revealed 88% of breakpoints within the switch α (35%), switch γ (23%) and switch μ (29%) region, whereas 13% of breakpoints were identified in the joining region (Figure 1).
MYC breakpoint cluster analysis
Based on the structure of MYC, the allowable range of DNA nucleotides was 4555 bp, to just before the second exon, which contains the first translated codon. The scan statistic identified two distinct clusters, one from 1892 to 2452 and the other from nucleotides 2717 to 3496, containing 60 of 78 (77%) assessed breakpoints (P=0.004). The remaining 18 (located between the nucleotides 381 and 4415 of MYC gene) did not partition into defined clusters (Figure 2 and Table 2).
MYC breakpoint clusters associated with various breakpoints of IGH
The IGH-specific distributions of the MYC breakpoints are graphed in Figure 3 and are shown to be significantly different (Kruskal–Wallis test, P=0.0007). Importantly, the pairwise comparison revealed no differences between the IGH switch μ and the JH region with respect to their corresponding MYC breakpoints. Therefore, these two IGH regions were combined to determine an association between breakpoints within the MYC clusters and the IGH areas using cross tabulation. A clear relationship could be demonstrated for IGH regions Sα and Sγ and the clusters 1 and 2 (P=0.002, contingency table). Whereas breakpoints 5′ of cluster 1 were found to be over-represented with a link to IGH region switch γ (P=0.0013), MYC breakpoints 3′ of cluster 2 are strongly related to the IGH loci switch α (P=0.0025). Moreover, MYC breakpoints within the two clusters and in between were significantly associated to the combined IGH loci switch μ and JH (P=0.001). Thus, the breakpoints within cluster 1 and with smaller MYC nucleotide numbers are related to IGH region Sγ, whereas breakpoints within cluster 2 and with higher MYC nucleotide numbers are related to IGH region Sα. (Table 2 and Figure 4).
Searching for breakpoint-associated sequence motifs
In the search for known recombination-facilitating motifs (Supplementary Table 4), a polypyramidine tract was repeatedly found near translocation breakpoints on MYC. Such pyrimidine tracts are known to facilitate integration of simian virus (SV 40).28 Furthermore, an undirected motif search using de novo motif finding algorithms (Gibbs motif sampler and MEME) provided additional evidence for a pyrimidine tract motif, or its reverse complement, a GAGA motif. Figure 5 depicts the sequence logo of the composite motif (and its reverse complement) from 100 runs of Gibbs Motif Sampler. MEME runs yield qualitative similar sequence motifs (data not shown). A poly-pyrimidine motif of this sort is known to form paranemic (base unpaired) structure, which is known to form Z-DNA (left handed helix) and other structure in supercoiled DNA as occurs during transcription (Figure 5).
Determination of der(8) reciprocal IGH/MYC breakpoints in a subset of patients
To study the exact configuration the reciprocal breakpoints on der(8) we isolated the IGH/MYC fusion site in a subset of patients. The cases were selected on the availability of DNA and quality of the reciprocal PCR products obtained.
We wanted to know whether the breakpoints are perfectly reciprocal or whether deletions or insertions of nucleotides can be found. The sequences derived from 17 patients revealed four types of translocations: (i) perfectly reciprocal at least for MYC (two cases), (ii) with loss of material in both fusion partners (four cases), (iii) with gain of material derived either from IGH or MYC (four cases) or (iv) with loss of material of IGH and/or MYC and gain of material of unknown origin (seven cases) (Figure 6). These sequences without any homology to known human sequences seem to be incorporated during the translocation process by chance, since they are not derived from inverted duplications of MYC or IGH regions. Possibly the nucleotides incorporated at these sites originate from the nucleolytic processing of broken DNA ends during the NHEJ process or can be attributed to the action of pol μ or pol λ, both involved in the filling of small gaps before DNA end joining (reviewed in Hefferin M L29). The different types of translocation do not seem to be associated with one of the MYC breakpoint clusters identified in this study. In addition to the accession numbers of either MYC/IGH or IGH/MYC breakpoints the flanking sequences of all breakpoints are provided in Supplementary Table 5.
Primary involvement of the central nervous system or bone marrow was diagnosed in 10 of our 37 patients, respectively. An abdominal mass was seen in 58 of our patients. Of the 78 BL patients 18 patients suffered a recurrence of disease and three patients perished from complications of chemotherapy (Table 1). Among the 18 patients with a relapse (17 out of 76 from the BFM study group), the IGH breakpoint was evenly located in the switch α (five patients), switch γ (six patients) and switch μ (six patients), whereas from the 10 patients with a break in the JH region, only one patient relapsed (Table 1). We next asked whether the der14 MYC/JGH breakpoint configuration is associated with specific clinical parameters. For this purpose, we categorized the patients into three different MYC/JGH breakpoint groups so that at least 15 patients belong to each category (Table 3). No statistically significant findings emerged from these analysis, neither staging according to Murphy30 nor lactate dehydrogenase (LDH) levels before therapy were associated with one of the defined der14 MYC/IGH categories.
Notably, out of the four patients with the rare der14 breakpoint configuration (5′ to MYC cluster 1 and IGH switch γ) three relapsed. In contrast, all five patients survived who had the der14 breakpoint configuration 3′ to MYC cluster 2 and IGH switch α. For the two patients who were not enrolled in the BFM study only sparse clinical information was available.
As part of the maturation process, B cells enter the GC and become susceptible to enzymatic activities that alter the genome by hypermutation, Ig class switching and secondary V(D)J rearrangements. The GC is a crucible of somatic genetic change and hence a plausible site for oncogenetic errors.4, 9, 31, 32 In accordance with other studies, we found that most breakpoints within the IGH region in sporadic BLs with t(8;14)/MYC-IGH translocations map to the switch regions and only a minority to the JH locus (Figure 1 and Supplementary Table 6). We demonstrate evidence of two clusters within the MYC breakpoint region (Figure 2). Given that different nucleases, RAG or AID, are responsible for the various IGH cleavage sites, the fact that the JH breakpoints and switch μ breakpoints did not differ with respect to their corresponding MYC breakpoint regions is unexpected and may suggest that a third mechanism is responsible for DNA cleavage in MYC; or that RAG and AID may cause cleavage in MYC breakpoint regions equivalently, possibly due to structural attributes of the MYC promoter region (see below).
Duquette,19 recently demonstrated that AID targets to G-loops, co-transcriptional RNA: DNA hybrids on the C-rich strand and single-stranded regions and G4 DNA on the G-rich strand.19 Similar distributions of RAG induced JH and AID induced switch μ breakpoints does not necessarily argue against a prominent role of these G-loops. The clusters on MYC were quite wide and diffuse (560 and 779 bp in a 4555 bp region) compared to three sharply circumscribed peaks (10–15 bp in a region of 150 bp) described previously within Mbr of BCL-2 and attributed to RAG activity.15, 16 This diffuse nature of MYC clusters additionally suggests that the mechanism for DNA cleavage is not restricted to a site-specific mechanism such as RAG-mediated DSB.
Interestingly, the breakpoints within the MYC clusters are significantly associated with breaks in the various IGH regions. Whereas breakpoints 5′ of MYC cluster 1 were preferentially paired with breaks in the IGH region switch γ (P<0.01), breakpoints 3′ of MYC cluster 2 were associated with the most 3′ located IGH loci, switch α (P<0.02). MYC breakpoints within the two clusters were significantly associated to the combined IGH loci switch μ and JH (P<0.001) (Figures 3 and 4). The significant association between particular IGH switch regions and different MYC breakpoint regions also may suggest that another cleavage activity exists for the MYC locus. Could such an association between the MYC and IGH switch breakpoints be explained by the action of AID-recombinase exclusively? Isotype-specific AID co-factors that target AID to their specific switch regions may clearly contribute to the MYC breakpoint pattern. For the physiological CSR process, there must be factors that explain isotype-specific switching activities and thus provide an accessory layer of specificity for the CSR reaction, potentially at the level of chromatin organization. Transcription is absolutely required for AID-mediated CSR and AID deaminates only single-stranded DNA. Thus, the MYC gene may be transcribed at a different rate leading to distinct DNA or chromatin structure in an isotype-specific pattern, leading to isotype-specific open regions of MYC chromatin. Notably the clusters in MYC represent the coverage of 2–4 nucleosomes. Therefore, an obvious explanation for our data is that the distinctive collections of transcription factors in B cells switching to γ or α modulates transcription of different regions of MYC. There is also some other specificity to CSR switching activities.33, 34 B-cells that lack the trans-activation domain of the c-Rel are able to carry out only μ to α but not μ to ɛ CSR.35 Plasmid switching assays also revealed the existence of different switching activities that mediate μ to γ3, μ to α, μ to γ1 and μ to ɛ.34, 36, 37 However, all these isotype related switching activities seem to act very specifically whereas the MYC/IGH breakpoint pattern found in our study is only an association, albeit highly statistically significant.
There are also significant differences between CSR and V(D)J recombination in terms of repairing DNA DSB. For instance, 53BP1 is recruited to sites of DNA damage and is fully dispensable for V(D)J recombination, but required for CSR.38 Similarly, in the absence of the damage-response protein H2AX, CSR is very much impaired but the V(D)J recombination is not.39, 40, 41 However, again, these functional differences between the CSR and the V(D)J recombination cannot be recapitulated in our correlative study of the MYC/IGH breakpoints. The presence of polypyrimidine tracts in correspondence with the MYC clusters further indicates the fragility of the locus. This sequence motif can form slippage structures with extruded single-strand loops, and triple helices (H-DNA).42, 43 Secondary structural features of DNA are known to attract activity of the nuclease activity of RAG and AID and may be involved in other nucleases such as topoisomerase II.44, 45 We did not find any significant matches to the V(D)J RSS motif; however, this does not completely rule out its potential role.
An analysis of DNA motifs (Supplementary Table 4) did not reveal any significant associations between known motifs and breakpoints within the MYC gene. Using an ‘undirected’ motif search, we scanned for motifs that appeared more common in the near vicinity (+100 bp) and discovered the presence of the polypyrimidine track considered above, roughly a repeating ‘CT’ dinucleotide motif. Such features have been associated with non-B DNA structures particularly slippage with loop exclusion, and triplex;42 however, it should be noted that this feature was in proximity and not directly at the breakpoints. Stem-loop structures, similar in some respects to DNA loops that are formed in dinucleotide repeat slippage structures, are known to attract CSR (AID) which are relevant to the formation of MYC breaks described herein.46
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We are indebted to the technical staff of the Department of Pediatric Hematology and Oncology, Giessen, especially to Jutta Schieferstein and Franziska Müller for excellent technical assistance. We thank Martin Zimmermann (Department of General pediatrics, Medical School, Kiel, Germany) for statistical analysis of the clinical features. The study was funded in part by grants from the network of competence ‘Pediatric Oncology’ of the Bundesministerium für Bildung und Forschung (BMBF) No. 01GI9963 and the Forschungshilfe Station Peiper. AB and UF were supported by a Grant from the German Cancer Association, the Else-Kröner-Fresenius-Foundation and the Research Foundation of the University of Munich, Germany. JW is a Scholar of the Leukemia and Lymphoma Society of America.
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