Molecular characterization of the MLL-SEPT6 fusion gene in acute myeloid leukemia: identification of novel fusion transcripts and cloning of genomic breakpoint junctions

Abstract

One of the MLL fusion partners in leukemia is the SEPT6 gene, which belongs to the evolutionarily conserved family of genes of septins. In this work we aimed to characterize at both the RNA and DNA levels three acute myeloid leukemias with cytogenetic evidence of a rearrangement between 11q23 and Xq24. Molecular analysis led to the identification of several MLL-SEPT6 fusion transcripts in all cases, including a novel MLL-SEPT6 rearrangement (MLL exon 6 fused with SEPT6 exon 2). Genomic DNA breakpoints were found inside or near Alu or LINE repeats in the MLL breakpoint cluster region, whereas the breakpoint junctions in the SEPT6 intron 1 mapped to the vicinity of GC-rich low-complexity repeats, Alu repeats, and a topoisomerase II consensus cleavage site. These data suggest that a non-homologous end-joining repair mechanism may be involved in the generation of MLL-SEPT6 rearrangements in acute myeloid leukemia.

Introduction

Abnormalities of 11q23, resulting in fusion of the mixed lineage leukemia (MLL) gene with numerous translocation partners, are found in primary acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), as well as in secondary, topoisomerase II inhibitor–related leukemia.1 The MLL gene codes for a multi-domain protein that is a major regulator of class I homeobox (HOX) gene expression.2 HOX genes play a key role in the regulation of hematopoietic development and altered patterns of MLL activity might cause abnormal HOX gene expression in hematopoietic stem cells, resulting in blockage of hematopoietic maturation and, eventually, leukemia.3

To date, more than 50 MLL fusion partners have been cloned.4 Five of these, SEPT2, SEPT5, SEPT6, SEPT9, and SEPT11, code for septins4,5 and belong to an evolutionarily conserved family of genes with 13 members identified so far.6 As a consequence, the septins are the protein family most frequently involved in rearrangements with MLL, suggesting that their involvement in MLL-related leukemia is anything but a chance event.5,6 Septins are conserved GTP-binding proteins that assemble into homo- and hetero-oligomers and filaments with key roles in cell division cytoskeletal dynamics and secretion.6

To our knowledge, the fusion between MLL and SEPT6 has so far only been described in 10 AML patients.7–13 However, the genomic breakpoint junction was only characterized in 2 patients.9,10 We present a detailed RNA and DNA analysis in 3 new AML patients with the MLL-SEPT6 rearrangement, one of them showing a novel in-frame fusion transcript.

Design and Methods

The study comprised three cases of childhood AML in which Xq24 and 11q23 rearrangements were detected by karyotyping and/or molecular cytogenetic analyses of the leukemic cells. Molecular studies involved RT-PCR, LD-PCR, HN-PCR, sequencing, and bioinformatic analyses. For detailed information on patients, methods and results see Online Supplementary Appendix.

Results and Discussion

Karyotyping and molecular cytogenetics

The 3 AML patients showed cytogenetic evidence of a rearrangement involving the long arm of chromosome 11 (11q23), the first with additional material of unknown origin in 11q23 and Xp11 (patient 1), the second as a translocation between 11q23 and Xq24, and the third as an insertion of 11q13q23 into Xq24 (Table 1, Figure 1). FISH analysis on leukemic metaphases was performed, demonstrating in all cases a break in MLL (Figure 1) and the presence of MLL sequences in Xq (cytogenetically cryptic in case 1). The known localization of SEPT6 in Xq24 prompted further analysis with BACs mapped to this band in patients 2 and 3. In patient 2, the breakpoint was mapped to clone CTD-2334F19, suggesting a breakpoint in or near the 5′ region of the SEPT6 gene. In patient 3, the breakpoint was mapped to the overlapping region of the two BAC clones RP11-379J1 and CTD-2334F19, which suggested that the break occurred in the 5′ region of the SEPT6 gene. In patient 1, BAC analysis could not be performed due to lack of material. Rearrangements recombining 11q23 and Xq24 resulting in MLL-SEPT6 fusions are usually complex as a result of the opposite orientation of MLL and SEPT6 on the respective chromosome arms. At least four different types of chromosomal rearrangements have been described that can generate the MLL-SEPT6 in-frame fusion.7–10 The combined chromosome banding and molecular cytogenetic investigations of our 3 patients confirm that complex, sometimes cryptic, chromosome rearrangements are common in AML patients with MLL-SEPT6 rearrangements (Table 1).

Characterization of MLL-SEPT6 fusion transcripts

RT-PCR followed by sequencing analysis led to the identification of MLL-SEPT6 fusion transcripts in all 3 cases. In patient 1, two major PCR fragments of 719 bp and 605 bp were detected (Online Supplementary Figure 1A). Sequencing analysis revealed a fusion of MLL exon 7 and MLL exon 8 with SEPT6 exon 2. In addition, a minor band of 541 bp detected in this patient (Online Supplementary Figure S1A) was shown by sequencing analysis to correspond to an out-of-frame fusion between MLL exon 7 and SEPT6 exon 2 with splicing of 74 bp corresponding to MLL exon 6. RT-PCR analysis of patient 2 revealed one major band of 473 bp and a minor band of 399 bp (Online Supplementary Figure S1B). Sequencing analysis revealed the presence of two novel MLL-SEPT6 chimeric transcripts: an in-frame fusion between MLL exon 6 and SEPT6 exon 2 and an out-of-frame fusion of MLL exon 5 to SEPT6 exon 2 (Online Supplementary Figure S1D). The novel MLL-SEPT6 in-frame fusion variant is expected to give rise to a chimeric fusion protein, where the N terminus of MLL fused to almost the entire open reading frame of SEPT6, except for the first nine amino acids.

Figure 1.G-banding and FISH analyses of the leukemic cells of patients 2 and 3. (A) Partial karyotype of case 2, with arrows indicating breakpoints of rearranged chromosomes. (B) FISH and (C) inverted DAPI images of metaphase plate from case 2. The hybridization was performed using a locus-specific, break-apart probe for MLL (green and red signals) and the BAC clone CTD-2334F19 (blue signal). The blue signal has moved to the derivative chromosome 11, indicating that the breakpoint is in the 5′ of the SEPT6 gene. Arrows indicate derivative chromosomes, arrow heads are pointing to the normal chromosome 11. (D) Partial karyotype of case 3. (E) FISH and (F) inverted DAPI images of a metaphase plate from case 3. Hybridization was performed using the MLL probe and the clone CTD-2334F19. The BAC clone (blue colour) splits giving two signals on the rearranged X chromosome, indicating that the breakpoint is inside that clone.

In patient 3, RT-PCR analysis showed the presence of two PCR fragments of 605 bp and 541 bp (Online Supplementary Figure S1C). Sequencing analysis demonstrated fusions between MLL exon 7 and SEPT6 exon 2, with the smaller fragment showing, as in patient 1, an out-of-frame splicing of MLL exon 6. The 10 cases of MLL-SEPT6 rearrangement described so far showed fusions between SEPT6 exon 2 and MLL exon 7 (3 cases), MLL exon 8 (3 cases), both MLL exons 7 and 8 (3 cases), or MLL exon 9 (one case).8–13 The novel MLL-SEPT6 chimeric transcript we here describe between MLL exon 6 and SEPT6 exon 2 may be called type IV, after the three fusion types previously identified (Table 1).

SEPT6 belongs to an evolutionarily conserved family of genes that encode a P loop-based GTP-binding domain flanked by a polybasic domain and, in most cases, a coiled-coil-region.6 The SEPT6 protein possesses all the three domains and, as previously reported in cases of gene fusion involving MLL and other septins (MLL-SEPT2, MLL-SEPT5, MLL-SEPT9, and MLL-SEPT11), almost the entire open reading frame of SEPT6, containing all the three septin function-defining domains, is fused with the N-terminal moiety of MLL. A relevant role of septins in MLL-related leukemia, besides activation of the MLL protein by dimerization, is therefore a possibility that should not be ruled out.5,6 Additional support for this hypothesis comes from the observation that all 13 patients reported so far with MLL-SEPT6 rearrangement were children (age range; 0–29 months) with AML (the FAB-typed included one M1, five M2, four M4 and one M5; Table 1). Since the majority (65%) of pediatric patients with MLL rearrangements have ALL,4 we hypothesize that the SEPT6 domains of the MLL-SEPT6 chimeric protein contribute to myeloblastic leukemogenesis in children. In fact, the MLL fusion with the other septins (SEPT2, SEPT5, SEPT9, and SEPT11) is also preferentially associated with myeoloblastic rather than lymphoblastic leukemogenesis.4,5,14 In all 3 cases studied we observed the presence of out-of-frame alternative splicing variants, something that has not been previously reported7–13 and whose biological relevance is not clear.

Table 1.Clinical, karyotyping, FISH and RT-PCR data on all known acute myeloid leukemia-patients with MLL-SEPT6, and classification of the fusion variants.

Characterization of MLL-SEPT6 genomic breakpoints

The genomic breakpoints in all cases occurred in the MLL 8.3 kb breakpoint cluster region (BCR) and in SEPT6 intron 1 (Online Supplementary Figure 2). In patient 1, a total of six suggestive HN-PCR fragments were gel extracted and sequenced. Sequencing of the amplification products showed that the breakpoint was located 231 bp downstream of MLL exon 8 and 476 bp downstream of SEPT6 exon 1 (Online Supplementary Figure 2D). The HN-PCR study of case 2 revealed a total of 11 suggestive HN-PCR fragments that were gel extracted and sequenced. Sequencing analysis showed that the genomic breakpoints were located 629_634 bp downstream of MLL exon 6 and 14410_14415 bp upstream of SEPT6 exon 2 (Online Supplementary Figures 2B and 2E). In this case, the exact position of the genomic breakpoint could not be determined due to the presence of an identical 5-bp microhomology sequence (TGGGA) at the MLL-SEPT6 genomic junction. In patient 3, an LD-PCR fragment of 2179 bp was detected (Online Supplementary Figure 2C). Interestingly, partial direct sequencing of the amplification product revealed the same genomic breakpoint junction as detected in patient 1, with fusion of nucleotide 231 downstream of MLL exon 8 with nucleotide 476 downstream of SEPT6 exon 1 (Online Supplementary Figure 2D).

We searched for topoisomerase II consensus cleavage sites in the vicinity of the breakpoint regions in MLL introns 6 and 8 and SEPT6 intron 1. We found one sequence with 100% homology with the topoisomerase II consensus cleavage site located in SEPT6 intron 1 (GTTTTCCTGTTGTTGTTT), nucleotide position 9533_9550 bp downstream of SEPT6 exon 1. We also searched the breakpoint junctions (15 bp either side) for repetitive DNA sequence elements and motifs known to be associated with site specific recombination, cleavage, and gene rearrangement, but none could be found. Translocations may or may not involve gain or loss of genetic material at the genomic breakpoint junctions. Patients 1 and 3, as well as the two previously reported cases, showed no nucleotide(s) deletion or duplication at the breakpoint junction.9,10 In our patient 2, the exact position of the genomic breakpoint could not be determined due to an identical 5-bp microhomology region at the MLL-SEPT6 genomic junction, so it is unknown whether duplications and/or deletions occurred in this particular patient. The identification of identical microhomologies at genomic junctions suggests that the non-homologous DNA end-joining (NHEJ) pathway may be involved in this rearrangement.15 In patients 1 and 3, the genomic junction mapped near a 484 bp LINE1 repeat in MLL intron 8, whereas the genomic breakpoint in SEPT6 intron 1 mapped near two GC-rich low complexity repeats. In patient 2, the MLL intron 6 genomic breakpoint occurred inside a 298 bp Alu repeat, whereas the breakpoint junction in the SEPT6 intron 1 mapped near a 300 bp Alu repeat. Although repetitive sequences may occur near or spanning breakpoint junctions by chance, it is plausible that introns with a high density of repetitive sequences, such as SEPT6 intron 1, are vulnerable to breaking and non-homologous pairing that can lead to gene fusions such as MLL-SEPT6. Strikingly, although the breakpoints in the large SEPT6 intron 1 seem to be distributed all over the intronic region (our patient 2, and cases 6 and 11 in the literature; Table 1), patients 1 and 3 showed exactly the same genomic breakpoint both in MLL and SEPT6. Since the genomic breakpoint junctions in these 2 patients were cloned by different methods (HN-PCR and LD-PCR), on separate occasions, and taking the strictest anti-contamination measures, these findings can only be explained by the presence of a hot-spot for recombination at the said sites. Supporting this hypothesis, in addition to the above-mentioned high density of repetitive sequences in SEPT6 intron 1, is the detection of a topoisomerase II consensus site-specific cleavage in the same intron. Identical genomic breakpoints or breakpoint clustering within very narrow regions have been reported before, namely in MLL-AF4,16 MLL-AF9,17 and TCF3-PBX118 leukemias. Interestingly, the presence of an MLL intron 8 genomic breakpoint in patient 3 does not seem to translate into fusion transcripts that include MLL exon 8, since the only in-frame fusion transcript detected showed a fusion between MLL exon 7 and SEPT6 exon 2. Splicing of MLL exon 8, which includes the first of the four zinc fingers of the first zinc finger domain of the MLL gene, has been previously described in acute leukemia with MLL rearrangements, both in cases with translocation [(t(4;11), t(9;11), and t(11;19)]19,20 and tandem duplication,21 changing the structural and possibly the functional features of the first zinc finger region of the MLL protein.

The MLL genomic breakpoints in MLL-SEPT6 AML patients seem to occur preferentially in the telomeric half (between introns 7 and 11) of the MLL BCR. This is characteristic of infant AML (the MLL-SEPT6 cases with genomic breakpoint characterization have an age at diagnosis from 0 to 17 months) and topoisomerase II inhibitor-related secondary leukemia, and a putative association with in utero exposure to topoisomerase II inhibitors has been hypothesized.22 The detection of a topoisomerase II recognition sequence in SEPT6 intron 1 also supports the hypothesis that exposure to topoisomerase II inhibitors, can result in double-strand DNA breaks that trigger the error-prone non-homologous end-joining pathway, which in turn can lead to formation of the MLL-SEPT6 fusion oncogene.

Footnotes

• Funding: this research was supported by grants from the “Associação Portuguesa Contra a Leucemia” (2006-30.2.AP/MJ) and the “Comissão de Fomento da Investigação em Cuidados de Saúde” (P.I. 26/2007).
• The online version of this article contains a supplementary appendix.
• Authorship and Disclosures NC designed and performed the research, analyzed the data and wrote the paper. FM and JS performed the research, analyzed the data and wrote the paper. MP, CC, SL, and SB performed the research and analyzed the data. LN, AG, and AEA clinically assessed the patients. SH and MRT analyzed the data and wrote the paper. The authors reported no potential conflicts of interest.

• Received December 6, 2007.
• Revision received February 11, 2008.
• Accepted February 13, 2008.