Background
Abnormalities of 11q23 involving the
MLL gene are found in approximately 10% of human leukemias [
1].
MLL rearrangements are present in >70% of infant leukemias, irrespective of the immunophenotype being more consistent with acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML), but are less frequent in leukemias from older children [
2].
MLL translocations are also found in approximately 10% of adult AML, and can also be found in a proportion of patients with therapy-related leukemia after treatment for other malignancies with topoisomerase II inhibitors [
3]. Although clinically and morphologically heterogeneous,
MLL-rearranged ALL and AML show unique gene expression profiles [
4,
5].
To date, nearly 100 different chromosome bands have been described in rearrangements involving 11q23 and 64 fusion genes have been cloned and characterized at the molecular level [
6]. The most common
MLL fusion partners are
AFF1/AF4 (4q21),
MLLT3/AF9 (9p23),
MLLT1/ENL (19p13.3),
MLLT10/AF10 (10p12),
MLLT4/AF6 (6q27),
ELL (19p13.1),
EPS15/AF1P (1p32),
MLLT6/AF17 (17q21), and
SEPT6 (Xq24) [
6]. Usually,
MLL rearrangements result from the non-homologous-end-joining (NHEJ) DNA repair pathway following DNA damage [
7]. Reciprocal chromosomal translocations are the most frequent events associated with the genetic recombination of
MLL, but other mechanisms have been identified, including internal partial tandem duplication (
MLL-PTD), chromosome 11 deletions or inversions, and several types of complex
MLL rearrangements [
6]. Occasionally, chromosomal translocation or deletion have been described to originate
MLL spliced fusions, which arise by fusing the 5'
MLL region to downstream located partner genes [
8]. In the present study, we have identified the
CT45A2 gene as a novel fusion partner of
MLL in a pediatric patient with
de novo biphenotypic acute leukemia (BAL), as a result of a cryptic insertion of 11q23 material in Xq26 resulting in a spliced
MLL fusion.
Methods
Patient Data
A 6-year-old boy was admitted to the Portuguese Oncology Institute (Porto, Portugal) with a history of fever, asthenia and cutaneous pallor. Peripheral blood analysis revealed anemia (Hb 6.3 g/dl) and bicytopenia. Bone marrow analysis revealed the presence of 51% of blasts with the immunophenotype CD3+, CD13+, CD33+, and CD117+, which lead to the diagnosis of biphenotypic phenotype (T/myeloid) acute leukemia. No blasts were detected in the cerebrospinal fluid. He was treated according to the ELAM 02 protocol (aracytine, mitoxantrone and methotrexate) and entered complete remission after induction chemotherapy. Seven months later he was submitted to allogeneic bone marrow transplantation with umbilical cord hematopoietic progenitors, but the patient showed evidence of relapse after one year. Treatment with the AML relapse protocol was started, but only partial remission had been achieved four months later. The patient underwent a haploidentical transplant with his mother's peripheral blood cell progenitors, but the disease relapsed again and the patient died nine months later.
Chromosome Banding and Molecular Cytogenetics
The diagnostic bone marrow sample was cultured for 24 hours in RPMI 1640 medium with GlutaMAX-I (Invitrogen, London, UK) supplemented with 20% fetal bovine serum (Invitrogen, London, UK). Chromosome preparations were made by standard methods and banded by trypsin-Leishman. Karyotypes were described according to the International System for Human Cytogenetic Nomenclature [
9]. Fluorescence in situ hybridization (FISH) analysis was performed using the LSI MLL Dual-Color, Break-Apart Probe and LSI IGH/BCL2 Dual-Color, Dual Fusion Probe (Vysis, Downers Grove, USA), respectively, in previously stained metaphases that have been destained and processed for FISH as described [
10].
RNA and DNA Extraction
High molecular weight DNA and RNA were extracted from the bone marrow sample using 1 ml of Tripure isolation reagent (Roche Diagnostics, Indianapolis, USA), according to the manufacturer's instructions.
Long-Distance-Inverse Polymerase Chain Reaction (LDI-PCR)
The DNA sample was treated and analyzed as previously described [
11,
12]. Briefly, 1 μg of genomic DNA was digested with restriction enzymes and self-ligated to form DNA circles. Amplification was performed with specific primers for fusion sequences on der(11) and der(X). LDI-PCR reactions were performed as described [
12] and according to the manufacturer's recommendations (PCR Extender System, 5 Prime, Hamburg, Germany). Amplified products were analyzed on a 1% agarose gel (SeaKem LE Agarose, Rockland, USA). PCR amplimers were isolated from the gel and subjected to DNA sequence analyses to obtain the patient-specific fusion sequences. After sequencing, unknown sequences were characterized by blasting the human genome database (Genomic BLAST,
http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Reverse-Transcription Polymerase Chain-Reaction (RT-PCR)
For cDNA synthesis, 1 μg of RNA was subjected to reverse transcription with random hexamers using the Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, USA), according to the manufacturer's instructions. RT-PCR assay for detection of MLL-CT45A2 fusion transcripts was performed with a forward primer (MLL-S; 5'-GAGGATCCTGCCCCAAAGAAAAG-3') located in MLL exon 8 (GenBank accession no. NM_005933) and a reverse primer (CT45A2-AS; 5'- GGCCATCCTCTGCCTTTTC-3') located in CT45A2 exon 2 (GenBank accession no. NM_152582). Additional primers in the MLL breakpoint cluster region (exons 9 to 13) and CT45A2 open reading frame (exons 3 to 5) were used to exclude the presence of additional splice variants (data not shown). PCR reactions were performed in a 50 μl reaction volume containing 2 μl of synthesized cDNA, 5 μl of 10× GeneAmp PCR buffer II (100 mM Tris-HCl pH 8.3, 500 mM KCl) (Applied Biosystems, Foster City, USA), 5 μl of 25 mM MgCl2, 0.4 μl dNTP mix (25 mM each dNTP) (Applied Biosystems), 0.4 mM of each primer (Metabion, Martinsried, Germany), and 1 unit of AmpliTaq Gold DNA Polymerase (Applied Biosystems). Reaction tubes were kept on ice at all times to prevent non-specific amplification and incubated for 5 min at 94°C, followed by 35 cycles of 30 sec. at 95°C, 1 min at 63°C, and 1.5 min at 72°C, followed by a final elongation of 10 min at 72°C on a GeneAmp PCR System 9700 (Applied Biosystems). Amplified products were analyzed on a 2% agarose gel (SeaKem LE Agarose) and the results were visualized in an image analyzer ImageMaster VDS (Amersham Biosciences, Little Chalfont, UK).
Search for DNA sequence motifs known to be associated with site-specific recombination, mutation, cleavage, and gene rearrangement, and repetitive sequence elements spanning or in the vicinity of deletion and insertion breakpoints [
13,
14] was performed with SEQ tools [
15] and RepeatMasker [
16]. Search for repetitive sequence homology was performed with ClustalW [
17].
Discussion
We have identified a novel fusion partner of
MLL, the
CT45A2 gene, which is a member of the Cancer/Testis (CT) gene family cluster localized at Xq26.3. In our pediatric patient with
de novo biphenotypic acute leukemia, the
MLL-CT45A2 fusion resulted from a cryptic insertion of 11q23 material into Xq26.3. The insertion of the 5'
MLL region upstream of the
CT45A2 gene leads to
MLL-CT45A2 fusion transcripts. In our case, a weaker transcript exhibited
MLL exons 1-8 fused to
CT45A2 exon 2 and consecutive exons. This transcript is out-of-frame and produces only a truncated version of the MLL protein. The stronger PCR product, however, represented an in-frame fusion transcript containing
MLL exons 1-9 fused to the intact
CT45A2 transcript, a process known as spliced
MLL fusion since the chimeric
MLL-CT45A2 is only generated at the RNA level [
8]. Interestingly, the chimeric mRNA contains 6 bp from the 5-UTR of
CT45A2 exon 2 resulting in two additional amino acids. To our knowledge, this is the first description of a spliced
MLL fusion resulting from an insertion event. Indeed, spliced
MLL fusions were previously described in leukemia patients with the translocations t(1;11) (
MLL-EPS15), t(4;11) (
MLL-AFF1), t(9;11) (
MLL-MLLT3), t(11;15) (
MLL-MPFYVE), t(11;19) (
MLL-MLLT1) and t(11;22) (
MLL-SEPT5) [
6,
8,
19], as well as in a single leukemia patient with an intrachromosomal 11q23 deletion (
MLL-DCPS) [
11]. Spliced
MLL fusions can occur either by transcriptional read-through followed by a subsequent splice event or by trans-splicing [
11]. Regardless of the underlying mechanism, the chimeric
MLL-CT45A2 fusion is only produced at the RNA level. A reciprocal
CT45A2-MLL transcript does not exist because the
FXYD2 gene, transcribing in the opposite direction, is located upstream of the remaining
MLL exons 10-37. However, we cannot exclude that the recently described gene-internal promoter upstream of
MLL exon 12 is able to produce a 5'-truncated MLL protein of about 230 kDa [
18].
Another consequence of this spliced fusion is that the expression of the
CT45A2 gene, usually restricted to testicular tissue [
20], is activated. CT genes encode a heterogeneous group of immunogenic proteins (CT antigens) that were initially identified as immunogenic tumor antigens and whose expression is almost restricted to the normal testis and a percentage of various tumor types, including melanoma and carcinomas of the bladder, lung and liver [
20‐
24]. The combination of restricted normal tissue expression, spontaneous immunogenicity and frequent tumor expression has made these antigens attractive targets for cancer vaccines [
21,
24]. The CT45 gene family comprises six members (
CT45A1 to
CT45A6) located in Xq26.3 that are near-identical gene copies, suggesting the occurrence of recent gene duplications, but whose function remains to be elucidated [
20]. The phenotypic consequences of
CT45A2 expression in the leukemia cells of this patient are currently unknown. However, as CT45A2 exhibits the typical CT antigen immunogenic profile, it would be worthy to further investigate the possible humoral and cell-mediated immune responses to this protein.
Detailed analysis of the genomic breakpoint junctions in our patient revealed the presence of filler-DNA nucleotides and an inverted repeat flanking the breakpoint junction 5' of
CT45A2. The repair of chromosomal double-strand breaks (DSBs) occurs by two types of DNA repair pathways: homologous recombination and non-homologous end-joining (NEHJ) [
25,
26]. The presence of filler-DNA at breakpoints junctions are typical hallmarks of NHEJ [
27], and inverted repeats may facilitate the formation of secondary structure intermediates between DNA ends at translocation breakpoints [
13]. Moreover, all breakpoint regions were found to be rich in repetitive sequences, particularly LINE/L1 elements and SINE/Alu repeats. Although repetitive sequences may occur near or spanning breakpoint junctions by chance, it is plausible that introns or genomic regions with a high density of repetitive sequences, such as
MLL intron 9, are more vulnerable to breaking and non-homologous pairing that can lead to gene fusions. However, since the chromosomal breakpoints in our patient were always located outside of these repetitive DNA sequences, it is unlikely that they could be directly involved in the recombination events.
The insertion of 11q23 in Xq26.3 was associated with the deletion of the 3' region of
DDX26B (encompassing exons 9-16), leading to a premature termination of ddx26b open-reading frame (ORF). The DDX26B [DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 26B] protein is a helicase that belongs to the DEAD/DEAH box family of proteins that are considered to be RNA helicases, which have been described to be necessary for, or involved in, many different processes of RNA metabolism [
28]. In eukaryotic cells, in particular, these range from transcription to degradation of RNA, and include pre-mRNA splicing, mRNA export, ribosome biogenesis, translation, initiation, and gene expression in organelles [
28]. Since our patient is a male, the only functional copy of
DDX26B is disrupted as a result of the insertion, leading to the absence of the DDX26B protein in the cell or, in alternative, the presence of a truncated protein. Since the particular function of DDX26B in myeloid cells is not known, the phenotypic impact of this abnormality cannot be predicted. The insertion was also associated with loss of the only copy of the
CT45A1 gene present in the cell. However, since
CT45A1 expression is restricted to normal testis, the cellular impact of its loss is not expected to be relevant. Similarly, the phenotypic impact, if any, of the insertion in Xq26.3 of 16 additional 11q23 genes (
FXYD6,
TMPRSS13,
IL10RA,
APOO2962.2,
TMPRSS4,
SCN4B,
SCN2B,
AMICA1,
MPZL3,
MPZL2,
CD3E,
CD3D,
CD3G,
UBE4A,
ATP5L, and
AP001267.2) can also not be predicted.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
NC designed and performed the research, analyzed the data and drafted the manuscript. CM designed and performed the research, analyzed the data and drafted the manuscript. JS performed the research and analyzed the data. LT, SL, and CC performed the chromosome banding and molecular cytogenetic studies. MP performed sequencing analysis. SB performed RT-PCR analysis. LN clinically assessed the patient. RM and MRT coordinated the study and participated in manuscript writing. All authors read and approved the final manuscript.