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[Cancer Research 61, 2665-2669, March 15, 2001]
© 2001 American Association for Cancer Research

Molecular Biology and Genetics

t(11;14)(q23;q24) Generates an MLL-Human Gephyrin Fusion Gene along with a de facto Truncated MLL in Acute Monoblastic Leukemia1

Naruo Kuwada, Fumihiko Kimura, Takuya Matsumura, Takuya Yamashita, Yukitsugu Nakamura, Naoki Wakimoto, Takashi Ikeda, Ken Sato and Kazuo Motoyoshi2

Third Department of Internal Medicine, National Defense Medical College, Saitama 359-8513, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 PATIENT AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
More than 20 different partner genes with MLL have been cloned from leukemia cells with translocations involving chromosome 11 band q23 (11q23). All reported partner genes fused in-frame to MLL and the fusion cDNA encode chimeric MLL proteins with a significant portion derived from the partner genes. We analyzed one patient with de novo acute monoblastic leukemia with t(11;14)(q23;q24) and identified that a human homologue of gephyrin (human gephyrin) fused with MLL. Gephyrin is a rat glycine receptor-associated protein, which forms submembranous complexes and anchor glycine or {gamma}-aminobutyric acidA receptors to microtubules. Alternative splicing of human gephyrin gene created two different forms of fusion cDNA. In one form, human gephyrin gene fused in-frame to MLL exon 9, and the chimeric product had COOH terminus of human gephyrin protein, including the tubulin binding site. In the other, the reading frame terminated shortly after the fusion point. As a result, only seven amino acids with no known function were attached to the NH2 terminus of MLL protein. The functional significance of this de facto truncated MLL gene product is not clear.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
PATIENT AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recurring chromosomal aberrations play a crucial role in the tumorigenesis of a variety of human neoplasms, including hematological malignancies. Chromosomal translocations involving chromosome 11q23 have been observed in both acute myeloid and lymphoblastic leukemias as well as in therapy-related leukemias (1, 2, 3, 4, 5, 6) . Molecular cloning of translocation breakpoints revealed that a gene called MLL (also called ALL-1, HRX, or HTRX) is rearranged and has its breakpoints clustered within an 8.3-kb region (7) . This gene encodes a protein with a predicted molecular mass of 431 kDa (8 , 9) . The wild-type MLL protein has three AT-hook DNA binding domains, transcriptional activation and repression domains, multiple zinc finger domains known as plant homeodomain fingers, and a region homologous to mammalian DNA methyltransferases (10 , 11) . The SET [Su-(var)3–9, enhancer of zeste, and trithorax] domain in the COOH terminus of MLL, along with the plant homeodomain fingers, are conserved with the Drosophila trx3 gene. trx is a positive regulator of homeobox gene expression and is involved in segment determination in Drosophila. MLL is thought to be the mammalian homologue of the Drosophila homeobox regulating gene, trx, because mice with a mutant Mll gene (the mouse orthologue of MLL) develop homeotic transformations (12) .

Chromosome translocations involving MLL create in-frame fusion products of the NH2 terminus of MLL with different partner proteins (13, 14, 15) . More than 20 partner genes have been cloned to date (10 , 16, 17, 18, 19) . Moreover, partial tandem duplications of MLL appear to be frequently encountered in adult acute myeloid leukemia, particularly in association with trisomy 11 (20) . All partner genes are fused in-frame to MLL, resulting in novel fusion proteins, rather than simply a truncation of MLL. As to oncogenesis derived from MLL translocations, a two-hit model has been suggested (12) : (a) MLL fusion products cause haplo-insufficiency of the MLL COOH-terminal portion; and (b) partner genes could confer gain-of-function activity. Several groups have tried to elucidate the effects of these two events using various forms of MLL fusion or truncated MLL constructs (21, 22, 23, 24, 25, 26, 27) .

In the present study, we have identified human gephyrin as a novel fusion partner of MLL from a patient who presented with a de novo acute monoblastic leukemia with t(11;14)(q23;q24). This rare chromosomal translocation has been reported in a limited number of leukemia patients (28 , 29) . gephyrin was originally recognized as a rat protein that copurified with glycine receptors in inhibitory neurons of the central nervous system (30) . It is a cytoplasmic protein that is known to anchor glycine or {gamma}-aminobutyric acidA receptors to subsynaptic microtubules (31 , 32) . We were able to identify two MLL-human gephyrin fusion products, presumably attributable to the alternative splicing of human gephyrin. In one product, MLL was fused in-frame to the human gephyrin gene, and the resultant fusion protein contained the NH2 terminus of MLL and the COOH terminus of the human gephyrin protein. However, in the second product, only seven amino acids were attached to the NH2-terminal portion of the MLL protein, generating the shortest partner amino acid sequence among those reported. This fusion product may be considered as a de facto truncated form of MLL. On the basis of these findings, we discuss here possible mechanisms by which these MLL-human gephyrin fusion products contribute to leukemogenesis.


   PATIENT AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 PATIENT AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient.
A 67-year-old female was diagnosed with acute monoblastic leukemia with hyperleukocytosis. Chromosomal analysis showed t(11;14)(q23;q24). She received chemotherapy consisting of idarubicin and cytarabine with leukapheresis, but on day 2, she died of renal and respiratory failure. Autopsy revealed marked systemic infiltration of leukemic cells.

Southern Blot Analysis.
High molecular weight DNA was extracted from bone marrow cells of the donor using the GENOMIX kit (Talent s.r.l., Trieste, Italy). After digestion with a restriction enzyme, BamHI, the DNA was subjected to electrophoresis on a 0.7% agarose gel, transferred to a charged nylon filter, and hybridized with a cDNA probe. A 0.9-kb BamHI fragment derived from MLL cDNA was labeled by the random primer method (High Prime; F. Hoffmann-La Roche Ltd., Basel, Switzerland) and used as a probe.

Panhandle PCR.
High molecular weight DNA was extracted from peripheral blood cells using Qiagen Genomic-tip 100/G and Genomic DNA Buffer set (Qiagen, Hilden, Germany). Five µg of genomic DNA were analyzed by panhandle PCR as reported previously (33) .

3' RACE.
Total cellular RNA was extracted from peripheral blood cells of the patient using Isogen (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The RNA was subjected to the rapid amplification of cDNA ends technique using a 3' RACE kit (Life Technologies, Inc., Grand Island, NY). The gene-specific primers used were MLL primer 1 (5'-TCC TCC ACG AAA GCC CGT CGA G-3') from MLL exon 8 and reverse primer (5'-AGC AAA CAG AAA AAA GTG GCT CCC-3') from MLL exon 9.

Genomic PCR and RT-PCR.
Five hundred ng of genomic DNA were amplified by PCR in a total volume of 25 µl with the Taq PCR core kit (Qiagen). After 35 rounds of PCR (30 s at 94°C, 30 s at 56°C, and 2 min at 72°C), 5 µl of PCR product were electrophoresed on a 3% agarose gel. The primers used for genomic PCR were forward primer (5'- TGA TTC CTG TCC ATA GAA ATG CAA ATA AGT-3'), reverse control primer (5'-TGG TGA CTT CTT CTT GGT AAA TGA TAC GGA-3') from chromosome 14 sequence (accession no. AL049835.3), and reverse primer (described above). For RT-PCR, total RNA (1 µg) was reverse transcribed to cDNA in a total volume of 20 µl with random nonamers and 5 units of reverse transcriptase (RNA PCR kit version 2; TaKaRa Shuzo Co., Ltd., Kyoto, Japan). The cDNA (0.5 µl) was amplified by 35 rounds of PCR (30 s at 94°C, 30 s at 55°C, and 1 min at 72°C). Oligonucleotides used were MLL primer 1 (described above) and GSP2 (5'-TGC TGG GAA GGG GGG TAA ATT GT-3'), from human gephyrin exon 11, for MLL-human gephyrin fusion; gephyrin 552F (5'-AGA TGT CAC TCC AGA GGC CAC A-3'), from human gephyrin exons 4 and 5, and MLL exon 11 (5'-GGC ACA GAG AAA GCA AAC CA-3') for human gephyrin-MLL fusion; GAPDH F1 (5'-ACA TCG CTC AGA CAC CAT GG-3') and GAPDH R1 (5'-GTA GTT GAG GTC AAT GAA GGG-3') for GAPDH as a control. A nested PCR reaction using a 0.5-µl aliquot of the first reaction because template DNA enhanced the yield. The primers used for nested PCR were reverse primer (described above) and GSP3 (5'-TTG AGC AAG GAC TCG CCC CAT T-3'), from human gephyrin exon 11, for MLL-human gephyrin fusion; gephyrin exon 7 (5'-AAG ATT TGC CTT CCC CAC CT-3'), from human gephyrin exon 7, and MLL exon 10 (5'-TGC CAT TGG AGA GAG TGC TGA G-3') for human gephyrin-MLL fusion. Conditions for nested PCR were the same as in the initial RT-PCR, and the PCR products were electrophoresed as outlined above.

Nucleotide Sequencing.
The PCR products were cloned into a TA cloning vector (Invitrogen Corp., Carlsbad, CA). The nucleotide sequences of PCR products were determined by the fluorescently labeled dideoxy terminators (BigDye Terminator Cycle Sequencing FS Ready Reaction kit; Applied Biosystems, Tokyo, Japan) on a 377 Applied Biosystems automated sequencer. To obtain entire sequences, some PCR products were truncated using a Deletion Kit for Kilo-Sequencing (TaKaRa Shuzo Co., Ltd.).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 PATIENT AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Genomic DNA Cloning of the t(11;14).
Southern blot analysis of DNA from the leukemic cells digested with BamHI demonstrated two rearranged bands by the use of an MLL probe (Fig. 1)Citation . To identify the partner gene, we carried out panhandle PCR. A sequencing analysis of the 4.2-kb PCR product revealed that the MLL gene was fused to an unknown sequence in intron 9 (according to the exon-intron structure reported by Nilson et al. (8) . In a BLAST database search,4 this unknown sequence matched the genomic sequence from chromosome 14 (Fig. 2)Citation . We verified the fusion by genomic PCR (data not shown). Furthermore, the breakpoint was not found within Alu repetitive sequences. We could not find the partner gene within this PCR product because no EST clones matched the unknown sequence.



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  		Fig. 1. Southern blot of DNA was digested with BamHI and probed with the 0.9-kb fragment of the MLL gene. Lane C, normal control; Lane Pt, leukemic cells from the patient. Two rearranged bands (arrows) were detected in the patient sample.

 


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  		Fig. 2. Panhandle PCR products from der(11) chromosome. The MLL gene was truncated in intron 9, and the 3' sequence was replaced by the chromosome 14 genome sequence, AL049835.3. The breakpoint of the MLL gene is located between nucleotide positions 748 and 749 (numbering according to the 11q23-breakpoint sequence with accession no. HSU04737). In the chromosome 14 genomic sequence, the breakpoint is found between positions 163453 and 163454 of AL049835.3.

 
3' RACE Analysis.
To explore the fusion partner gene at the cDNA level, we performed 3' RACE. The two clones obtained both had partner sequences fused to MLL exon 9. One clone, having the shorter partner sequence, was named clone A, and the second, having the longer sequence, was named clone B. In a database search, these partners showed high homology to a rat cDNA sequence (accession no. X66366) for gephyrin, a glycine receptor-associated protein (Fig. 3A)Citation . Recently, a human cDNA sequence almost identical to rat gephyrin cDNA was reported (Ref. 34 ; KIAA1385, accession no. AB037806). We herein refer to this gene as human gephyrin. However, clone B contains an insert that does not have any corresponding sequence either in rat or human gephyrin cDNA. The insert sequence was assumed to be an exon sequence in an alternatively spliced form of human gephyrin, because it is found in the chromosome genome sequence (Fig. 4)Citation with splicing donor and acceptor sites (Fig. 3B)Citation and in a human EST (accession no. AA377065) from the small intestine cDNA library.



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  		Fig. 3. 3' RACE products. A, two clones containing the MLL gene and partner gene sequences. The partner gene sequence shows high homology with rat gephyrin cDNA and human gephyrin cDNA KIAA1385. Clone B contains an insert indicated by the hatched box. This insert did not have a corresponding sequence either in rat or human gephyrin cDNA. B, the insert sequence in clone B. The intron sequence predicted by the genome sequence is shown in italics. The insert sequence has splicing donor and recipient sites on either ends (boldface italics).

 


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  		Fig. 4. Structure of human gephyrin. Top, genome structure; each line represents a genome contig. The breakpoint is shown by an arrow on AL049835.3. Middle, human gephyrin was supposed to consist of 22 exons (boxes on dashed line). The box with (*) between exons 7 and 8, the insert sequence of 3' RACE clone B. Bottom, putative domain structure of human gephyrin protein. a.a., amino acid(s).

 
Human Homologue of gephyrin.
We performed public database searches for information about human gephyrin. The cDNA clone KIAA1385 is highly homologous to rat gephyrin cDNA as well as clones A and B. This clone has 4193 bp with an open reading frame of 2304 bp. However, the start codon has not as yet been identified. By analogy to the rat gephyrin sequence, the start codon is assumed to be located within nucleotides 1122–1124, and the open reading frame may encode 736 amino acids. The predicted amino acid sequence of KIAA1385 and the rat gephyrin amino acid sequence show >99% identity. These findings suggest that KIAA1385 is the human homologue of rat gephyrin cDNA. The entire sequence of KIAA1385 has corresponding chromosome 14 genome sequences in the GenBank database (Fig. 4)Citation . A homology search within other organisms revealed that gephyrin has two domains (MogA and MoeA domains) that are homologous to proteins of the molybdenum cofactor synthesis pathway. Sequence inspection identified a proline-rich motif between amino acids 219 and 233 and a tubulin binding site between amino acids 320 and 331. Furthermore, human gephyrin appears to be ubiquitously expressed, with high expression in brain, liver, and kidney as identified in the HUGE database.5

Analysis of Fusion Products.
We verified the MLL-human gephyrin fusion by RT-PCR and obtained products of predicted sizes only within the patient sample (Fig. 5A)Citation . Analysis of the fusion point revealed an in-frame fusion between MLL exon 9 and the human gephyrin homologue in clone A (Fig. 5B)Citation . In clone B, the reading frame terminated shortly downstream to the fusion point. These findings suggest that there should arise two chimeric proteins from MLL and human gephyrin fusion. We named these proteins MLL-gephyrin A and MLL-gephyrin B, respectively, according to the 3' RACE clones. The MLL-gephyrin A protein consists of AT-hooks, a methyltransferase, and transcription repressor domains of MLL, in addition to the human gephyrin MoeA domain and the tubulin binding motif. The MLL-gephyrin B protein contains the NH2 terminal portion of MLL and seven amino acids from the partner gene.



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  		Fig. 5. Structure of the MLL-human gephyrin fusion proteins. A, detection of the fusion transcripts by RT-PCR. Lane M, size marker; Lane P, leukemic cells from the patient; Lane C, normal control. Lanes 1 and 2 contain positive control amplifications of GAPDH. Both the MLL-human gephyrin and human gephyrin-MLL fusion transcripts were found in the patient sample (Lanes 3 and 5). B, the putative fusion proteins suggested by 3' RACE clones were designated MLL-gephyrin A and B, respectively. In MLL-gephyrin A, MLL exon 9 was fused in-frame to human gephyrin exon 8. In MLL-gephyrin B, the reading frame terminates briefly after the fusion point.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 PATIENT AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Cloning of the t(11;14) breakpoint of a de novo acute monoblastic leukemia has demonstrated that MLL fused to the human gephyrin gene. The t(11;14)(q23;q24) is an extremely rare chromosomal abnormality with only two reports in the literature (28 , 29) . However, MLL rearrangement was not examined in these cases. The breakpoint of the MLL gene was within the breakpoint cluster region described previously. Neither MLL nor the chromosome 14 genome sequence has Alu sequences near the genomic fusion point; therefore, it is unlikely that homologous recombination has caused this translocation.

The human gephyrin gene is highly homologous to the rat gephyrin gene. The rat gephyrin gene encoding a Mr 93,000 protein was cloned in 1992 (30) . Gephyrin is known to be a receptor-associated protein linking the ß subunit of glycine receptors to the subsynaptic cytoskeleton. It is essential for the localization of inhibitory glycine or {gamma}-aminobutyric acidA receptors to presumptive postsynaptic sites (31 , 32) . In this process, the gephyrin protein is thought to form clusters under the plasma membrane. gephyrin contains two domains that are homologous to the Escherichia coli proteins in the molybdenum cofactor synthesis pathway. Gene targeting studies demonstrate that gephyrin is required both for synaptic clustering of glycine receptors in the spinal cord and for molybdoenzyme activity in nonneural tissues. Furthermore, gephyrin has been shown to interact with RAFT1 (FRAP) (Ref. 35 ). RAFT1 is an ATM-related protein that appears to participate in mitogen-stimulated signaling, and RAFT1 mutants that could not associate with gephyrin failed to signal to downstream molecules. The COOH-terminal portion of gephyrin, including the proline-rich motif and the tubulin binding site, was necessary for this interaction, but the precise RAFT1 binding site has as yet to be identified. The gephyrin-binding region of RAFT1 shares sequence similarity with the intracellular loops of the ß subunit of the glycine receptor. Several other proteins that associate with gephyrin have been reported. Collybistin, a newly identified brain-specific GDP/GTP exchange factor, induces submembrane clustering of gephyrin (36) . Profilin, an actin monomer-binding protein that stimulates actin polymerization, binds to the proline-rich motif of gephyrin (37) .

To date, >20 different partner genes for MLL have been reported (10 , 16, 17, 18, 19) . Many of the partners are nucleoproteins that show homology to transcriptional factors or have known roles in transcriptional regulation. This has led to the hypothesis that MLL translocations create chimeric transcription factors. Recently, however, several fusion partners that appear to be cytoplasmic proteins have been reported. For example, AF6 is a putative ras effector and component of tight junctions (38 , 39) that has been shown to bind both ras and the tight junction protein ZO-1. Moreover, AF6 is thought to mediate the clustering of Eph receptors at postsynaptic specialization sites in the adult rat brain (40 , 41) . Therefore, AF6 and gephyrin may act similarly through interactions with receptor molecules. However, it is unclear whether these properties actually contribute to leukemogenesis, because the MLL-AF6 fusion product was located in the nucleus (42) . Subcellular localization of the MLL-human gephyrin fusion proteins and the function that the cytoplasmic family of fusion partners gives to MLL fusion products are of future interest.

The 3' RACE analysis resulted in two forms of fusion products. Different forms of fusion products have been observed in patients with MLL-AF4, MLL-AF9, MLL-CBP, and MLL-ENL fusions (43, 44, 45, 46, 47) . In a case with complex chromosomal abnormality, two partner genes were found to make dual fusion products with MLL (48) . Moreover, a single aberration, cryptic t(11;17), resulted in two in-frame MLL fusion cDNAs in a patient because of alternative splicing of the MLL gene (17) . In our case, the two fusion clones might have been caused by an alternately spliced human gephyrin gene. The rat gephyrin gene has seven different cDNA forms isolated from spinal and brain cDNA libraries (30) . The insert sequence in our clone B was detected in the chromosome 14 genome sequence and in a human EST clone. In addition, it has splice donor and acceptor sites on either end of the genome sequence. These observations suggest that the insert sequence represents an exon sequence in an alternatively spliced form of human gephyrin.

The fusion product encoded by clone A has the NH2 terminus of MLL and the COOH terminus of the human gephyrin homologue. The tubulin binding site is retained, allowing the MLL- gephyrin A product to interact with microtubules. RAFT1 and glycine receptors may also bind to the fusion protein. Interactions with these and other proteins may confer some function to the MLL NH2-terminal portion. Although the region required for cluster formation remains unknown, oligomerization is another possible property of the fusion product. In MLL-bacterial lacZ fusion knock-in mice, the artificial fusion gene causes leukemia, as do the naturally occurring fusion genes (49) . lacZ has tetramer formation features and could contribute to leukemogenesis through oligomerization of the MLL-lacZ fusion protein.

In clone B, the reading frame terminates immediately after the breakpoint. The partner gene provides only seven amino acids containing no known features to the NH2 terminus of the MLL protein. This is the shortest fusion partner portion of those reported. In fact, this fusion product may be equivalent to a truncated MLL protein.

MLL gene rearrangement may have two effects on tumorigenesis (12) . One is the removal of the MLL COOH-terminal sequence, which may cause haplo-insufficiency of MLL and work on the wild-type MLL as a dominant-negative form. The second is the positive contribution of gaining specific fusion partners. These hypotheses have been examined by introducing various fusion genes and truncated MLL. In myelomonocytic or monocytic cell lines, truncated MLL as well as MLL-partner gene fusion products can affect cell growth, differentiation, homeobox gene expression, or cell cycle (21, 22, 23) . However, the truncated MLL gene could not induce leukemia when it was introduced into mouse embryonic stem cells or bone marrow cells (24, 25, 26, 27) . In knock-in mice, the Mll-AF9 fusion gene caused leukemia after a myeloproliferative state (25) . The late onset of overt tumors suggests that additional genetic events are required for leukemogenesis.

In our case, further study is required to elucidate which of the MLL fusion products may be responsible for leukemogenesis. However, the clone B product is less likely to be sufficient for transformation. All cloned 11q23 translocations have produced in-frame fusion genes containing significant amounts of the partner sequences. This observation implies selection for a translatable or functional transcript in the process of leukemogenesis (10) . Furthermore, there may be ongoing MLL fusion events in healthy individuals. MLL-AF4 fusion products and multiple MLL partial tandem duplication products have been detected in healthy individuals by RT-PCR (50 , 51) . In the latter, the estimated mean number of duplication positive cells in healthy blood donors is 1 in <=5000 cells. However, the MLL disruption may have to survive three rounds of selection before causing leukemia: (a) in-frame fusion with a significant portion of the partner gene is needed; (b) this must occur in hematopoietic cells within a developmental window of vulnerability (10) . It is at this point that cells with MLL aberrations gain proliferative or survival advantage; and (c) additional genetic hits liberate leukemic transformation. According to this hypothesis, the short form of the MLL-human gephyrin fusion may be an example of a nonfunctional MLL fusion product that we were able to identify incidentally, by virtue of the leukemia caused by the longer form of the MLL-human gephyrin fusion protein. Our patient revealed a novel MLL-partner gene fusion along with a de facto truncated MLL gene. Further studies on this abnormality will contribute to understanding MLL-mediated leukemogenesis.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a grant from the Research Foundation For Community Medicine. Back

2 To whom requests for reprints should be addressed, at National Defense Medical College, Third Department of Internal Medicine, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. Phone: 81-42-995-1617; Fax: 81-42-996-5202; E-mail: motoyosi{at}me.ndmc.ac.jp Back

3 The abbreviations used are: trx, trithorax; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EST, expression sequence tag. Back

4 Internet address: http://www.ncbi.nlm.nih.gov/BLAST. Back

5 Internet address: http://www.kazusa.or.jp/huge. Back

Received 7/ 6/00. Accepted 1/16/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 PATIENT AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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