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Biochemical and Biophysical Research Communications 280, 301–306 (2001)

doi:10.1006/bbrc.2000.4124, available online at http://www.idealibrary.com on

olecular Characterization of the Genomic Breakpointunction in the t(11;18) (q21;q21) Translocationf a Gastric MALT Lymphoma

umiko Sato,*,† Yoshimitsu Akiyama,* Toru Tanizawa,† Toshikatsu Shibata,‡ Kiyoshi Saito,§higeo Mori,¶ Ryuichi Kamiyama,† and Yasuhito Yuasa*,1

Department of Molecular Oncology and †School of Allied Health Sciences, Tokyo Medical and Dental University,aculty of Medicine, Bunkyo-ku, Tokyo 113-8519, Japan; ‡Department of Pathology, Tsuchiura-Kyodo Hospital,suchiura, Ibaragi 300-0053, Japan; §Department of Pathology, International Medical Center of Japan,hinjuku-ku, Tokyo 162-8655, Japan; and ¶Division of Pathology, Institute of Medical Sciences,niversity of Tokyo, Minato-ku, Tokyo 108-8639, Japan

eceived November 30, 2000

tions (5–8). The t(11;18) (q21;q21) translocation, fusingtn

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The t(11;18) translocation, fusing the API2 andALT1 genes, is one of the most frequent chro-osomal translocations associated with mucosa-

ssociated lymphoid tissue (MALT) lymphomas. Theranslocation breakpoints have been cloned and char-cterized at the mRNA sequence level. Although theenomic organization of the API2 gene has been de-cribed, hitherto the genomic sequence of MALT1 re-ains unknown. To gain some insight into the mech-

nism that generates this translocation, we cloned andequenced an API2–MALT1 fused transcript as well asenomic DNA of the t(11;18) translocation from aALT lymphoma. We localized the API2 breakpointithin intron 7. Nucleotide sequence analysis re-

ealed that the genomic breakpoint junction possesseshe consensus heptamers of immunoglobulin V(D)J re-ombination signal sequences, all the matches beingompletely present on the API2 allele and five of sevenatches on the MALT1 allele. These data suggest that

he translocation in the MALT lymphoma might haveeen mediated in part by an aberrant V(D)J recombi-ation event. © 2001 Academic Press

Key Words: MALT lymphoma; translocation; API2;ALT1; V(D)J recombination; heptamer.

Marginal zone B-cell lymphomas of mucosa-ssociated lymphoid tissue (MALT) type are the mostommon subtype of lymphomas arising at extranodalites such as the stomach, thyroid, salivary gland, andung (1–4). In MALT lymphomas, the recurrent abnor-

alities are trisomies of chromosomes 3, 7, 12, and 18,nd t(11;18) (q21;q21) and t(1;14) (p22;q32) transloca-

1 To whom correspondence and reprint requests should be ad-ressed.

301

he API2 and MALT1 genes, is a characteristic cytoge-etic abnormality in MALT lymphomas (9–14).The API2 gene is a member of the human IAP (in-

ibitor of apoptosis) family, and the protein coded byPI2 has been shown to be highly expressed in lym-hoid cells (15). Common features of the gene familyembers include one to three copies of a motif termed

he baculovirus IAP repeat (BIR) domain in the-terminal region, a caspase recruitment domain

CARD), and a single RING finger domain close to the-terminus (15). API2 and API1, which are members of

he IAP family exhibiting a 70% homology in theirmino acid sequences, directly interact with and in-ibit caspases-3 and -7, and thus inhibit apoptosis (16).reakpoints in the API2 gene in MALT lymphomasave been frequently found between the third BIR andARD domains, and much less frequently between theARD and RING domains (10–14).The MALT1 gene encodes a 813-amino acid protein,

nd contains two immunoglobulin (Ig)-like C2 domainsnd a domain similar to the murine Ig g chain VDJ4equence (10, 11). The function of the MALT1 protein isnknown. The genomic organization of the MALT1ene has not been reported. Breakpoints in the MALT1ene in MALT lymphomas have been found to be vari-ble compared with those in API2 (10–14).The BLAST search with the predicted MALT1 pro-

ein revealed homologies with some cell surface glyco-roteins. The MALT1 cDNA sequence shows a homol-gy with that of CD22b, which is expressed in B cells.D22 appears to be a heterodimer consisting of 130-Da (a) and 140-kDa (b) glycoproteins with proteinores of 80 and 100 kDa, respectively. The CD22 geneovers 22 kb of DNA and comprises 15 exons, and its

0006-291X/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

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ocus is located within band 19q13.1 (17). Moreover,mino acids 308–476 of the MALT1 protein exhibited aomology with the laminin 5a3b subunit (18). Lamininis an isoform within the laminin family of proteins

onsisting of three distinct polypeptides, i.e., the a-3,-3 and g-2 chains (19). Laminin 5a3b is located on8q11.2.Recurrent chromosomal translocations acquired in aultistep process of transformation leading to the evo-

ution of autonomous cell clones are well recognized inodal B-cell lymphomas (20). These translocationsharacterize distinct disease subtypes, and involveenes controlling cell proliferation and apoptosis. Theiological relevance of these translocations is under-cored by the fact that a number of known and putativerotooncogenes have been identified through the studyf genes located at these translocation breakpoints21). The precise molecular mechanisms underlyinghromosomal translocations remain largely unknown.n some cases there is compelling evidence that theranslocation is caused by aberrant V(D)J recombinasectivity, as evidenced by the presence of crypticeptamer/nonamer sequences (22, 23). However, sincehe sequences of the genomic breakpoint junction inhe t(11;18) (q21;q21) translocation have not been re-orted, the molecular mechanisms underlying translo-ation in MALT lymphomas were unknown.

The t(11;18) translocation has not been frequentlyetected in gastric MALT lymphomas (12). Therefore,omologs of the MALT1 and/or API2 genes might bessociated with translocation in some gastric MALTymphomas.

Here we searched for API2–MALT1 fusion tran-cripts in 10 gastric lymphomas, including the MALType, using the reverse transcription (RT)-PCR

Primer Sets and

Primer sets Sense primer

PI2–MALT1Set 1a API2-U 59-TTAATGCTGCCGTGGAAATGGGC-39Set 2a API2-U

PI2–CD22 bSet 3 API2-U

PI2–laminin5a3bSet 4 API2-U

PI1–MALT1Set 5 API1-Ub 59-GTTAAATCTGCCTTGGAAATGGGC-3ALT1–API2Set 6 MALT1-U 59-GATCGAGACAGTCAAGATAGCAAG-3

a We used primer set 1 for detection of API2–MALT1 fusion transcrf MALT1, we also designed MALT1-D2 as an antisense primer for

b Primers for the CD22b (Accession No. X59350) and laminin 5a3here there are homologies with MALT1. A primer for API1 (AccessiPI2 primer was located. CD22b-D, codons 790–798; laminin5a3b-D

302

ethod. Then we also examined the presence or ab-ence of fused transcripts of API2–CD22b, API2–aminin 5a3b, and API1–MALT1. Since we found aPI2–MALT1 fusion transcript in one MALT lym-homa, we further analyzed it at the genomic sequenceevel.

ATERIALS AND METHODS

Patient samples. A total of 10 frozen tissue samples (6 MALTymphomas and 4 diffuse large B-cell lymphomas) obtained fromatients with gastric B-cell non-Hodgkin lymphomas were analyzedn this study. The pathologic diagnosis in each case was establishedccording to the REAL classification (24).

RNA and DNA extraction and cDNA synthesis. RNA was ex-racted from frozen tissue sections using an RNeasy Mini kitQIAGEN, Dusseldorf, Germany) following the manufacturer’s rec-mmendation. One microgram of total RNA was used for RT. RT waserformed using Superscript II reverse transcriptase (Life Technol-gies, Inc., Grand Island, NY) according to the manufacturer’s in-truction.For the preparation of high-molecular-weight DNA, tissue sectionsere processed with the standard phenol/chloroform extraction pro-

ocol that includes digestion with proteinase K (100 mg/ml, Boeh-inger Mannheim, Indianapolis, IN).

PCR. Each RT-PCR comprised 35 cycles of denaturation for 1in 94°C, annealing for 2 min at 63 or 65°C, and extension for 1 min

sets 1, 5, and 6) or 3 min (sets 2, 3, and 4) at 72°C, followed by finalxtension for 10 min at 72°C (Table 1).For long and accurate (LA)-PCR (Takara, Kyoto, Japan) for exami-

ation of genomic translocation sequences, PCR comprised 30 cycles of4°C for 30 s and 65°C for 20 min, followed by final extension for 10 mint 72°C. Amplification of the GAPDH gene was examined for qualityontrol with a primer pair (sense primer, 59-GACCACAGTCCATGC-ATCAC-39; antisense primer, 59-GTCCACCACCCTGTTGCTGTA-39).

Sequencing. The PCR products were sequenced directly with aycle sequencing kit (Takara, Kyoto, Japan) using end-labeled prim-rs. Approximately 100 ng of API2–MALT1 cDNA products wereequenced using 2 pmol of a forward (API2-U) or reverse (MALT1-

-PCR Conditions

Antisense primer

Annealingtemperature

(°C)

MALT1-D1 59-CAGGATGACCAAGATTATTTAATTC-39 63MALT1-D2 59-TGAGGTGGCTGCAGAACTCTGG-39 65

CD22 b-Db 59-TGAGTGGAGGATCTGAACCGTGG-39 65

laminin5a3b-Db 59-CTCGGAGAGTTGTGAGATTGGTG-39 65

MALT1-D1 63

API2-D 59-AGAGTTTCTGAATACAGTGGCTGC-39 65

. To detect translocations with a breakpoint in the C-terminal region2.ccession No. X84900) genes were designed to cover all the regionso. NM001167) was designed in the correspondent region where the

odons 736–744; API1-U, codons 396–403.

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1) primer and a Thermosequenase kit (Amersham Pharmacia Bio-ech, Buckinghamshire, England). The cycling parameters were 30 st 94°C, 30 s at 65°C, and 1 min at 72°C for 15 cycles, and electro-horesis was performed on 6% polyacrylamide gels containing 15%ormamide and 7 mol/liter urea.

As for sequencing of the genomic DNA, the LA-PCR product wasloned into the pT7Blue T-vector (Novagen, Madison, WI) and thenequenced with a cycle sequencing kit (Takara).

ESULTS

etection of API2–MALT1 Fusion Transcripts

Total RNA was isolated from each specimen and ana-yzed by RT-PCR for API2–MALT1 fusion transcripts.ach cDNA sample was examined with primer pair setsand 2 (Table 1). While all 10 lymphoma specimens

ontained detectable GAPDH mRNA, an API2–MALT1usion transcript, 206 bp in size, was identified in 1 (case

FIG. 1. RT-PCR detection and molecular structure of the API2–Musion transcripts. RT-PCR with primer set 1 revealed a specific producases 2 and 3 are shown). The size of the amplified fragment is 206hen analyzed with primer set 2 (center). RT-PCR products with a G

dentities are indicated at the top. (B) The structure of the API2M001165) is shown at the top with three BIR domains separated

tructure of MALT1 (Accession No. AB026118) is shown at the bottochain VDJ4 sequence at the C-terminal. The arrows with nam

PI2–MALT1 transcript (Table 1). The breakpoints in API2 and MALhe third BIR domain and fused in-frame to MALT1. The nucleotnderneath the fusion cDNA (API2–MALT1).

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, the MALT type) of the 10 (10%) gastric lymphomasFig. 1A) with primer set 1. Primer set 2 also producedusion cDNA, 1453 bp in size, in only the same case (Fig.A). This case was a male Caucasian, aged 43 years, andelicobacter pylori-positive.The nucleotide sequence analysis revealed that the

mplified fragment was a fusion product of API2 andALT1 (Fig. 1B). The API2 breakpoint seems to lie

etween exons 7 and 8, because the API2 sequence inhe fused cDNA ends at the 39 end of exon 7. It shoulde noted that the chimeric protein retains three BIRomains. The MALT1 sequence starts from codon 309n the fused transcript. An important finding was thatwo genes were fused in-frame and were predicted toroduce a chimeric protein comprising API2 andALT1, as shown schematically in Fig. 1B.

LT1 fusion transcripts. (A) Gel electrophoresis of the API2–MALT1in case 1. The remaining cases did not show any amplified fragments(left). The same case also showed an amplified fragment (1453 bp)

DH primer pair that amplifies 457 bp are also shown (right). SampleALT1 fusion cDNA. The cDNA structure of API2 (Accession No.om the C-terminal RING domain by a CARD domain. The cDNAwith two Ig-like C2 domains and a domain similar to the mouse Igindicate the locations of the RT-PCR primers used to detect theare indicated by large arrows (2). The API2 cDNA is truncated afterand amino acid sequences of the breakpoint junction are shown

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etection of Hypothetical Fusion Transcripts

To detect hypothetical fusion transcripts of API2–D22b, API2–laminin 5a3b, or API1-MALT1, we used

hree primer pairs (sets 3–5, Table 1). Fusion tran-cripts were not found in all the 10 cases (data nothown).

loning and Analysis of the API2–MALT1 GenomicBreakpoint Junction

The genomic breakpoint junction in case 1 was am-lified with primer set 1, the size of product beingbout 8 kbp (Fig. 2A). To examine the breakpoint, weesigned three primers (Nos. 1–3) in API2 intron 7 as9 primers (Fig. 2B), and amplified case 1 DNA withALT1-D1 as the 39 primer. All the three primers

ielded the PCR products, indicating that the break-oint is located in the downstream of primer No. 3. Tourther approach the breakpoint, we designed threeore 59 primers (Nos. 4–6) as shown in Fig. 2B. Sincee could detect amplified fragments using up to primero. 5 as the 59 primer (Fig. 2A), the breakpoint was

FIG. 2. PCR detection and molecular structure of the genomic ACR. LA-PCR with primer set 1 (left), and API2 No. 5 and MALT1-D

ragments are approximately 8 and 3 kbp, respectively. (B) The strure shown as straight lines. The arrows (3) indicate the locations oxamine the breakpoint of the genomic API2, we used six sense primn intron 7 according to Accession No. AP001167. The alternative sp

304

hown to be located within a region of 280 bp in sizeetween primer Nos. 5 and 6. The PCR product withrimers No. 4 and MALT1-D1 was cloned and se-uenced (Fig. 3). The sequence of the 59 portion of thereakpoint region completely corresponded to that ofPI2 intron 7. When the sequence of the 39 portion of

he breakpoint region was applied for the BLASTearch, we found a homology with EST AW968625.his sequence contained an alternative splicing smallxon (11 amino acids) of the MALT1 gene (Fig. 2B),uggesting that the 39 portion of the breakpoint regionas derived from the MALT1 intron (13).Analyses of the nucleotide sequences determined

rom the germline API2 and MALT1 alleles in theicinity of the breakpoint revealed characteristic fea-ures. The API2 sequence contains a CACAGTG hep-amer identical to the V(D)J recombination signal se-uences recognized in Ig or T-cell receptor (TCR) geneearrangements (23, 25). There are five of sevenatches for the heptamer-like sequence in the MALT1

ene, as indicated in Fig. 3. No nonamer-like sequencesere found in either gene.

–MALT1 DNA. (A) Detection of the genomic API2–MALT1 DNA byght) revealed specific fragments in case 1. The sizes of the amplifiedre of the genomic API2–MALT1 DNA. Exons are boxed, and intronse PCR primers used to detect the genomic API2–MALT1 DNA. To(Nos. 1–6), indicated by arrows or arrowheads, which we designed

ng exon of MALT1 (11 amino acids) is indicated by a triangle (ƒ).

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ISCUSSION

In this study, the API2–MALT1 translocation wasetected in one case (10%). Although both API2–ALT1 and MALT1–API2 fusion transcripts may po-

entially be produced by the t(11;18) (q21;q21) translo-ation, RT-PCR analysis only showed the presence ofhe API2–MALT1 transcript (sets 1 and 2), that is, noeciprocal MALT1–API2 transcript was found (set 6,able 1) (data not shown). The former protein mayherefore play an important role in lymphoma forma-ion.

The cDNA sequence of the case 1 API2–MALT1 fu-ion transcript was the same as those reported in sev-ral cases earlier, suggesting that this breakpoint maye a hot spot (10, 12). In these cases the 59 end of theusion transcript contained all three BIR domains en-oded by API2, suggesting that if a chimeric protein isroduced by the API2–MALT1 fusion transcript, itould have an antiapoptotic function. On the contrary,

he CARD and RING domains of API2 were not presentn the cDNA product. The function of the MALT1 por-ion in the context of the API2–MALT1 fusion tran-cript is unknown.As the b subunit of CD22 exhibits an amino acid

equence homology with MALT1, and CD22 is a hu-an B-lymphocyte-restricted antigen that is expressed

n B-cells as a membrane protein, we supposed thatD22b might be associated with translocation in someALT lymphomas. Although CD22b was expressed in

ur cases, API2–CD22b transcripts were not detected.ikewise, neither API2–laminin 5a3b nor API1–ALT1 transcript was detected. These data suggest

hat CD22b, laminin 5a3b, or API1 may not be asso-iated with translocation in MALT lymphomas.

To gain some insight into the mechanism underlyingPI2–MALT1 translocation, we cloned and sequenced

FIG. 3. Sequences flanking the breakpoint junction in case 1. Thehe pT7Blue T-vector and then sequenced using primers No. 5 and MAnd the case 1 genomic DNA are shown. Nucleotide sequences matchy shadowed boxes. On the other hand, there are 5/7 matches for th

305

he case 1 breakpoint junction at the genomic DNAevel. The 59 sequence of the breakpoint correspondedo that of API2 intron 7. On the other hand, the MALT1reakpoint was located in the intron that contains thelternative splicing exon.In many translocations involving Ig or TCR genes,

ryptic V(D)J heptamer/nonamer consensus se-uences in the immediate vicinity of the breakpointsan be found (26, 27). The significant V(D)J hep-amer consensus sequence (CACAGTG) was found onhe API2 side of the breakpoint in our case. However,his heptamer sequence was not present in any otherPI2 intron sequences. Therefore, it may be reason-ble that this region in intron 7 is a hot spot forecombination events in MALT lymphomas. Whene searched for another consensus heptamer se-uence, CACTGTG, in the whole API2 genomic se-uence, there was only one site containing this se-uence in exon 3. However, there have been no fusionranscript cases involving this area so far. Since thiseptamer sequence is located between the first andecond BIR domains, these data suggest that morehan one BIR sequence may be necessary for lym-homagenesis. On the contrary, the heptamer-likeequence near the MALT1 breakpoint region showednly five of seven matches. Similar matches havelso been reported in ETV6 –AML1 translocations28).

Although translocations of most, if not all, B-cellymphomas involve Ig loci, t(11;18) translocations of

ALT lymphomas involve API2 and MALT1, but notg loci (23). Since the MALT1 protein exhibits a homol-gy with Ig domains, MALT1 may be a member of theg superfamily (11), and might involve the transloca-ional event through an unknown mechanism otherhan the heptamer.

R product with primers API2 No. 4 and MALT1-D1 was cloned into1–GD1 as shown in Fig. 2B. The sequences of API2, EST AW968625the V(D)J heptamer consensus sequence (CACAGTG) are indicatedeptamer in MALT1, as indicated by boxes.

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(11;18) translocation, further studies are necessary onore cases of MALT lymphomas with translocations.

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