Long-distance Inverse PCR Research Articles

Involvement of the CEBP Gene Family in Four IGH@ Chromosomal Translocations in B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL).

Chromosomal translocations involving the immunoglobulin heavy chain locus (IGH@) are rare in BCP-ALL (comprising ~3% of cases) and involve different breakpoints from those found in lymphoma and myeloma. Cytogenetics, metaphase and interphase FISH, using a split-signal probe specific for IGH@, identified four translocations: t(14;19)(q32;q13) (n = 10), t(8;14)(q11;q32) (n = 8), t(14;14)(q11;q32) (n =3) and t(14;20)(q32;q13) (n=2), among a series of BCP-ALL patients. Sequential FISH mapping of these translocations showed that four different members of the CEBP gene family flanked these breakpoint regions. Long-distance inverse PCR with primers specific for the IGHJ6 gene segment allowed molecular cloning of many of these breakpoints and confirmed the involvement of the CEBPA (19q13, n= 4), CEBPD (8q11, n=6), CEBPE (14q11, n =2) genes. Breakpoints were located within the 3′ UTR of CEBPA and either 3′ UTR or the 5′ region of CEBPE, whereas breakpoints in 8q11 were ~30kb centromeric of CEBPD. Over-expression of the respective target genes was shown by QRT-PCR. Evidence that the IGH@ locus directly contributed to gene deregulation was demonstrated by lower expression of CEBPA in 9/17 derived BCP-ALL cell lines without t(14;19)(q32;q13). CEBP proteins have been implicated in the differentiation of myeloid progenitors and CEBPA mutations have been detected in a subset of sporadic and familial acute myeloid leukemia, suggesting that CEBPA may act as a tumor suppressor gene in these malignancies. However, no mutations within the 5′ region of CEBPA comparable to those seen in AML were detected in any cases with t(14;19)(q32;q13), although one case did exhibit deletion of six nucleotides resulting in the loss of 2 amino acids. Moreover, the deregulated expression of CEBPA and other CEBP genes in a subset of BCP-ALL indicates that they may act as dominant oncogenes in B-cell precursors. Constitutive expression of CEBPA and CEBPB in normal lymphoid progenitors has previously been shown to induce myeloid differentiation whilst CEBPE is involved in the terminal differentiation of neutrophils. We showed that expression of full-length CEBPE in the IL3-dependent B-cell precursor cell line Ba/F3 did not result in enhanced proliferation or protection from a variety of apoptotic stimuli, but instead resulted in expression of myeloid differentiation antigens. Most CEBP genes are intronless, different protein isoforms with differing functions being generated translationally. 2-D western blot analysis of nuclear extracts from the SEM BCP-ALL cell line showed selective expression of the 32kd CEBPA protein isoform only, an isoform that lacks the anti-mitotic activity of full-length CEBPA. These data implicate deregulation of the CEBP gene family via juxtaposition to the IGH@ in the pathogenesis of a subset of BCP-ALL. They demonstrate for the first time, the involvement of four members of the same gene family in a single subtype of hematological disease. The transformation of B-cell progenitors may occur through the selective expression of shorter CEBP protein isoforms that lack the ability to induce myeloid differentiation.

A LDI-PCR Based Method Allows the Identification of Any MLL Rearrangement.

Chromosomal rearrangements of the MLL gene are responsible for 5–10% of all acute leukemias, biphenotypic leukemias and myelodysplastic syndromes. 5–10% of these MLL aberrations are therapy-related leukemias with the frequent fusion partners ELL, MLLT3, MLLT10 and MLLT2. The large number of known and unknown MLL fusions (>80) renders a precise diagnosis a demanding task. Even though all MLL rearrangements are associated with high risk acute leukemias, the outcome (favorable or poor) depends on the partner gene. Thus, the identification of MLL gene fusions is necessary for rapid clinical de-ci-sions resulting in specific therapy regimens. After their cytogenetic identification, only the most common MLL fusions MLLT2, MLLT3, MLLT1, MLLT4, ELL and MLLT10 (80%) are investigated by RT-PCR analysis, whereas infrequent or unknown MLL rearrangements (20%) are more or less excluded from further analysis. Therefore we established a LDI-PCR (long-distance inverse PCR) based method that uses small amounts of genomic DNA to determine any type of MLL associated chromosomal abnormality. With this diagnostic tool more than 400 prescreened und unscreened adult and pediatric samples (AML/ALL) from different European diagnostic centers have been analyzed. During this study more than 200 MLL rearrangements were characterized for their precise localization of genomic break-points. The identified MLL rearrangements consist of 25 different fusion genes including the following eight novel MLL partner genes: ACACA, ARGHEF17, BCL9L, MAML2, SELB, SMAP1, and TIRAP. The identified MLL aberrations are basically reciprocal translocations, but also deletions, inversions, insertions and interstitial duplications have been determined. Combining the data of our study and data retrieved from the literature, a total of 88 partner genes can now be annotated. 52 fusion partners (60%) have been characterized on the molecular level whereas at least 36 (40%) remain to be identified. These results demonstrate that this new diagnostic tool in combination with split-signal FISH is qualified for the rapid analysis of known as well as unknown MLL partner genes. Furthermore, the determined patient specific fusion sequences are useful for minimal residual disease (MRD) monitoring of MLL associated acute leukemias. The use of these MRD markers will contribute to improve the treatment and outcome of acute leukemia patients. A first prospective study was already initiated by the German ALL study group and verified the reliability of these genomic markers for MRD monitoring.

T(3;14)(p14.1;q32) involving IGH and FOXP1 is a novel recurrent chromosomal aberration in MALT lymphoma

The three chromosomal translocations t(11;18)(q21;q21), t(14;18)(q32;q21), and t(1;14)(p22;q32) are associated with MALT lymphoma. In a case of MALT lymphoma of the thyroid, we observed t(3;14)(p14.1;q32) by cytogenetic analysis. Fluorescence in situ hybridization studies showed that the immunoglobulin heavy chain locus (IGH) was rearranged on chromosome 14. Long-distance inverse polymerase chain reaction identified FOXP1 as the partner gene on chromosome 3. To determine the frequency of the t(3;14)(p14.1;q32), two fluorescence in situ hybridization assays were established to screen 91 MALT lymphomas, all of which were negative for the above-mentioned three translocations, and eight splenic and six nodal marginal zone lymphomas. Overall, nine MALT lymphomas (10%) harbored t(3;14)(p14.1;q32) comprising tumors of the thyroid (three of six), ocular adnexa (four of 20), and skin (two of 20), whereas those of the stomach (n = 20), salivary gland (n = 20), and lung (n = 5) were negative as well as the splenic and nodal marginal zone lymphomas. Most t(3;14)(p14.1;q32) + MALT lymphomas harbored additional genetic abnormalities, such as trisomy 3. Further studies revealed that the three known translocations and t(3;14)(p14.1;q32) are mutually exclusive. Real-time quantitative reverse transcriptase polymerase chain reaction showed upregulation of FOXP1 in cases with t(3;14)(p14.1;q32) or trisomy 3. This study identifies FOXP1 as a new translocation partner of IGH in a site-dependent subset of MALT lymphomas.

T(3;14)(P14.1;Q32) Involving IGH and FOXP1 Is a Recurrent Chromosomal Aberration in MALT Lymphoma.

Translocations t(11;18)(q21;q21), t(14;18)(q32;q21), and t(1;14)(p22;q32) are known to occur in MALT lymphoma. In a patient with MALT lymphoma of the thyroid a novel t(3;14)(p14.1;q32) was observed by cytogenetic analysis. Subsequent fluorescence in situ hybridization studies showed that the immunoglobulin heavy chain locus (IGH) was rearranged on chromosome 14. Long-distance inverse polymerase chain reaction identified FOXP1 as the partner gene on chromosome 3. Two FISH-assays were established to screen 90 MALT lymphomas, 8 splenic and 6 nodal marginal zone lymphomas for this aberration. Overall, 8 MALT lymphomas (9%) harbored t(3;14)(p14.1;q32); the aberration was detected in MALT lymphomas of the ocular adnexa (4 of 20), thyroid (2 of 5), and skin (2 of 20), but not in the stomach (n=20), salivary gland (n=20), and lung (n=5). Splenic and nodal marginal zone lymphomas were t(3;14)(p14.1;q32) negative. Further 38 MALT lymphomas with known t(11;18)(q21;q21), t(14;18)(q32;q21), or t(1;14)(p22;q32) did not reveal a t(3;14)(p14.1;q32). Most t(3;14)+ MALT lymphomas (7 of 8) harbored additional genetic abnormalities, such as +3. Real-time quantitative RT-PCR showed up-regulation of FOXP1 in cases with t(3;14)(p14.1;q32) or trisomy 3. This study identifies FOXP1 as a new translocation partner of IGH in MALT lymphomas.

Rapid amplification of immunoglobulin heavy chain switch (IGHS) translocation breakpoints using long-distance inverse PCR

Molecular cloning of immunoglobulin heavy chain (IGH) translocation breakpoints identifies genes of biological importance in the development of normal and malignant B cells. Long-distance inverse PCR (LDI-PCR) was first applied to amplification of IGH gene translocations targeted to the joining (IGHJ) regions. We report here successful amplification of the breakpoint of IGH translocations targeted to switch (IGHS) regions by LDI-PCR. To detect IGHS translocations, Southern blot assays using 5' and 3' switch probes were performed. Illegitimate Smu rearrangements were amplified from the 5' end (5'Smu LDI-PCR) from the alternative derivative chromosome, and those of Sgamma or Salpha were amplified from the 3' end (3'Sgamma or 3'alpha LDI-PCR) from the derivative chromosome 14. Using a combination of these methods, we have succeeded in amplifying IGHS translocation breakpoints involving FGFR3/MMSET on 4p16, BCL6 on 3q27, MYC on 8q24, IRTA1 on 1q21 and PAX5 on 9p13 as well as BCL11A on 2p13 and CCND3 on 6p21. The combination of LDI-PCR for IGHJ and IGHS allows rapid molecular cloning of almost all IGH gene translocation breakpoints.

Coexistent Rearrangements of c-MYC,BCL2, andBCL6 Genes in a Diffuse Large B-Cell Lymphoma

We present a patient with stage III de novo diffuse large B-cell lymphoma. The lymphoma cells showed mature B-cell immunophenotype but lacked surface immunoglobulin (Ig) expression. Long-distance and long-distance inverse polymerase chain reaction assays to detect the oncogene/Ig gene rearrangement revealed that the cells carried 3 independent fusion genes, namely, c-MYC/Ig heavy chain gene (IgH), BCL2/IgH, and Ig lambda light chain gene/BCL6. Thus, the lymphoma cells concurrently carried t(8;14)(q24;q32), t(14;18)(q32;q21), and t(3;22)(q27;q11), which developed in association with class switching, V/D/J recombination, and somatic hypermutation, respectively. The lymphoma responded to chemoradiotherapy, and the patient has been well for 2 years, suggesting that multiple oncogene rearrangements may not necessarily be associated with poor clinical outcome.

Etoposide induces chimeric Mll gene fusions.

MLL gene fusions are the hallmark of more than 70% of therapy-related leukemias (t-ML) associated with topoisomerase II inhibitors (e.g., etoposide) and cause leukemia in murine transgenic models. To determine whether Mll genomic fusions can occur after exposure to topoisomerase II inhibitors, we developed a long-distance inverse PCR DNA-based assay for chimeric Mll fusions in mouse embryonic stem cells. We detected Mll fusions at a higher frequency following 100 microM etoposide for 8 h (16x10(-6) cell(-1)) than in no-drug controls (1.0x10(-6) cell(-1), P=0.0002) or after treatment with a comparably cytotoxic exposure to the antimicrotubule drug vincristine (1.0x10(-6) cell(-1), P=0.0047). The fusion points in Mll chimeric products induced by etoposide were localized to a 1.5 kb region between exons 9 and 11, analogous to the MLL breakpoint cluster region in human leukemia. All 49 Mll fusion partners analyzed matched known genomic murine sequences, with 40 (82%) matching annotated genes covering eighteen murine autosomes. One partner was Runx1, the murine homologue of the transcription factor AML-1, a target of human translocations in therapy-related leukemia. These findings indicate that etoposide triggers the formation of Mll gene fusions, a critical step for the development of treatment-induced leukemic transformation.

BCL6 gene translocation in follicular lymphoma: a harbinger of eventual transformation to diffuse aggressive lymphoma

AbstractFollicular lymphoma (FL) is characterized by a relatively indolent clinical course, but the disease often transforms into a more aggressive large cell lymphoma with a rapidly progressive clinical course. In the present study, we analyzed 41 cases of FL known to have subsequently transformed to aggressive lymphoma and an additional 64 FL samples from patients not subsequently transformed. We studied BCL6 gene rearrangement by the methodology of long-distance inverse polymerase chain reaction (LDI-PCR). Of the 41 cases known to transform, 16 (39.0%) harbored BCL6 translocation or deletion at the time of FL diagnosis. Among 64 cases not known to transform, BCL6 translocation was detected in 9 (14.1%). The prevalence of BCL6 translocation in the group known to transform was significantly higher (P = .0048). Among the transformation cases, the partners of the BCL6 translocation were identified in 13 cases and included IGH, CIITA, U50HG, MBNL, GRHPR, LRMP, EIF4A2, RhoH/TTF, and LOC92656 (similar to NAPA), whereas in the control group the BCL6 partners were IGH, CIITA, SIAT1, and MBNL. In 13 cases paired specimens before and after transformation were available. Among these paired specimens, a loss (3 cases) or a gain (1 case) of BCL6 translocation was observed after the transformation. Analysis of clonality showed that all of these cases represented the evolution of a subclone of the original tumor population. Our study demonstrated that BCL6 translocation is not necessary for transformation but that BCL6 translocation in FL may constitute a subgroup with a higher risk to transform into aggressive lymphoma.

MALT1 is deregulated by both chromosomal translocation and amplification in B-cell non-Hodgkin lymphoma

AbstractThe MALT1 gene was identified through its involvement in t(11;18)(q21;q21), seen in 30% of cases of mucosa-associated lymphoid tissue (MALT) lymphoma. Here, we show that deregulated MALT1 expression may occur in B-cell non-Hodgkin lymphoma (B-NHL) of various histologic subtypes either through translocation to the immunoglobulin heavy chain (IGH) locus or by genomic amplification. First, 2 cases, one case of MALT lymphoma and another of aggressive marginal zone lymphoma (MZL) with t(14;18)(q32;q21), cytogenetically identical to the translocation involving BCL2, were shown by fluorescence in situ hybridization (FISH) to involve MALT1, which lies about 5 Mb centromeric of BCL2. Molecular cloning of both by long-distance inverse polymerase chain reaction showed breakpoints lying 1 to 2 kilobase (kb) centromeric of the first 5′ MALT1 exon; both cases showed MALT1 overexpression at either RNA or protein levels. Second, we examined the structure and gene expression profile of genomic amplifications involving 18q21 in a panel of 40 B-NHL cell lines using comparative genomic hybridization to microarrays (array CGH) and gene expression profiling techniques. Using array CGH, 2 peaks of genomic amplification were observed, one centered around BCL2 and the other around MALT1.Ofthe 3 cell lines with MALT1 amplification, 2 showed MALT1 overexpression as assessed by gene profiling, quantitative reverse transcription–polymerase chain reaction (QRT-PCR), and Western blotting. To determine if comparable events occurred in primary MALT and splenic MZL tumors, 40 cases were analyzed by FISH or QRT-PCR; genomic amplification and MALT1 overexpression were seen in 2 cases. Together, these data implicate MALT1 as a dominant oncogene that may play a role in the pathogenesis of B-NHL.

Non-immunoglobulin/ BCL6 Gene Fusion in Diffuse Large B-cell Lymphoma: Prognostic Implications

3q27 translocation affecting the BCL6 gene is one of the most common chromosomal abnormalities in diffuse large B-cell lymphoma (DLBCL). BCL6 translocation can involve not only one of the three immunoglobulin gene (Ig ) loci but also another non- Ig chromosomal locus. 5'-rapid amplification of cDNA ends and long-distance inverse polymerase chain reaction (PCR) methods have identified a total of 13 recurrent non- Ig partner genes to date. As the result of non- Ig / BCL6 translocation, many types of regulatory sequences of each partner gene substitute for the 5' untranslated region of the BCL6 and the rearranged BCL6 is presumed to be under the control of the replaced promoter activity. BCL6 translocation occurs more frequently in extranodal DLBCL than in node-based disease. However, the impact of BCL6 translocation on the treatment outcome of DLBCL has been the subject of controversy. We found that survival of DLBCL patients with non- Ig partners was inferior to that of those with Ig / BCL6 translocation, suggesting that non- Ig / BCL6 fusion is a poor prognostic indicator of DLBCL. We next created BCL6 expression plasmids containing a series of non- Ig / BCL6 fusion genes. COS-7 cells transiently transfected with these plasmids expressed high levels of Bcl-6 protein and showed characteristic punctate nuclear staining. These findings suggested that non- Ig / BCL6 translocation plays a pathogenetic role in a proportion of DLBCL.

Cyclin D3 is a target gene of t(6;14)(p21.1;q32.3) of mature B-cell malignancies

AbstractChromosomal translocation t(6;14)(p21.1;q32.3) has been reported as a rare but recurrent event not only in myeloma and plasma cell leukemia but also in diffuse large B-cell non-Hodgkin lymphoma (B-NHL) (diffuse large B-cell lymphoma [DLBCL]) and splenic lymphoma with villous lymphocytes (SLVL); however, the nature of the target gene(s) has not been determined. This study identified t(6;14)(p21.1;q32.3) in 3 cases of transformed extranodal marginal zone B-NHL, in 1 case of SLVL, and in 1 case of a low-grade B-cell lymphoproliferative disorder. In a sixth case, a CD5+ DLBCL, the translocation was identified by molecular cloning in the absence of cytogenetically detectable change. Two chromosomal translocation breakpoints were cloned by using long-distance inverse polymerase chain reaction methods. Comparison with the genomic sequence for chromosome 6p21.1 showed breakpoints approximately 59 and 73.5 kilobases 5′ of the cyclin D3 (CCND3) gene with no other identifiable transcribed sequences in the intervening region. Although Southern blotting with derived genomic 6p21.1 probes failed to detect other rearrangements, fluorescent in situ hybridization assays, using BAC (bacterial artificial chromosome) clones spanning and flanking theCCND3 locus, along with probes for IGHconfirmed localization of 6p21.1 breakpoints within the same region, as well as fusion of the CCND3 and IGH loci. Furthermore, in all cases, high-level expression of CCND3was demonstrated at RNA and/or protein levels by Northern and Western blotting and by immunohistochemistry. These data implicateCCND3 as a dominant oncogene in the pathogenesis and transformation in several histologic subtypes of mature B-cell malignancies with t(6;14)(p21.1;q32.3) and suggest thatCCND3 overexpression seen in about 10% of DLBCL cases may have a genetic basis.

Establishment and characterization of a new human myeloma cell line, KYδ-1, producing the δ/κ type immunoglobulin

We describe the establishment and characterization of a new multiple myeloma (MM) cell line, KYδ-1, which expressed δ/κ type immunoglobulin (Ig). The patient was a 65-year-old woman with MM, who presented extramedullary dissemination, lymphadenopathy and short survival. The KYδ-1 cell line was derived from the pleural fluid obtained in the terminal phase of the disease. The cells expressed δ/κ Ig in the cytoplasm, and CD10, CD29, CD33, CD38, CD44, CD54, and HLA-DR antigens on the cell surface. Chromosomal analysis revealed two independent translocations, t(3;14)(p21;q32) and t(3;11)(p21;q13), which were confirmed by fluorescence in situ hybridization using chromosome painting probes. Reverse transcriptase-mediated polymerase chain reaction (PCR) and Northern blot analyses demonstrated overexpression of the CCND1 gene, suggesting alteration of the BCL1- CCND1 locus. We thus performed long-distance inverse PCR using nested primers for the Cα constant region of immunoglobulin heavy chain gene ( IGH) and obtained a clone that encompassed the 11q13/ IGH fusion. Nucleotide sequencing determined that the fusion occurred at the Sα2 switch region and at the centromeric side of the major translocation cluster of BCL1. The other IGH allele consisted of a VDJ complex that was adjacent to the Cδ constant gene, indicating that a class switch-like mechanism from the Cμ to Cδ was involved in the production of the Ig δ heavy chain. Point mutations within the P53 and N-RAS genes were presumably related to the rapidly progressive disease in this particular MM patient.

Prenatal origin of acute lymphoblastic leukaemia in children

There is little current insight into the natural history of childhood leukaemia or the timing of relevant mutational events. TEL-AML1 gene fusion due to chromosomal translocation is frequently seen in the common form of childhood acute lymphoblastic leukaemia. We investigated whether this abnormality arises prenatally. We identified, by reverse-transcriptase PCR screening of blood or bone marrow, TEL-AML1 fusion in 12 children, plus a pair of identical twins, aged 2-5 years from Italy and the UK, who had newly diagnosed acute lymphoblastic leukaemia. We amplified and sequenced the genomic TEL-AML1 fusion gene with a long-distance inverse PCR method. Primers were designed that could be used in short-range PCR to screen for patient-specific, leukaemia clone-specific TEL-AML1 genomic fusion sequences in neonatal blood spots from each child. We initially identified TEL-AML1 fusion sequences in blood spots from the identical twins, diagnosed with concordant acute lymphoblastic leukaemia at age 4 years, who shared a single or clonotypic TEL-AML1 sequence that suggested prenatal origin in one twin. Three children were excluded because control genes could not be amplified. Of the other nine patients, six had positive blood spots. Blood spots that were classified as negative were uninformative. Our findings showed that childhood acute lymphoblastic leukaemia is frequently initiated by a chromosome translocation event in utero. Studies in identical twins show however that such an event is insufficient for clinical leukaemia and that a postnatal promotional event is also required.

Molecular Cloning of Translocation t(1;14)(q21;q32) Defines a Novel Gene (BCL9)at Chromosome 1q21

AbstractAbnormalities of chromosome 1q21 are common in B-cell malignancies and have been associated with a poor response to therapy. The nature of the involved gene(s) on chromosome 1q21 remains unknown. A cell line (CEMO-1) has recently been established from a patient with precursor-B–cell acute lymphoblastic leukemia (ALL), which exhibited a t(1;14)(q21;q32). To identify the gene involved in this translocation, we have cloned both rearranged IGHJ alleles using long-distance inverse polymerase chain reaction (LDI-PCR). TwoIGHJfragments were amplified from CEMO-1 DNA and sequenced. One allele showed novel sequences upstream of JH5with no homology to either IGHor any other sequences on the databases. Using a single-copy Xho I fragment immediately 5′ ofJH5, PAC clones were isolated and mapped to chromosome 1q21 on normal metaphases by fluorescence in situ hybridization (FISH), confirming that this allele represented the t(1;14)(q21;q32) breakpoint. Sequence analysis of the 1q21 XhoI fragment showed identity with an expressed sequence tag (EST), and this probe was therefore used to probe Northern blots. Two transcripts of 6.3 kb and 4.2 kb expressed at low level in mRNA from all tissues were detected: a third transcript of 1.6 kb was expressed only in thymus, spleen, and small intestine. Full-lengthBCL9cDNA clones were obtained from a normal human fetal brain cDNA library supplemented by 5′ and 3′ RACE. Sequence analysis predicted a protein of 1394 amino acids containing 18% proline, 11% glycine, 11% serine, and 6% methionine, but no recognizable protein motifs or significant homologies to any other known proteins. The CEMO-1 1q21 breakpoint fell within the 3′ UTR of the BCL9gene. Low-level expression of BCL9was detected in Epstein-Barr virus-transformed normal B cells by Northern blot; in contrast, abundant BCL9expression was observed in CEMO-1, indicating that deregulated expression of this gene was one pathological consequence of the translocation. Screening of a panel of 39 B-cell malignancies with 1q abnormalities by Southern blot showed one additional case with a breakpoint in the 3′ UTR ofBCL9, indicating that this was a recurrent breakpoint. FISH analysis using an 850-kb YAC spanning BCL9 identified a further case with t(1;22)(q21;q11) causing juxtaposition of BCL9 to theIGλ locus. Other breakpoints were heterogeneous, falling both centromeric (10 cases) and telomeric (10 cases) of the BCL9gene. These data suggest that BCL9may be the target of translocation in some B-cell malignancies with abnormalities of 1q21 and that deregulated BCL9expression may be important in their pathogenesis.

Molecular Cloning of Translocation t(1;14)(q21;q32) Defines a Novel Gene (BCL9) at Chromosome 1q21

Abnormalities of chromosome 1q21 are common in B-cell malignancies and have been associated with a poor response to therapy. The nature of the involved gene(s) on chromosome 1q21 remains unknown. A cell line (CEMO-1) has recently been established from a patient with precursor-B-cell acute lymphoblastic leukemia (ALL), which exhibited a t(1;14)(q21;q32). To identify the gene involved in this translocation, we have cloned both rearranged IGHJ alleles using long-distance inverse polymerase chain reaction (LDI-PCR). Two IGHJ fragments were amplified from CEMO-1 DNA and sequenced. One allele showed novel sequences upstream of JH5 with no homology to either IGH or any other sequences on the databases. Using a single-copy Xho I fragment immediately 5' of JH5, PAC clones were isolated and mapped to chromosome 1q21 on normal metaphases by fluorescence in situ hybridization (FISH), confirming that this allele represented the t(1;14)(q21;q32) breakpoint. Sequence analysis of the 1q21 Xho I fragment showed identity with an expressed sequence tag (EST), and this probe was therefore used to probe Northern blots. Two transcripts of 6.3 kb and 4.2 kb expressed at low level in mRNA from all tissues were detected: a third transcript of 1.6 kb was expressed only in thymus, spleen, and small intestine. Full-length BCL9 cDNA clones were obtained from a normal human fetal brain cDNA library supplemented by 5' and 3' RACE. Sequence analysis predicted a protein of 1394 amino acids containing 18% proline, 11% glycine, 11% serine, and 6% methionine, but no recognizable protein motifs or significant homologies to any other known proteins. The CEMO-1 1q21 breakpoint fell within the 3' UTR of the BCL9 gene. Low-level expression of BCL9 was detected in Epstein-Barr virus-transformed normal B cells by Northern blot; in contrast, abundant BCL9 expression was observed in CEMO-1, indicating that deregulated expression of this gene was one pathological consequence of the translocation. Screening of a panel of 39 B-cell malignancies with 1q abnormalities by Southern blot showed one additional case with a breakpoint in the 3' UTR of BCL9, indicating that this was a recurrent breakpoint. FISH analysis using an 850-kb YAC spanning BCL9 identified a further case with t(1;22)(q21;q11) causing juxtaposition of BCL9 to the IGlambda locus. Other breakpoints were heterogeneous, falling both centromeric (10 cases) and telomeric (10 cases) of the BCL9 gene. These data suggest that BCL9 may be the target of translocation in some B-cell malignancies with abnormalities of 1q21 and that deregulated BCL9 expression may be important in their pathogenesis.