Human Molecular Genetics, 2001, Vol. 10, No. 12 1275-1285
© 2001 Oxford University Press
B-cell neoplasia associated gene with multiple splicing (BCMS): the candidate B-CLL gene on 13q14 comprises more than 560 kb covering all critical regions
Abteilung Organisation Komplexer Genome (H0700), Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany and 1Innere Medizin III, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany
Received 17 January 2001; Revised and Accepted 4 April 2001.
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ABSTRACT |
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Deletions in chromosomal band 13q14.3 occur in >50% of B-cell chronic lymphocytic leukemias (B-CLL) and mantle cell lymphoma, indicating the localization of a tumor suppressor gene involved in the pathomechanism of these diseases. Within a 400 kb recurrently deleted segment at least two minimally deleted subregions had been reported. For the two genes residing in the proximal subregion, initially named LEU1 and LEU2, a pathogenic role has not yet been established. We report here that LEU1 is only a small portion of a large gene, which spans all previously reported critical subregions including the distal subregion. This gene, designated B-cell neoplasia-associated gene with multiple splicing (BCMS), is composed of at least 50 exons spanning
560 kb of genomic DNA and is expressed in more than 20 RNA splicing variants. While tissue-specific expression of RNA variants was observed, there was no evidence for the expression of a variant specific for B-CLL. Sequence analysis of the RNA variants suggests that BCMS transcripts belong to the group of non-coding RNAs. The alignment of the gene with all critical subregions provides a strong argument for BCMS being the most likely candidate for the tumor suppressor gene in 13q14 involved in the leukemogenesis of B-CLL. Due to the limited understanding of functional RNAs, however, it remains difficult to prove the pathogenic role of BCMS. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B-cell chronic lymphocytic leukemia (B-CLL) is characterized by a progressive accumulation of CD5+ B-lymphocytes associated with autoimmunity and immunodeficiency (1). It is the most common leukemia in adults in Western countries. While the pathomechanism of this disease is not well understood (2), genomic aberrations were shown to predict patient survival (3). The most frequent chromosomal aberrations are loss of material on chromosome band 13q14 in >50% of cases (4) followed by deletions affecting 11q22–q23 and trisomy of chromosome 12 (5–7). Similarily, deletion of 13q14 is the most frequent chromosome aberration (
60%) in mantle cell lymphoma (MCL), affecting the same genomic subregion (8). In B-CLL translocations at 13q14 were shown to be accompanied by loss of the marker D13S25 located 1.6 cM distal to the RB1 gene (9). Detailed deletion analyses revealed a segment flanked by D13S273 and D13S25 as the critical region (10,11). High resolution contigs of genomic fragments spanning the D13S273–D13S25 interval were generated independently by several groups (4,10,12–16) and utilized to assess the critical subregions (Fig. 1). We identified two minimally deleted subregions within a 400 kb segment defined by the two flanking markers PAC-272–3 and PAC-48 (4). The proximal subregion overlaps in part with the regions reported by Kalachikov et al. (13), Liu et al. (14) and Corcoran et al. (15), while the distal subregion coincides with a portion of the larger deleted segments published by Bullrich et al. (10) and Bouyge-Moreau et al. (12). These two subregions are represented by PAC-272–3 (120 kb in length) and PAC-58 (80 kb in length), respectively. Their extensions and positions are indicated in Figure 1. Within the proximal critical subregion, three expressed genes were identified (14,17), whereas the coding information within the distal subregion remained obscure. Kapanadze et al. (17) identified a gene encoding a zinc-finger domain of the RING type termed RFP2 (originally named LEU5), which resides in the proximal deleted subregion (17). It shares homologies with known tumor suppressor genes like the RET finger protein and BRCA1. However, mutation analysis of RFP2 did not reveal any point mutations in the open reading frame (ORF) (unpublished data; 17). Approximately 32 kb distal to RFP2, two genes were identified: originally termed LEU1, t5 or cDNA 170C-70 [now B-cell neoplasia-associated gene with multiple splicing, (BCMS)] and LEU2, t4 or cDNA 1B4 [now BCMS upstream neighbor (BCMSUN)], 1.2 and 2.1 kb in length, respectively (14). Their orientation is head-to-head with their 5' ends being only 193 bp apart. The existence of an independently expressed homolog of BCMSUN on 1p22–p31 (BCMSUN-like) has been reported (18). Deletion of a genomic segment containing the first exons of BCMS and BCMSUN was found in 170 cases with chromosomal loss affecting 13q14. However, in this series no inactivating mutations were found within the second allele (17,19). We report here on the analysis of the critical subregions within 13q14.3 disclosing a large gene, which covers the whole range of critical subregions identified so far. Detailed analysis revealed that this gene is subject to extensive alternative splicing with LEU1 being only one of the smallest splicing variants of the gene, which is now termed BCMS. Based on its primary structure and localization, BCMS seems to be the most likely candidate for the tumor suppressor gene in 13q14.3 relevant for B-CLL pathogenesis.
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RESULTS |
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Genomic organization of BCMS
The initial step in cloning BCMS was exon trapping from PAC-48 and PAC-58 (Fig. 1). This resulted in the identification of BCMS exons 12, 17, 19 and 28 derived from PAC-48, and exon 21 derived from PAC-58. To complete the coding information, 5'- and 3'-RACE-PCR experiments were performed using primers, which were based on the sequences of the trapped exons. RACE-PCR products varied considerably in length pointing to the possibility of alternatively spliced RNA products. After cloning of the RACE products, each fragment was sequenced uncovering a number of splicing variants. Additional splicing variants were obtained by RT–PCR from RNA of a series of human tissues (peripheral blood, leukocytes, testis, fetus, brain, small intestine, heart, pancreas) and cell lines (20–22) with primers originating from the new exon sequences. Detailed cDNA analyses revealed a total of 41 different mRNAs composed of 50 exons (Table 1). Since many RACE reactions were performed in both directions starting from internal exons, it is possible that a few 5'- and 3'-RACE fragments are part of the same splice variant. The minimal number of different splicing variants of BCMS is 25 (Fig. 2).
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Repeated RACE experiments and subsequent RT–PCRs showed that the exons of the previously published LEU1 gene represent 5' exons of the BCMS gene. Comparison of the BCMS cDNA sequences with known genomic sequences compiled in public DNA databases by BLAST searches utilizing the NCBI service, allowed us to determine genomic distances between exons and to order alternatively used exons from different transcript variants. Altogether, 49 of the 50 exons were fine mapped, while for one exon (11*) the genomic position remains tentative due to gaps in the published genomic sequences (Fig. 2). The exon–intron boundary sequences are listed in Table 2.
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Despite numerous RACE and SMART-RACE experiments designed to extend the 5'-coding sequence, no exon upstream of the first exon of LEU1 was detected. The 3'-RACE experiments indicated at least 10 alternative 3'-terminal exons containing polyadenylation signals linked to poly(A) stretches. One transcript variant (variant H, Fig. 2) was apparently primed from a genomic poly(A) stretch and lacks a polyadenylation signal motif within its 3'-terminal exon 26 (Table 2). While the possibility of other downstream exons present in yet unidentified splicing variants cannot be excluded, our data indicate a total length of BCMS of
560 kb. The largest genomic distance between exons was nearly 160 kb (exons 44–45). The exons range in size from 53 (exon 2) to 1251 bp (exon 4*). In total we determined nearly 11 kb (10934 bp) of cDNA sequence for BCMS distributed on cDNA clones ranging in size from 2.1 kb (variant AL) to 464 bp (variant C). Exons 22–26 and 40–43 are joined on the genomic sequence level. In other variants, individual exons from these groups are singled out (e.g. exon 20 in variant H, exons 24 and 41 in variant J and exons 23 and 24 in variant I) indicating their status as independent exons. The largest segment that is spliced out from the primary transcript is >160 kb in length (variant G, between exons 29 and 45). Table 3 lists the tissues from which the variants were isolated.
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Only 14 of the 50 exons of BCMS possess continuous ORFs: exons 1, 4, 5, 11*, 6, 8, 13, 17, 22, 28, 34, 36, 37 and 46. Notably, no transcript was identified which consists only of a combination of these exons, i.e. all transcripts contain internal exons without contiguous ORFs. Thus, BCMS is unlikely to be translated into a protein. In this context, it is important to note that the suggested ORF of 72 amino acids of the LEU1 gene published by Liu et al. (14) is disrupted in two splicing variants identified in the present study (variants B and C, Fig. 2). The corresponding ORF-disrupting exons 2 and 3 were observed in B-CLL lymphocytes as well as in eight different normal tissues.
Expression analysis of BCMS transcripts
To analyze the expression of BCMS transcripts we used RT–PCR experiments (Fig. 3) as well as poly(A)+ multiple tissue northern blots (Fig. 4). RT–PCRs with primers P8 and P5 showed a variety of different splicing variants of the BCMS gene with a tissue-specific expression pattern (Fig. 3). Composition of the BCMS variants was assessed by DNA sequence analysis of the gel-purified fragments. Note the degree of variation of splicing products in the different samples. In the northern blot analyses, probing with the LEU1 sequence, which corresponds to variant A representing the BCMS exons 1 and 4, revealed a 1.1 kb band equivalent to the previously published LEU1 signal (Fig. 4A). This analysis uncovered a strong expression in heart, skeletal muscle, testis and pancreas and a weaker expression in placenta, lung, liver, kidney, spleen, thymus, prostate, uterus, small intestine and colon (Fig. 4A). Additional bands were 1.7, 2.6, 2.8 and 4.4 kb in length. The 1.7 and 4.4 kb bands coincide with the 18S and 28S rRNA, respectively, i.e. they represent background signals (data not shown). The 2.6 kb transcript is expressed at low levels in heart, placenta, liver, spleen, thymus, prostate, testis, uterus and colon. The longest transcript, 2.8 kb in length, detected with variant A as a probe is expressed in heart, skeletal muscle, pancreas, spleen, prostate and testis. Hybridizing with cDNA variant AK containing exons 1, 2, 17, 28, 29, 33, 40 and 42 reveals a different expression profile with additional bands on the northern blots (Fig. 4B). The 1.1 kb transcript exhibits a similar expression pattern as observed on the variant A probed blot. Additional transcripts are detected in skeletal muscle (800 bp) and pancreas (600 bp). The pancreas-specific transcript would match with our smallest splicing variant C containing exons 1, 2 and 3. Pancreatic tissue exhibits additional mRNAs of the BCMS gene: one faint 900 bp band and a transcript of 1.15 kb corresponding to our variant B containing exons 1, 2 and 4. The hybridization shows an 6 kb band in heart, placenta, skeletal muscle and in pancreas. Both the skeletal muscle and pancreatic RNAs exhibit a very faint pattern of additional bands ranging between 1.4 and 4.4 kb. This points to the existence of several additional splicing variants of BCMS, which was expected from the results of the RACE experiments.
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The differences in the detected splicing variants using different probes from the same gene imply that there is at least one more start of transcription in addition to exon 1, since otherwise all variants would have been detected by using exon 1 as a probe. Reverse transcription of RNA sequences upstream of exon 17 also indicated the existence of a transcription start site downstream of exon 1 (not shown). Variant I containing exons 19, 20, 21, 22, 25, 27, 28, 29, 33, 36, 37 and 39, i.e. only a 3'-portion of the gene, revealed two very strongly expressed transcripts in pancreatic tissue 1.1 and 1.7 kb in size (Fig. 4C). Variant I also detects a 5.5 kb transcript in heart and skeletal muscle. When only exon 40 was used as a probe the 1.1 kb transcript appeared in placenta and pancreatic tissue, whereas the 6 kb transcript exhibits the strongest expression in heart and skeletal muscle and a weaker expression in peripheral blood leukocytes (Fig. 4D). In skeletal muscle RNA a number of splicing variants were observed. In summary, there are a number of BCMS transcript variants expressed in a tissue-specific manner. The transcript sizes vary between
600 bp (matching with variant C) and 6 kb.
Analysis of BCMS in B-CLL patients
The assessment of a pathogenic function of BCMS in B-CLL is particularly demanding, because there are no ORFs spanning several exons and thus, it is difficult to prove inactivation of a gene product by point mutations. We performed DNA sequence analyses of the genomic sequences of the most frequently occurring exons including exon–intron boundaries, namely of exons 17, 28, 29, 33 and 40, in a series of B-CLL samples with monoallelic deletions affecting 13q14 including marker D13S25 (20 cases) and control samples (10 cases). No B-CLL-specific sequence alteration was observed. Similarly, RT–PCR analysis in these patient and control samples, as well as in FACS-sorted CD19/CD20-positive B-cells, showed expression of BCMS in B-CLL, but did not uncover a leukemia-specific expression of splicing variants.
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DISCUSSION |
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The most frequently deleted region in B-CLL and MCL is located in 13q14.3 distal to RB1 (4,5,8,9). Detailed analysis of the deleted region in a series of B-CLLs revealed two commonly deleted subregions 120 and 80 kb in size characterized by PAC-272–3 and PAC-58, respectively (4). Three genes were described in the 120 kb subregion, of which LEU1 is the most distal one, i.e. the gene closest to the 80 kb subregion (14,17). In this study we demonstrate that LEU1 consitutes only the 5' end of a much larger gene, named BCMS, which extends to the marker D13S294 and spans the entire 80 kb subregion. Thus, the two deleted subregions contain coding information for the same gene, offering an easy explanation for the occurrence of two distinct commonly lost genomic regions. Alignment of the gene structure with the previously reported commonly deleted regions (Fig. 1) showed that all regions affect at least part of BCMS resolving the presumed discrepancies between the data reported from different groups (4,10,12–16). While LEU1 was originally considered a candidate for the putative tumor suppressor gene of B-CLL (14), it was regarded a less likely candidate later on based on its location proximal to the commonly deleted subregions defined by various groups (10,12,16).
With 50 identified exons and a length of 560 kb, BCMS belongs to the very large human genes. Expression analysis by reverse transcription uncovered an enormously high variability of splicing variants. This variability concerns selection, number as well as combination of exons, with a particularly high number of alternative 3'-terminal exons. Therefore, in accordance with the previously named genes BCL1–BCL10 (OMIM), the gene was called BCMS. The splicing signal profiles for the BCMS exons are given in Table 2. The vast majority of the recognition motifs are corresponding to the human splice site consensus (23). In alternatively spliced genes the commonly observed rules for splicing, such as intron size and homology to the consensus exon–intron boundary sequences, are often violated (24). The deviations from the consensus sequences observed for the splicing donor and acceptor sites in BCMS are in agreement with this notion on genes expressing multiple splice variants.
Alternative splicing has often been observed to result in tissue-specific variants of the coding information. Northern blot analysis of multiple tissues revealed characteristic distributions of RNA variants. Tissue-specific expression of splicing variants can be the basis for a functional variation of the protein encoded by the respective gene. However, no ORF of significant length could be observed in BCMS transcripts and since all transcripts contain internal exons without a contiguous ORF, modulation of protein function cannot serve as an explanation for the multiple splicing of BCMS transcripts. While it cannot be formally excluded that a short ORF of BCMS codes for a biologically active peptide, the highly variable splicing, which consistently results in disruption of contiguous coding information argues strongly against a role of short ORFs from which biologically active translation products would derive.
Indeed, BCMS is most likely transcribed into a non-coding RNA (ncRNA). This group of ncRNAs has recently gained considerable interest, as the number of identified functional RNAs is steadily increasing. Most of these RNAs are small RNA polymerase III products, but following the identification of the X-inactive specific transcript (XIST) RNA involved in the inactivation of mammalian X chromosomes, a number of longer RNA polymerase II transcripts were reported (25–27). BCMS appears to add a particularly long gene to this new group of genes. While the increasing availability of genomic DNA sequences fosters the identification of new genes via gene searching algorithms, these approaches generally fail to identify genes of ncRNAs, since the computational analyses rely mainly on the identification of ORFs. As long as the knowledge on common motifs of functional RNA concerning the primary, secondary or tertiary structure remains limited, isolation of ncRNAs will be restricted to detailed locus-specific studies. On the other hand, due to the lack of a broader understanding of the mechanisms in which ncRNAs function, it is extremely difficult to prove a pathogenic effect of alterations in the respective genes. Accordingly, DNA sequence analysis has not yet provided a clue for a pathogenic role of this gene. Analysis of the 5' end of BCMS, formerly considered as the LEU1 gene, in patient samples led Rondeau et al. (19) to rule out LEU1 as a B-CLL relevant gene.
As no genomic alteration within the critical region on 13q14 has so far been identified, to which a pathogenic effect in B-CLL can be unequivocally assigned, the possibility of a pathogenic function of the genes within the critical region through haploinsufficiency needs to be considered. The phenomenon of haploinsufficiency can be explained by protein dosage effects or by a functional null zygosity. Since BCMS is unlikely to code for a protein, its pathogenic contribution might be based on somatic inactivation of the second allele via epigenetic factors. However, our investigation of the expression of BCMS in 20 B-CLL patients with monoallelic deletion of the gene has so far not revealed evidence for such a phenomenon.
The extremely high variability of splicing products of BCMS provokes the question, whether there are splicing variants specific for certain cell types or for transformed cells. While the northern analysis revealed tissue-specific occurence of several variants, RT–PCR analysis of peripheral blood lymphocytes from B-CLL patients and probands did not uncover a B-CLL-specific variant.
Recent analyses of the commonly deleted subregions in 13q14.3 and the genes residing therein have not yet revealed clues for the pathomechanisms of B-CLL. Following detailed analysis, LEU1, initially a strong candidate gene, was even ruled out by some investigators (19). However, our data demonstrating the existence of a large gene, of which LEU1 is only a minor portion of the 5' end, spanning all seemingly divergent critical subregions reported by different groups, provide a strong argument for reviving the expressed sequences from this region at D13S272 as candidate. Indeed, from our data it could be concluded that BCMS is a very good candidate for the tumor suppressor gene in 13q14 relevant for B-CLL. However, without a better understanding of the function of RNA polymerase II ncRNA products such as the BCMS transcript, the pathogenic role of BCMS remains undefined.
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MATERIALS AND METHODS |
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RNA and DNA preparation
Total RNA and genomic DNA were isolated from the cell lines Granta-519 (DSMZ no: ACC 342), EHEB (DSMZ no: ACC67) and JVM-2 (DSMZ no: ACC12) (20–22) as well as from mononuclear cell preparations of B-CLL patients and of control persons (obtained after Ficoll density gradient centrifugation) with the Trizol reagent (Gibco BRL) according to the manufacturer’s protocols.
Exon trapping
Exon amplification was performed using the 
GET vector (28).
RACE and SMART-RACE
The 5'- and 3'-sequences of cDNAs were extended using standard RACE protocols with oligonucleotide primers derived from sequences of trapped exons (4), published sequences (14) or sequences originated from new exons detected by RACE-PCR or RT–PCR.
RACE reactions were performed with the Marathon cDNA amplification kit (Clontech) using cDNA prepared from human testis, fetus, brain, heart, small intestine or leukocytes as template. The cDNAs were amplified with the AdvanTage cDNA polymerase mix (Clontech) or AmpliTaq Gold (PE Biosystems).
The SMART-RACE reactions were performed with cDNA from mononuclear cells of peripheral blood, human pancreas and heart (Clontech) and the B-cell lines Granta-519, EHEB and JVM-2 (20–22) using the SMART-PCR cDNA synthesis kit (Clontech) following the manufacturer’s instructions. First-strand synthesis was carried out with gene-specific primers and Superscript II reverse transcriptase (Gibco BRL). The cDNA sequences were amplified with the AdvanTage cDNA polymerase mix (Clontech).
RACE and SMART-RACE products were cloned using either the TA Cloning kit (Clontech) or the TOPO-XL cloning kit (Invitrogen) and subsequently analyzed by direct sequencing of colony PCR products using vector-specific primers. The sequences of all gene-specific primers used are listed in Table 4.
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RT–PCR and PCR
For RT–PCR analysis, first-strand cDNA was synthesized from total RNA (1–2 µg) using either oligo-dT or random-hexamere primers (RT–PCR kit, PE Biosystems; display Thermo-RT Kit, Display Systems Biotech). RT–PCR fragments were amplified with AmpliTaq polymerase, AmpliTaq Gold polymerase (PE Biosystems) or AdvanTage cDNA polymerase mix (Clontech) using gene-specific primers (Table 4).
For mutation analysis, PCR of genomic DNA was carried out with 20 ng of template DNA and 10 pmol of each primer (Table 4) in a total volume of 50 µl using AmpliTaq polymerase or AmpliTaq Gold polymerase.
Nucleotide sequence analysis
The nucleotide sequences of all the BCMS cDNA and genomic clones as well as the BCMS RT–PCR and PCR products were determined by cycle sequencing with the Big Dye Terminator chemistry (PE Biosystems) followed by electrophoresis on a Perkin-Elmer ABI-377 automated sequencer. The clones were sequenced using the standard M13 vector-primers and gene-specific primers.
Database analysis
BLAST searches against nr, dbEST, month and HTGS segments of GenBank were done at the NCBI server (http://www.ncbi.nlm.nih.gov//) (29).
Northern blot analysis
Multiple-tissue poly(A)+ northern blots (human MTN blot and human MTN blot IV, Clontech) were hybridized in Church solution at 62°C according to standard procedures. Probes were labeled with [
-32P]UTP using the STRIP-EZ RNA kit (Ambion) or the RNA labeling kit (Amersham Pharmacia) according to the manufacturer’s instructions.
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ACKNOWLEDGEMENTS |
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We thank Armin Pscherer (Heidelberg, Germany) and Eckhart Meese (Homburg, Germany) for critical discussions. The technical support by Jeremy Nickolenko, Petra Schramm and Ralf Klären (all Heidelberg) is gratefully acknowledged. The cell lines GRANTA-519 JVM-2 and EHEB were kindly provided by Hans G. Drexler (Braunschweig, Germany). This work was supported by grants from the Bundesministerium für Bildung und Forschung (01SF 9903/3 and 01KW 9935).
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FOOTNOTES |
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+ These authors contributed equally to this work
To whom correspondence should be addressed. Tel: +49 6221 424619; Fax: +49 6221 424639; Email: p.lichter@dkfz-heidelberg.de
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