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• W2041013986 abstract "The translocation t(2;7)(p11;q21) has repeatedly been documented in association with indolent B-cell lymphoproliferative disorders (BLPDs). However, the chromosomal breakpoints associated with this recurrent translocation have rarely been characterized. Using an approach based on long-range PCR, we mapped the t(2;7) breakpoints in five patients presenting with indolent B-cell neoplasia. The sequencing of these rearrangements revealed several striking parallels across the t(2;7) breakpoints. The junction sites on 2p11 consistently mapped to the heptamer recombination signal sequence (RSS) of an immunoglobulin kappa variable gene (IGK) within the Vκ3 family, while the breakpoints on 7q21 each localized to within 4 bp of an RSS-like element located approximately 0.5 kb upstream of the transcription start site of the cyclin-dependent kinase 6 gene (CDK6). These findings confirm the significant genetic overlap arising in BLPD-associated t(2;7) translocations, and implicate the deregulated expression of CDK6 as a common molecular mechanism involved in the emergence of clonal B-cell proliferations presenting with this recurrent abnormality. In addition, the successful mapping of the t(2;7) translocations in each of five patients using a simple PCR-based protocol highlights the potential diagnostic utility of this approach during characterization of cases harboring analogous rearrangements. The translocation t(2;7)(p11;q21) has repeatedly been documented in association with indolent B-cell lymphoproliferative disorders (BLPDs). However, the chromosomal breakpoints associated with this recurrent translocation have rarely been characterized. Using an approach based on long-range PCR, we mapped the t(2;7) breakpoints in five patients presenting with indolent B-cell neoplasia. The sequencing of these rearrangements revealed several striking parallels across the t(2;7) breakpoints. The junction sites on 2p11 consistently mapped to the heptamer recombination signal sequence (RSS) of an immunoglobulin kappa variable gene (IGK) within the Vκ3 family, while the breakpoints on 7q21 each localized to within 4 bp of an RSS-like element located approximately 0.5 kb upstream of the transcription start site of the cyclin-dependent kinase 6 gene (CDK6). These findings confirm the significant genetic overlap arising in BLPD-associated t(2;7) translocations, and implicate the deregulated expression of CDK6 as a common molecular mechanism involved in the emergence of clonal B-cell proliferations presenting with this recurrent abnormality. In addition, the successful mapping of the t(2;7) translocations in each of five patients using a simple PCR-based protocol highlights the potential diagnostic utility of this approach during characterization of cases harboring analogous rearrangements. The chromosomal translocation t(2;7)(p11∼13;q21∼22) is a recurrent genetic abnormality in B-cell lymphoproliferative disorders (BLPDs). The rearrangement was first observed in conjunction with splenic lymphoma with villous lymphocytes (SLVL) during a cytogenetic survey performed by Oscier et al.1Oscier D.G. Matutes E. Gardiner A. Glide S. Mould S. Brito-Babapulle V. Ellis J. Catovsky D. Cytogenetic studies in splenic lymphoma with villous lymphocytes.Br J Haematol. 1993; 85: 487-491Crossref PubMed Scopus (117) Google Scholar Since then, analogous translocations have repeatedly been documented during genetic studies of B-cell neoplasia, encompassing cases of both chronic lymphocytic leukemia (CLL)2Vahdati M. Graafland H. Emberger J.M. Karyotype analysis of B-lymphocytes transformed by Epstein-Barr virus in 21 patients with B cell chronic lymphocytic leukemia.Hum Genet. 1983; 63: 327-331Crossref PubMed Scopus (18) Google Scholar, 3Peterson L.C. Lindquist L.L. Church S. Kay N.E. Frequent clonal abnormalities of chromosome band 13q14 in B-cell chronic lymphocytic leukemia: multiple clones, subclones, and nonclonal alterations in 82 Midwestern patients.Genes Chromosomes Cancer. 1992; 4: 273-280Crossref PubMed Scopus (71) Google Scholar, 4Solé F. Woessner S. Florensa L. Montero S. Asensio A. Besses C. Sans-Sabrafen J. A new chromosomal anomaly associated with mature B-cell chronic lymphoproliferative disorders: del(7)(q32).Cancer Genet Cytogenet. 1993; 65: 170-172Abstract Full Text PDF PubMed Scopus (22) Google Scholar, 5Hayette S. Tigaud I. Callet-Bauchu E. Ffrench M. Gazzo S. Wahbi K. Callanan M. Felman P. Dumontet C. Magaud J.P. Rimokh R. In B-cell chronic lymphocytic leukemias, 7q21 translocations lead to overexpression of the CDK6 gene.Blood. 2003; 102: 1549-1550Crossref PubMed Scopus (42) Google Scholar, 6Fink S.R. Smoley S.A. Stockero K.J. Paternoster S.F. Thorland E.C. Van Dyke D.L. Shanafelt T.D. Zent C.S. Call T.G. Kay N.E. Dewald G.W. Loss of TP53 is due to rearrangements involving chromosome region 17p10p12 in chronic lymphocytic leukemia.Cancer Genet Cytogenet. 2006; 167: 177-181Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar and low-grade non-Hodgkin’s lymphomas.7Oscier D.G. Gardiner A. Mould S. Structural abnormalities of chromosome 7q in chronic lymphoproliferative disorders.Cancer Genet Cytogenet. 1996; 92: 24-27Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 8Corcoran M.M. Mould S.J. Orchard J.A. Ibbotson R.E. Chapman R.M. Boright A.P. Platt C. Tsui L.C. Scherer S.W. Oscier D.G. Dysregulation of cyclin dependent kinase 6 expression in splenic marginal zone lymphoma through chromosome 7q translocations.Oncogene. 1999; 18: 6271-6277Crossref PubMed Scopus (121) Google Scholar, 9Morgan R. Chen Z. Richkind K. Roherty S. Velasco J. Sandberg A.A. PHA/IL2: an efficient mitogen cocktail for cytogenetic studies of non-Hodgkin lymphoma and chronic lymphocytic leukemia.Cancer Genet Cytogenet. 1999; 109: 134-137Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 10Brito-Babapulle V. Gruszka-Westwood A. Platt G. Andersen C.L. Elnenaei M.O. Matutes E. Wotherspoon A.C. Weston-Smith S.G. Catovsky D. Translocation t(2;7)(p12;q21-22) with dysregulation of the CDK6 gene mapping to 7q21-22 in a non-Hodgkin’s lymphoma with leukemia.Haematologica. 2002; 87: 357-362PubMed Google Scholar, 11Sekikawa T. Takahara S. Kawano T. Nakada S. Ito K. Iwase S. Yamada H. Kobayashi M. Horiguchi-Yamada J. No V(H) somatic hypermutation was detected in B-cells of a patient with macroglobulinemia due to splenic marginal zone lymphoma.Int J Hematol. 2002; 76: 453-459Crossref PubMed Scopus (3) Google Scholar, 12Martín-Subero J.I. Harder L. Gesk S. Schlegelberger B. Grote W. Martinez-Climent J.A. Dyer M.J. Novo F.J. Calasanz M.J. Siebert R. Interphase FISH assays for the detection of translocations with breakpoints in immunoglobulin light chain loci.Int J Cancer. 2002; 98: 470-474Crossref PubMed Scopus (80) Google Scholar, 13Baró C. Salido M. Espinet B. Astier L. Domingo A. Granada I. Millà F. Carrió A. Costa D. Luño E. Hernández J.M. Campo E. Florensa L. Ferrer A. Salar A. Bellosillo B. Besses C. Serrano S. Solé F. New chromosomal alterations in a series of 23 splenic marginal zone lymphoma patients revealed by Spectral Karyotyping (SKY).Leuk Res. 2008; 32: 727-736Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 14Ambika S. Oo T.H. Translocation (2;7)(p11.2;q22) in a newly diagnosed low-grade B-cell non-Hodgkin lymphoma.Cancer Genet Cytogenet. 2008; 181: 65-66Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, 15Chen D. Law M.E. Theis J.D. Gamez J.D. Caron L.B. Vrana J.A. Dogan A. Clinicopathologic features of CDK6 translocation-associated B-cell lymphoproliferative disorders.Am J Surg Pathol. 2009; 33: 720-729Crossref PubMed Scopus (21) Google Scholar, 16Salido M. Baró C. Oscier D. Dierlamm J. Matutes E. Traverse-Glehen A. Berger F. Felman P. Thieblemont C. Gesk S. Athanasiadou A. Davis Z. Gardiner A. Milla F. Ferrer A. Mollejo M. Calasanz M.J. Florensa L. Espinet B. Luño E. Wlodarska I. Verhoef G. García-Granero M. Salar A. Papadaki T. Serrano S. Piris M.A. Solé F. Cytogenetic aberrations and their prognostic value in a series of 330 splenic marginal zone B-cell lymphomas: a multicenter study of the Splenic B-Cell Lymphoma Group.Blood. 2010; 116: 1479-1488Crossref PubMed Scopus (146) Google Scholar Despite the recurrent nature of this abnormality, however, sequence-level characterization of t(2;7) translocations has rarely been performed, and the degree of genetic overlap between discrete t(2;7) rearrangements remains unclear. The most comprehensive molecular mapping of t(2;7) translocations to date was performed in three cases of SLVL by Corcoran et al.8Corcoran M.M. Mould S.J. Orchard J.A. Ibbotson R.E. Chapman R.M. Boright A.P. Platt C. Tsui L.C. Scherer S.W. Oscier D.G. Dysregulation of cyclin dependent kinase 6 expression in splenic marginal zone lymphoma through chromosome 7q translocations.Oncogene. 1999; 18: 6271-6277Crossref PubMed Scopus (121) Google Scholar In each tumor sample, the t(2;7) breakpoint was found to juxtapose the cyclin-dependent kinase 6 gene (CDK6) on chromosome 7q21 with the immunoglobulin kappa locus (IGK) on 2p11. The breakpoint junctions on 2p11 consistently localized to the recombination signal sequence (RSS) of an IGK variable (Vκ) gene, thereby implicating the role of aberrant V(D)J recombination events during the formation of these translocation sites. Meanwhile, the breakpoints on 7q21 fell in close proximity to the transcription start site and promoter of CDK6, thus highlighting the potential dysregulation of this G1-phase cell cycle regulator during the emergence of the B-cell proliferations. This notion was subsequently validated by the confirmation of marked CDK6 overexpression in two of the SLVL tumor samples.8Corcoran M.M. Mould S.J. Orchard J.A. Ibbotson R.E. Chapman R.M. Boright A.P. Platt C. Tsui L.C. Scherer S.W. Oscier D.G. Dysregulation of cyclin dependent kinase 6 expression in splenic marginal zone lymphoma through chromosome 7q translocations.Oncogene. 1999; 18: 6271-6277Crossref PubMed Scopus (121) Google Scholar More recently, we characterized the t(2;7) breakpoint sequence in a patient presenting with a persistent but asymptomatic lymphocytosis of non-CLL-like clonal B cells–clinical features consistent with the diagnostic criteria of non-CLL-like monoclonal B-cell lymphocytosis.17Amato D. Oscier D.G. Davis Z. Mould S. Zheng J. Kolomietz E. Wang C. Cytogenetic aberrations and immunoglobulin VH gene mutations in clinically benign CD5- monoclonal B-cell lymphocytosis.Am J Clin Pathol. 2007; 128: 333-338Crossref PubMed Scopus (29) Google Scholar, 18Parker E. MacDonald J.R. Wang C. Molecular characterization of a t(2;7) translocation linking CDK6 to the IGK locus in CD5(-) monoclonal B-cell lymphocytosis.Cancer Genet. 2011; 204: 260-264Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar The mapping of the breakpoint in this case was achieved using a novel assay based on long-range PCR (L-PCR). In brief, consensus primers against the IGK variable genes were combined sequentially with primers targeting a candidate region on 7q21 that had initially been established via fluorescence in situ hybridization (FISH). Crucially, this approach gave rise to strong amplification across the t(2;7) breakpoint region, thereby enabling the translocation sequence to be established. In line with previous findings in SLVL, the 2p11 breakpoint in this case mapped directly to the RSS of a Vκ gene, while the breakpoint on 7q21 fell approximately 0.5 kb upstream of CDK6, and within just 3 bp of one of the t(2;7) junctions previously characterized by Corcoran et al.8Corcoran M.M. Mould S.J. Orchard J.A. Ibbotson R.E. Chapman R.M. Boright A.P. Platt C. Tsui L.C. Scherer S.W. Oscier D.G. Dysregulation of cyclin dependent kinase 6 expression in splenic marginal zone lymphoma through chromosome 7q translocations.Oncogene. 1999; 18: 6271-6277Crossref PubMed Scopus (121) Google Scholar Based on the striking parallels between the translocation breakpoints observed in these studies, we hypothesized that BLPD-associated t(2;7) rearrangements are characterized by genetic overlap, consistently linking Vκ genes on 2p11 with a putative breakpoint cluster region (BCR) on 7q21 located directly upstream of the coding region of CDK6. Furthermore, given the success of our L-PCR-based protocol in elucidating the breakpoint sequence in a patient lacking extensive preliminary cytogenetic characterization,18Parker E. MacDonald J.R. Wang C. Molecular characterization of a t(2;7) translocation linking CDK6 to the IGK locus in CD5(-) monoclonal B-cell lymphocytosis.Cancer Genet. 2011; 204: 260-264Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar we postulated that this approach may prove equally effective in mapping the t(2;7) breakpoint sequences in additional cases. In the present study, therefore, we set out to use this L-PCR assay to sequence the t(2;7) breakpoints in patients presenting with BLPDs. In addition to validating the merits of L-PCR during the mapping of t(2;7)/IGK-CDK6 rearrangements, the sequencing of such breakpoints would provide the most comprehensive molecular characterization of this recurrent translocation to date. Samples from five patients (designated P1 to P5) were incorporated into the present analysis. Clinical and molecular characteristics of patients P1,17Amato D. Oscier D.G. Davis Z. Mould S. Zheng J. Kolomietz E. Wang C. Cytogenetic aberrations and immunoglobulin VH gene mutations in clinically benign CD5- monoclonal B-cell lymphocytosis.Am J Clin Pathol. 2007; 128: 333-338Crossref PubMed Scopus (29) Google Scholar, 18Parker E. MacDonald J.R. Wang C. Molecular characterization of a t(2;7) translocation linking CDK6 to the IGK locus in CD5(-) monoclonal B-cell lymphocytosis.Cancer Genet. 2011; 204: 260-264Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar P2,14Ambika S. Oo T.H. Translocation (2;7)(p11.2;q22) in a newly diagnosed low-grade B-cell non-Hodgkin lymphoma.Cancer Genet Cytogenet. 2008; 181: 65-66Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar and P35Hayette S. Tigaud I. Callet-Bauchu E. Ffrench M. Gazzo S. Wahbi K. Callanan M. Felman P. Dumontet C. Magaud J.P. Rimokh R. In B-cell chronic lymphocytic leukemias, 7q21 translocations lead to overexpression of the CDK6 gene.Blood. 2003; 102: 1549-1550Crossref PubMed Scopus (42) Google Scholar have previously been reported. Patient P4 was incorporated into the present study after a 4-year history of Binet stage A/Rai stage I CLL. The patient presented with mutated immunoglobulin heavy chain variable (IGHV) genes (90.6% germline homology). Cytogenetic analysis revealed the presence of the abnormal karyotype 46,XX,t(2;7)(p13;q21)[16]/46,XX[9]. In addition, the involvement of the IGK locus was confirmed by FISH, during which aberrant signal patterns were observed in conjunction with >90% of peripheral blood nuclei. As of the most recent follow-up, conducted 8 months after the cytogenetic analysis, no treatment had been undertaken for this patient. Patient P5 presented with splenomegaly alongside a white blood cell count of 84 × 109/L (85% lymphocytes), a platelet count of 123 × 109/L, and a hemoglobin level of 10.3 g/dL, leading to a diagnosis of splenic marginal zone lymphoma (SMZL). Neoplastic B cells comprised 70.4% of peripheral blood leukocytes. The patient displayed the abnormal partial karyotype 46,XY,t(2;7)(p13;q21)[20], and involvement of the IGK and CDK6 loci in the t(2;7) translocation was confirmed using FISH. The patient was treated with fludarabine/cyclophosphamide, in part with rituximab, and was in complete remission at the time of the most recent follow-up evaluation (almost 3 years after initial diagnosis). Overall, the samples in the present study were from three female and two male patients presenting with indolent B-cell neoplasia at ages ranging from 50 to 75 years. Clonal B cells were of a CD5+ phenotype in patient P3 and a CD5− phenotype in patients P1 and P2; immunophenotypic data were lacking for patients P4 and P5. Notably, patients P1 and P2 were asymptomatic aside from the presence of clonal B cells, whereas more progressive hematological symptoms (including anemia and thrombocytopenia) were observed in patient P3 following an initial diagnosis of Binet stage A CLL. The presence of a t(2;7)(p11∼13;q21∼22) translocation was confirmed in each of the patients before the commencement of the present study, either by conventional karyotypic analysis, FISH, Southern blotting or a combination of these methods (Table 1),5Hayette S. Tigaud I. Callet-Bauchu E. Ffrench M. Gazzo S. Wahbi K. Callanan M. Felman P. Dumontet C. Magaud J.P. Rimokh R. In B-cell chronic lymphocytic leukemias, 7q21 translocations lead to overexpression of the CDK6 gene.Blood. 2003; 102: 1549-1550Crossref PubMed Scopus (42) Google Scholar, 14Ambika S. Oo T.H. Translocation (2;7)(p11.2;q22) in a newly diagnosed low-grade B-cell non-Hodgkin lymphoma.Cancer Genet Cytogenet. 2008; 181: 65-66Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, 17Amato D. Oscier D.G. Davis Z. Mould S. Zheng J. Kolomietz E. Wang C. Cytogenetic aberrations and immunoglobulin VH gene mutations in clinically benign CD5- monoclonal B-cell lymphocytosis.Am J Clin Pathol. 2007; 128: 333-338Crossref PubMed Scopus (29) Google Scholar, 18Parker E. MacDonald J.R. Wang C. Molecular characterization of a t(2;7) translocation linking CDK6 to the IGK locus in CD5(-) monoclonal B-cell lymphocytosis.Cancer Genet. 2011; 204: 260-264Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar wherein breakpoints were cytogenetically assigned to 2p11∼p13 and 7q21∼q22 based on chromosome banding patterns. DNA was extracted from peripheral blood (P1, P3, P4, and P5) or bone marrow (P2) according to standard procedures. Key clinical and cytogenetic characteristics of the patients are summarized in Table 1.Table 1Clinical, Diagnostic, and Cytogenetic Characteristics of Patients StudiedPatientDiagnosisSex/Age (years) at diagnosisTreatment?/Alive?Karyotypet(2;7) confirmationDNA SourceReferences∗Clinical, phenotypic and cytogenetic details for patients P1, P2, and P3 have been previously reported.P1MBLF/75No/yes46,XX,t(2;7)(p11;q22)[9]/46,XX[21]FISHPB17Amato D. Oscier D.G. Davis Z. Mould S. Zheng J. Kolomietz E. Wang C. Cytogenetic aberrations and immunoglobulin VH gene mutations in clinically benign CD5- monoclonal B-cell lymphocytosis.Am J Clin Pathol. 2007; 128: 333-338Crossref PubMed Scopus (29) Google Scholar, 18Parker E. MacDonald J.R. Wang C. Molecular characterization of a t(2;7) translocation linking CDK6 to the IGK locus in CD5(-) monoclonal B-cell lymphocytosis.Cancer Genet. 2011; 204: 260-264Abstract Full Text Full Text PDF PubMed Scopus (10) Google ScholarP2NHL stage IVF/65No/yes46,XX,t(2;7)(p11.2;q22)[5]/46,XX[15]–BM14Ambika S. Oo T.H. Translocation (2;7)(p11.2;q22) in a newly diagnosed low-grade B-cell non-Hodgkin lymphoma.Cancer Genet Cytogenet. 2008; 181: 65-66Abstract Full Text Full Text PDF PubMed Scopus (2) Google ScholarP3CLLM/61Yes/no45,XY,t(2;7)(p11∼12;q22),der(8)t(8;17)(p12;p11),−17[5]/47,XY,t(2;7),+5,+12[10]/46,XY[35]SBPB5Hayette S. Tigaud I. Callet-Bauchu E. Ffrench M. Gazzo S. Wahbi K. Callanan M. Felman P. Dumontet C. Magaud J.P. Rimokh R. In B-cell chronic lymphocytic leukemias, 7q21 translocations lead to overexpression of the CDK6 gene.Blood. 2003; 102: 1549-1550Crossref PubMed Scopus (42) Google ScholarP4CLLF/50No/yes46,XX,t(2;7)(p13;q21)[16]/46,XX[9]FISHPBP5SMZLM/57Yes/yes46,XX,t(2;7)(p13;q21)[20]FISHPBF, female; M, male; BM, bone marrow; MBL, monoclonal B-cell lymphocytosis; NHL, non-Hodkgin’s lymphoma; PB, peripheral blood; SB, Southern blot.∗ Clinical, phenotypic and cytogenetic details for patients P1, P2, and P3 have been previously reported. Open table in a new tab F, female; M, male; BM, bone marrow; MBL, monoclonal B-cell lymphocytosis; NHL, non-Hodkgin’s lymphoma; PB, peripheral blood; SB, Southern blot. A panel of seven primers was selected to target conserved sequences across the variable gene segments in each Vκ gene family on 2p11 (Figure 1A and Table 2).18Parker E. MacDonald J.R. Wang C. Molecular characterization of a t(2;7) translocation linking CDK6 to the IGK locus in CD5(-) monoclonal B-cell lymphocytosis.Cancer Genet. 2011; 204: 260-264Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 19Van Dongen J.J.M. Langerak A.W. Brüggemann M. Evans P.A. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. García-Sanz R. van Krieken J.H. Droese J. González D. Bastard C. White H.E. Spaargaren M. González M. Parreira A. Smith J.L. Morgan G.J. Kneba M. Macintyre E.A. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombination in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2497) Google Scholar As previously reported, this approach enabled up to 13 distinct IGK segments to be targeted by each Vκ-directed primer.18Parker E. MacDonald J.R. Wang C. Molecular characterization of a t(2;7) translocation linking CDK6 to the IGK locus in CD5(-) monoclonal B-cell lymphocytosis.Cancer Genet. 2011; 204: 260-264Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar This panel was then supplemented by an additional 13 primers on 7q21, selected to target the putative t(2;7)/IGK-CDK6 BCR upstream of the coding region of CDK6 (Figure 1B and Table 3). The 7q primers mapped to both the sense and antisense strands, and provided dense coverage in the regions directly upstream and downstream of the t(2;7) junction sites mapped in previous studies.8Corcoran M.M. Mould S.J. Orchard J.A. Ibbotson R.E. Chapman R.M. Boright A.P. Platt C. Tsui L.C. Scherer S.W. Oscier D.G. Dysregulation of cyclin dependent kinase 6 expression in splenic marginal zone lymphoma through chromosome 7q translocations.Oncogene. 1999; 18: 6271-6277Crossref PubMed Scopus (121) Google Scholar, 18Parker E. MacDonald J.R. Wang C. Molecular characterization of a t(2;7) translocation linking CDK6 to the IGK locus in CD5(-) monoclonal B-cell lymphocytosis.Cancer Genet. 2011; 204: 260-264Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar Oligonucleotide sequences were designed using Primer3 software,20Rozen S. Skaletsky H. Primer3 on the WWW for general users and for biologist programmers.Methods Mol Biol. 2000; 132: 365-386Crossref PubMed Google Scholar and selection parameters were chosen to ensure that primer length, melting temperature, and GC content were suitable for L-PCR (Jennifer Howe, personal communication) (Table 4).Table 2Design of L-PCR Primers on 2p11TargetSequenceGenomic targets (no.)Vκ15′-AAGGTTCAGCGGCAGTGGAT-3′12Vκ25′-CCTCCATCTCCTGCAGGTCTAGT-3′13Vκ3∗This primer gave rise to breakpoint-spanning amplification in each of the five patient samples considered in this study.5′-CCAGGCTCCTCATCTATGATGC-3′8Vκ45′-CTGCAAGTCCAGCCAGAGTGTT-3′1Vκ55′-CTGCAAAGCCAGCCAAGACA-3′1Vκ65′-CGAGGTTCAGTGGCAGTGGAT-3′3Vκ75′-GACCGATTTCACCCTCACAATTAA-3′1Each 2p11 primer targets a conserved sequence within the specified family of Vκ genes. The number of genomic targets designates the quantity of Vκ segments matched with >95% sequence homology by the corresponding oligonucleotide, as determined using the BLAT alignment tool on the UCSC Genome Bioinformatics browser (http://www.genome.ucsc.edu/cgi-bin/hgBlat) and V-BASE sequence directory (http://vbase.mrc-cpe.cam.ac.uk).∗ This primer gave rise to breakpoint-spanning amplification in each of the five patient samples considered in this study. Open table in a new tab Table 3Design of L-PCR Primers on 7q21PrimerStrandPosition relative to putative t(2;7) BCRSequenceA1+6.9 kb centromeric5′-GGCAGTACCAGTGCCAGCAATA-3′A2+6.5 kb centromeric5′-CAAGGCAGAGGCAGAAGAGAAGA-3′A3+4.5 kb centromeric5′-ACGGCATCCTCATTCTCCAGTC-3′A4+2.0 kb centromeric5′-AGAGCAGAGCTTCTGGCACTCA-3′A5+0.6 kb centromeric5′-TGCGAGTGTCAGTCGGCTCT-3′A6+0.3 kb centromeric5′-GCCTTCCGGAGAGAAATGAGG-3′A7+Spans putative BCR5′-ACTGTGTGACGTGGACGGATG-3′A8+3.2 kb telomeric5′-CTCAGCTGCACACATGGCCTA-3′S1−Spans putative BCR5′-CATCCGTCCACGTCACACAGT-3′S2∗These primers gave rise to patient-specific amplification in conjunction with one or more of the patient samples in this study. Each of these primers maps to the sense strand directly upstream of the coding region of CDK6.−0.2 kb telomeric5′-GAGGCAGAAAGGAAGCGAAGG-3′S3∗These primers gave rise to patient-specific amplification in conjunction with one or more of the patient samples in this study. Each of these primers maps to the sense strand directly upstream of the coding region of CDK6.−1.3 kb telomeric5′-CTAGCCATGATGTGCTGCTTCC-3′S4∗These primers gave rise to patient-specific amplification in conjunction with one or more of the patient samples in this study. Each of these primers maps to the sense strand directly upstream of the coding region of CDK6.−2.8 kb telomeric5′-GTAGAACCAATGGAAGCAGCATGT-3′S5∗These primers gave rise to patient-specific amplification in conjunction with one or more of the patient samples in this study. Each of these primers maps to the sense strand directly upstream of the coding region of CDK6.−5.7 kb telomeric5′-CACATGGTCTGATGCAGTGGAA-3′C1+5.5 kb telomeric5′-AGTGCCAGTTGGACTCCAGTGA-3′C2−13.7 kb telomeric5′-GAATTCCAGGAAGCCAAGATCTGA-3′C3−6.0 kb telomeric5′-TCCTGCAATGAGTGGATGTTTCA-3′Each primer has a single genomic target proximal to the putative t(2;7)/IGK-CDK6 BCR located upstream of the coding region of CDK6 (as indicated schematically in Figure 1B). This BCR corresponds to the shared 7q breakpoint site previously characterized in conjunction with both SLVL and non-CLL-like monoclonal B-cell lymphocytosis.8Corcoran M.M. Mould S.J. Orchard J.A. Ibbotson R.E. Chapman R.M. Boright A.P. Platt C. Tsui L.C. Scherer S.W. Oscier D.G. Dysregulation of cyclin dependent kinase 6 expression in splenic marginal zone lymphoma through chromosome 7q translocations.Oncogene. 1999; 18: 6271-6277Crossref PubMed Scopus (121) Google Scholar, 18Parker E. MacDonald J.R. Wang C. Molecular characterization of a t(2;7) translocation linking CDK6 to the IGK locus in CD5(-) monoclonal B-cell lymphocytosis.Cancer Genet. 2011; 204: 260-264Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar Note the high concentration of primers within 2 kb of this BCR.∗ These primers gave rise to patient-specific amplification in conjunction with one or more of the patient samples in this study. Each of these primers maps to the sense strand directly upstream of the coding region of CDK6. Open table in a new tab Table 4Constraints for Selection of L-PCR PrimersParameter∗Maximum Poly-X refers to the greatest permissible length of a mononucleotide repeat within a primer sequence.20 Each of these constraints was selected in order to obtain primers suitable for L-PCR-based amplification (Jennifer Howe, personal communication). All other primer selection parameters followed the standard constraints applied by Primer3 software.20MinimumOptimumMaximumPrimer size (bp)202224Melting temperature (°C)63.564.565.5GC content (%)455565Maximum Poly-X–≤23∗ Maximum Poly-X refers to the greatest permissible length of a mononucleotide repeat within a primer sequence.20Rozen S. Skaletsky H. Primer3 on the WWW for general users and for biologist programmers.Methods Mol Biol. 2000; 132: 365-386Crossref PubMed Google Scholar Each of these constraints was selected in order to obtain primers suitable for L-PCR-based amplification (Jennifer Howe, personal communication). All other primer selection parameters followed the standard constraints applied by Primer3 software.20Rozen S. Skaletsky H. Primer3 on the WWW for general users and for biologist programmers.Methods Mol Biol. 2000; 132: 365-386Crossref PubMed Google Scholar Open table in a new tab Each 2p11 primer targets a conserved sequence within the specified family of Vκ genes. The number of genomic targets designates the quantity of Vκ segments matched with >95% sequence homology by the corresponding oligonucleotide, as determined using the BLAT alignment tool on the UCSC Genome Bioinformatics browser (http://www.genome.ucsc.edu/cgi-bin/hgBlat) and V-BASE sequence directory (http://vbase.mrc-cpe.cam.ac.uk). Each primer has a single genomic target proximal to the putative t(2;7)/IGK-CDK6 BCR located upstream of the coding region of CDK6 (as indicated schematically in Figure 1B). This BCR corresponds to the shared 7q breakpoint site previously characterized in conjunction with both SLVL and non-CLL-like monoclonal B-cell lymphocytosis.8Corcoran M.M. Mould S.J. Orchard J.A. Ibbotson R.E. Chapman R.M. Boright A.P. Platt C. Tsui L.C. Scherer S.W. Oscier D.G. Dysregulation of cyclin dependent kinase 6 expression in splenic marginal zone lymphoma through chromosome 7q translocations.Oncogene. 1999; 18: 6271-6277Crossref PubMed Scopus (121) Google Scholar, 18Parker E. MacDonald J.R. Wang C. Molecular characterization of a t(2;7) translocation linking CDK6 to the IGK locus in CD5(-) monoclonal B-cell lymphocytosis.Cancer Genet. 2011; 204: 260-264Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar Note the high concentration of primers within 2 kb of this BCR. L-PCR was subsequently performed with the aim of providing sequence-level characterization of the t(2;7) breakpoint in each sample. The Vκ-directed consensus primers on 2p11 were combined sequentially with each of the BCR-targeted primers on 7q21. For each pair of primers, L-PCR was performed using both patient DNA and control DNA (HapMap reference sample NA10851), such that the observ" @default.
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