Sequencing of t(2;7) Translocations Reveals a Consistent Breakpoint Linking CDK6 to the IGK Locus in Indolent B-Cell Neoplasia  Edward P.K. Parker, Reiner Siebert, Thein H. Oo, Douglas Schneider, Sandrine Hayette, Chen Wang  The Journal of Molecular Diagnostics  Volume 15, Issue 1, Pages 101-109 (January 2013) DOI: 10.1016/j.jmoldx.2012.07.006 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Primer targets on 2p11 and 7q21. A: Schematic of the IGK locus at 2p11, indicating the relative positions of the 76 variable (Vκ) genes, five joining (Jκ) segments, constant region (Cκ), and associated enhancers.21 The Vκ segments are arranged in proximal and distal clusters, separated by a region of noncoding DNA spanning approximately 800 kb, and can be distinguished into seven families based on sequence homology. Accordingly, the panel of primers on 2p11 comprised seven consensus primers selected to target conserved sequences across multiple Vκ segments in each family (Table 2). The orientation of this primer hybridization is indicated for a single segment in the proximal Vκ gene cluster. Note that the primer amplifies toward the RSS of the Vκ segment. B: An additional panel of 13 primers was selected to target the proposed t(2;7)/IGK-CDK6 BCR on 7q21, comprising five sense primers (S1 to S5) and eight antisense primers (A1 to A8) (Table 3). The putative BCR (down arrow) aligns with the shared 7q breakpoint site previously observed in both SLVL and non-CLL-like monoclonal B-cell lymphocytosis.8,18 All primers are located within a maximum of 7 kb of the putative BCR, with dense coverage within 2 kb upstream and downstream of this site. The relative positions of the first two coding exons of the CDK6 locus (NM_001145306) are also indicated. The Journal of Molecular Diagnostics 2013 15, 101-109DOI: (10.1016/j.jmoldx.2012.07.006) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Characterization of the t(2;7) breakpoint in a representative patient sample (P5). A: L-PCR and subsequent gel electrophoresis were performed using DNA from patient P5 (Pt lanes) and control DNA (C lanes). Each pair of lanes corresponds to a different combination of test primers. A strong patient-specific band of approximately 5.8 kb was obtained by combining a consensus primer against the Vκ3 family of IGK variable genes with a 7q21 primer targeting the sense strand directly upstream of the putative t(2;7) BCR (primer S5). The oligonucleotide sequences of the L-PCR primers are listed in Tables 2 and 3, and their annealing points are indicated schematically in Figure 1. Control primers C1 and C2 on 7q21 were selected to provide amplification of approximately 8.3 kb. No amplification was observed in the absence of a DNA template (NT lane). The sequencing of this PCR product subsequently confirmed the successful elucidation of the t(2;7) breakpoint site in this patient sample (Table 6). B: The successful characterization of the proposed t(2;7) junction site in P5 was subsequently validated by performing L-PCR with additional primers adjacent to the putative 2p and 7q breakpoint sites. Patient-specific bands of the expected sizes were observed for each of these primer combinations, thereby confirming the accurate mapping of the t(2;7) breakpoint. The IGK-directed primers anneal 0.4 kb (V3-20i) and 1.1 kb (V3-20ii) upstream of the recombination signal of segment IGKV3-20 (implicated in the 2p11 breakpoint of patient P5). The sequences of these primers are listed in Tables 3 and 5. Analogous results were previously obtained for patient P1 (Parker et al18), and were also observed for each of patients P2, P3, and P4 (Supplemental Figure S1). The Journal of Molecular Diagnostics 2013 15, 101-109DOI: (10.1016/j.jmoldx.2012.07.006) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Schematic of the recurrent t(2;7) breakpoint. The t(2;7) translocations in each of the five patients in this series showed significant parallels. The 2p11 junctions consistently localized to the RSS located downstream of a Vκ3 gene segment within the proximal cluster of IGK variable genes, while the 7q21 breakpoints mapped to a narrow BCR located approximately 0.5 kb upstream of the transcription start site of CDK6. This recurrent t(2;7) breakpoint therefore juxtaposes the IGK-associated transcriptional enhancers (located telomeric to the translocation-associated Vκ segments) with the CDK6 locus, while leaving the coding region of this cell cycle regulator intact. Shown here are the relative positions of the 76 Vκ genes, five Jκ segments, and the Cκ region of the IGK locus on 2p11,21 as well as the eight exons and intervening introns of the CDK6 locus (NM_001145306). The IGK and CDK6 loci are shown both in their germline orientations (top) and after formation of the t(2;7) (bottom). The t(2;7) breakpoint was sequenced from the der(2) chromosome in each of the patients in this study. The Journal of Molecular Diagnostics 2013 15, 101-109DOI: (10.1016/j.jmoldx.2012.07.006) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Consistent amplification across the t(2;7) junctions using a single primer pair. Once the t(2;7) breakpoint had been elucidated in each of the patient samples (P1 to P5), it was evident that consistent amplification across the translocation junctions could be attained using just a single pair of test primers. Specifically, despite the involvement of several discrete Vκ segments within the t(2;7) breakpoints (Table 6), each of these segments could be targeted using a single Vκ3-directed consensus primer. In addition, the breakpoints on 7q21 mapped to within 8 bp of one another, such that a single primer adjacent to this 7q BCR could be used to target the t(2;7) breakpoint in each case (eg, primer S2). These findings highlight the striking genetic overlap between discrete BLPD-associated t(2;7) translocations. Notably, test primers did not give rise to amplification from control DNA (C lanes) lacking a t(2;7) rearrangement. In addition, no amplification was observed in the absence of a DNA template (NT lane). Oligonucleotide sequences of the test and control primers are listed in Tables 2 and 3. The Journal of Molecular Diagnostics 2013 15, 101-109DOI: (10.1016/j.jmoldx.2012.07.006) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions