VDJ-Switch Region Analysis in Multiple Myeloma Patients Reveals Homogeneity and Long-Term Stability of Switch Junctions, and Ongoing Mutation Upstream of Switch Mu.

Abstract

During class switch recombination (CSR) B cells change their immunoglobulin heavy chain (IgH) constant region genes by a deletional recombination mechanism involving well characterized switch (S) regions several Kb downstream of IgH variable region (VDJ) sequences. Unlike VDJ recombination, the junction formed by CSR between pre-switch S-mu (IgM) and post-switch S-gamma (IgG) is imprecise, and the key factors mediating this process are not fully understood. In multiple myeloma, a bone marrow cancer characterized by post-switch plasma cells bearing a unique clonotypic VDJ, switch regions have been shown to mediate translocations in a subset of MM patients, suggesting a role in transformation. In this study we cloned and sequenced the VDJ-hybrid immunoglobulin switch (VDJ-S) regions of post-switch multiple myeloma (MM) cells at diagnosis and a second time point to determine if the switch junction was normal, homogeneous within a time point, and stable over the course of malignancy.The VDJ-S of five stage III IgG MM patients were examined in this study. Each patient has a specific clonotypic VDJ region and downstream hybrid switch junction (S-mu/S-gamma) arising after class switch recombination (CSR) from IgM to IgG. For each patient two samples were tested: a diagnosis sample and a second sample taken between 1.6 and 4.2 years later. The 5–7 Kb VDJ-S was amplified by long distance PCR using bulk DNA samples from bone marrow and blood as template, CDR2 specific sense primers and S-gamma specific antisense primers. In each case a single DNA product was amplified, cloned, and sequenced with primers covering the VDJ-S region. Patient specific CDR2 primers were used to confirm that the fragments were clonotypic. Unique switch junctions for patients 1–5 were detected at positions 125, 251, 435, 590, and 750 of S-mu, respectively. This agrees with previous studies mapping MM switch junctions to the 5′ portion of S-mu. The sequence and mutation profile of the switch junctions, with more frequent mutations in the S-mu region upstream of the hybrid junction, suggests that these junctions arise from normal CSR. A single hybrid junction was detected in all patients, corresponding to the single VDJ-Switch fragment amplified by long-distance PCR. The switch junction sequences also remained constant between time point samples, suggesting switch junction stability. Interestingly, new mutations were observed in the 3.5 Kb region upstream of the switch region, in the second time point samples of 2/5 patients. Most notably, 19 new mutations were detected in one patient, including 3 bp and 64 bp deletions 135 and 223 bp downstream of the intronic enhancer, respectively. The significance of these mutations is unclear, considering previous studies showing mutations, insertions, and deletions in this region in normal CSR. Nevertheless, it seems this process continues in clonotypic cells of some patients throughout malignancy. This ongoing mutation may lead to ‘remodeling' of the VDJ-S region, which is significant in the context of immunoglobulin translocations where proximity with immunoglobulin enhancers may confer a selective advantage.