Rapid Molecular Cloning of Rearrangements of the IGHJ Locus Using Long-Distance Inverse Polymerase Chain Reaction

Clonal rearrangements of the Ig heavy chain (IGH) locus consisting of either intrachromosomal (VDJ) rearrangements or interchromosomal translocations are a consistent feature of all B-cell malignancies and may be used both diagnostically and to monitor response to therapy. Many of these rearrangements are targeted to the IGHJ segments, but only some can be amplified with regular polymerase chain reaction (PCR) techniques. To permit PCR amplification of potentially all IGHJ rearrangements, we have devised a method incorporating self-ligation of restriction endonuclease-digested DNA fragments with long-distance PCR (long-distance, inverse PCR LDI-PCR). We show here, using only 4 nested oligonucleotide primers, the successful amplification and DNA sequencing of all IGHJ rearrangements up to 5.4 kb in length from a panel of 13 cases and cell lines of various types of B-cell malignancy. In all cases, both VDJ and DJ IGH rearrangements and translocation breakpoints were amplified. Six cases exhibited t(14;18)(q32;q21). All translocation breakpoints were cloned and sequenced. Three cases exhibited a rearrangement to the BCL2 major breakpoint region (MBR). However, 2 other cases exhibited rearrangements between the MBR and the minor cluster region (mcr). These 2 cases broke within 44 bp of each other, confirming the presence of an additional 3' BCL2 breakpoint cluster region. The final case fell immediately 3' of the 3' UTR of the BCL2 gene adjacent to an Alu repeat. No other BCL2 breakpoints within this region have been reported. Four cases exhibited t(11;14)(q13;q32). All 3 cases with translocations targeted to the IGHJ segments were successfully amplified and sequenced, including 1 case in which the BCL1 translocation could not be detected by DNA blot using the currently available probes. All three translocation breakpointsfell outside the BCL1 major translocation cluster between 20 and 40 kb telomeric and showed no clustering. Two of the three fell within or adjacent to Alu repeat regions. LDI-PCR is a simple and robust technique that allows PCR amplification of nearly all IGHJ rearrangements.