Amplifying Igh translocations

To the editor:

PCR has been used to amplify chromosome translocation breakpoints between Myc and Igh switch regions in B cell tumors and mouse tissues^1,2,3,4. However, the Igh switch region is both long and redundant and this is a particularly challenging part of the genome to amplify accurately. To assay for Igh-Myc translocations Unniraman et al.⁵ used primers specific for Myc combined with consensus primers that hybridize in multiple locations in Igh S_μ and S_α switch region repeats and did 80 cycles of nested amplification followed by Southern blotting for detection⁵. When we did this assay using DNA extracted from lymph nodes of interleukin 6 (Il6)-transgenic mice, which have Igh-Myc translocations, we found that multiple species were present in ethidium bromide–stained gels and that some of the minor bands hybridized with internal Myc-specific probes (data shown). In contrast, nested PCR with a combination of Myc and Igh constant region–specific primers produced discrete bands in ethidium bromide–stained gels that also hybridized with the same internal Myc-specific probe^1,2,3,4 (data shown). Using the 80-cycle consensus primer PCR assay, Unniraman et al.⁵ were able to amplify Igh-Myc hybrid DNA from 30% of the gut samples from activation-induced deaminase (AID)–deficient mice but not from kidney or brain samples. The tissue samples that were positive were reproducibly positive only 40% of the time⁵. Nevertheless, cloning showed that some of the amplicons were hybrid molecules of Myc and Igh⁵. Unniraman et al.⁵ suggest that these amplicons come from B cells that experienced AID-independent Igh-Myc translocations and that these cell populations were expanded by pristane injection in an unknown but AID-dependent way. However, these amplicons are also found in mouse strains that fail to develop pristane-induced plasmacytoma and are therefore not predictive of plasmacytoma development¹.

To determine whether B cells are required for the production of these IgH-Myc amplicons using consensus primers, we assayed 40 gut samples from 10 C57BL/6 recombination activating gene 1–deficient (Rag1^−/−) mice, 40 gut samples from 10 BALB/c mice and 10 kidney samples from BALB/c mice. We found that 20% of the C57BL/6 Rag1^−/− gut samples, 52% of the BALB/c gut samples and 60% of the BALB/c kidney samples were positive in this assay, although there was substantial variability between samples, including differences in the pattern and intensity of hybridization (Fig. 1). Cloning showed that amplicons produced with the consensus primers from Rag1^−/− gut and BALB/c kidney samples had unique Igh-Myc hybrid sequences (data not shown), but these amplicons could not be detected with the specific primers in the same samples. We caution that amplicons produced using consensus Igh switch primers may or may not reflect the existence of a chromosome translocation and that in our experience, such amplicons are not dependent on the presence of mature B cell DNA.

Figure 1: Igh-Myc PCR and Southern blotting.

Top, ethidium bromide–stained agarose gels of PCR of BALB/c gut and kidney (Kid) and Rag1^−/− gut tissue samples using primers that hybridize with Igh switch region repeats and unique sequences in Myc¹. Bottom, Southern blots of the same samples using the internal Myc oligonucleotide as a probe⁵, after a 24-hour exposure. The Myc-hybridizing bands are not necessarily the prominent bands in the ethidium bromide gels. PCR and Southern blots were done as described⁵.