AID-Targeting and Hypermutation of Non-Immunoglobulin Genes Does Not Correlate with Proximity to Immunoglobulin Genes in Germinal Center B Cells

Upon activation, B cells divide, form a germinal center, and express the activation induced deaminase (AID), an enzyme that triggers somatic hypermutation of the variable regions of immunoglobulin (Ig) loci. Recent evidence indicates that at least 25% of expressed genes in germinal center B cells are mutated or deaminated by AID. One of the most deaminated genes, c-Myc, frequently appears as a translocation partner with the Ig heavy chain gene (Igh) in mouse plasmacytomas and human Burkitt's lymphomas. This indicates that the two genes or their double-strand break ends come into close proximity at a biologically relevant frequency. However, the proximity of c-Myc and Igh has never been measured in germinal center B cells, where many such translocations are thought to occur. We hypothesized that in germinal center B cells, not only is c-Myc near Igh, but other mutating non-Ig genes are deaminated by AID because they are near Ig genes, the primary targets of AID. We tested this “collateral damage” model using 3D-fluorescence in situ hybridization (3D-FISH) to measure the distance from non-Ig genes to Ig genes in germinal center B cells. We also made mice transgenic for human MYC and measured expression and mutation of the transgenes. We found that there is no correlation between proximity to Ig genes and levels of AID targeting or gene mutation, and that c-Myc was not closer to Igh than were other non-Ig genes. In addition, the human MYC transgenes did not accumulate mutations and were not deaminated by AID. We conclude that proximity to Ig loci is unlikely to be a major determinant of AID targeting or mutation of non-Ig genes, and that the MYC transgenes are either missing important regulatory elements that allow mutation or are unable to mutate because their new nuclear position is not conducive to AID deamination

Analysis of Expressed and Non-Expressed IGK Locus Rearrangements in Chronic Lymphocytic Leukemia

Immunoglobulin κ (IGK) locus rearrangements were analyzed in parallel on cDNA/genomic DNA in 188 κ- and 103 λ-chronic lymphocytic leukemia (CLL) cases. IGKV-KDE and IGKJ-C-intron-KDE rearrangements were also analyzed on genomic DNA. In κ-CLL, only 3 of 188 cases carried double in-frame IGKV-J transcripts: in such cases, the possibility that leukemic cells expressed more than one κ chain cannot be excluded. Twenty-eight κ-CLL cases also carried nonexpressed (nontranscribed and/or out-of-frame) IGKV-J rearrangements. Taking IGKV-J, IGKV-KDE, and IGKJ-C-intron-KDE rearrangements together, 38% of κ-CLL cases carried biallelic IGK locus rearrangements. In λ-CLL, 69 IGKV-J rearrangements were detected in 64 of 103 cases (62%); 24 rearrangements (38.2%) were in-frame. Four cases carried in-frame IGKV-J transcripts but retained monotypic light-chain expression, suggesting posttranscriptional regulation of allelic exclusion. In all, taking IGKV-J, IGKV-KDE, and IGKJ-C-intron-KDE rearrangements together, 97% of λ-CLL cases had at least 1 rearranged IGK allele, in keeping with normal cells. IG repertoire comparisons in κ- versus λ-CLL revealed that CLL precursor cells tried many rearrangements on the same IGK allele before they became λ producers. Thirteen of 28 and 26 of 69 non-expressed sequences in, respectively, κ- or λ-CLL had < 100% homology to germline. This finding might be considered as evidence for secondary rearrangements occurring after the onset of somatic hypermutation, at least in some cases. The inactivation of potentially functional IGKV-J joints by secondary rearrangements indicates active receptor editing in CLL and provides further evidence for the role of antigen in CLL immunopathogenesis