Ig and non-Ig gene mutation patterns in IµHABcl6 GC B-cell lymphomas. (A) Sequence analysis of AID target genes was performed on genomic DNA from mature B-cell tumors that developed in IµHABcl6 (n = 3), IµHABcl6 Ung^−/−Msh2^−/− (n = 4), IµHABcl6 Ung^−/− (n = 3), and IµHABcl6 Msh2^−/− (n = 2) mice. With exception of the IµHABcl6 Ung^−/− FLs, all tumors in this graph are DLBCLs. A total of ∼632 kb of sequencing data were obtained. No mutations were detected in any pre-B-cell lymphomas, consistent with their AID-independent stage of development (not shown). Mutation frequencies for JH4, Pim1, and RhoH are shown as indicated. Bars denote mutation frequency on the y-axis with black and white fill indicating clonal and nonclonal mutations, respectively. Statistical significance was assessed by Pearson’s χ² test. NS, not significant; *P < .05; **P < .01. (B) The pattern of nucleotide substitutions detected in the IgH JH4 regions from mature B-cell tumors with the indicated genotypes was compared with that of Peyer’s patch GC B cells from healthy 4-month-old IµHABcl6 and IµHABcl6 Ung^−/−Msh2^−/− mice. Nucleotides in the left column are mutated to the nucleotides in the top row. If the same nucleotide substitution occurred at the same site in multiple clones, it was counted only once. Percentage of mutations occurring at a specific nucleotide base is calculated in the last column. Total mutation frequency and hotspot fold over random (calculated as the ratio of observed-to-expected frequency of hotspot C/G to T/A mutations) is below each box. (C) Analysis of CSR and subsequent mutational analysis of Ig 5′Sµ region was performed on ex vivo activated healthy B cells (white bars) and mature B-cell lymphomas (black bars) from the indicated genotypes. Numbers on the x-axis denote the percentage of cells that underwent CSR from IgM to IgG1 (determined by flow cytometry). (D) Clonal mutation plots of Pim1, Pax5, and RhoH. Genomic loci are shown with untranslated (open boxes) and coding (filled boxes) regions. An ∼1-kb region (brackets and dashed lines) downstream of the major transcriptional start site (arrows) was sequenced. Analysis of ∼96 kb of total sequencing data from 12 B-cell tumors (7 DLBCL, 3 FL, and 2 pre-B cell) revealed 6 unique clonal mutations within Pim1, Pax5, and RhoH. All mutations were found in DLBCLs from IµHABcl6 Ung^−/−Msh2^−/− mice. Mutations (underlined) and surrounding nucleotides are shown. Bolded sequences indicate an AID hotspot motif. The [AG]₉ and [A]₁₀ microsatellite repeats within the RhoH locus are indicated.