miR-217 expression in B cells enhances SHM. (A-B) Quantification of mutations in the J_H4 intronic sequence of GC (Fas⁺GL7⁺) B cells isolated from pooled Peyer’s patches of 6 control and 6 miR-217^TG mice: (A) Sanger sequencing. Segment sizes in the pie charts are proportional to the number of sequences carrying the number of mutations indicated at the periphery. Mutation frequencies are indicated below each chart, and the total numbers of independent sequences analyzed are indicated in the chart centers. Statistical significance was determined by a 2-tailed Student t test (P = .028). (B) NGS quantification of the total J_H4 mutation frequency and of the mutation frequency at cytosines within the RGYW/WRCY AID hotspots in J_H4 sequences in GC B cells from control (open bars) and miR-217^TG mice (filled bars). The mutation frequency in the J_H4 sequence of AID^−/− splenic B cells is shown in hatched bars. A total of 300 000 kb/genotype was sequenced. Statistical significance of the mean mutation frequency at each position was determined by a paired Student t test (P = 1.9 × 10⁻⁹ for total mutation frequency and P = .002 for mutation frequency at WRCY hotspots).