A high-resolution landscape of mutations in the BCL6 super-enhancer in normal human B cells

Abstract

The super-enhancers (SEs) of lineage-specific genes in B cells are off-target sites of somatic hypermutation. However, the inability to detect sufficient numbers of mutations in normal human B cells has precluded the generation of a high-resolution mutational landscape of SEs. Here we captured and sequenced 12 B cell SEs at single-nucleotide resolution from 10 healthy individuals across diverse ethnicities. We detected a total of approximately 9,000 subclonal mutations (allele frequencies <0.1%); of these, approximately 8,000 are present in the BCL6 SE alone. Within the BCL6 SE, we identified 3 regions of clustered mutations in which the mutation frequency is ∼7 × 10^-4 Mutational spectra show a predominance of C > T/G > A and A > G/T > C substitutions, consistent with the activities of activation-induced-cytidine deaminase (AID) and the A-T mutator, DNA polymerase η, respectively, in mutagenesis in normal B cells. Analyses of mutational signatures further corroborate the participation of these factors in this process. Single base substitution signatures SBS85, SBS37, and SBS39 were found in the BCL6 SE. While SBS85 is a denoted signature of AID in lymphoid cells, the etiologies of SBS37 and SBS39 are unknown. Our analysis suggests the contribution of error-prone DNA polymerases to the latter signatures. The high-resolution mutation landscape has enabled accurate profiling of subclonal mutations in B cell SEs in normal individuals. By virtue of the fact that subclonal SE mutations are clonally expanded in B cell lymphomas, our studies also offer the potential for early detection of neoplastic alterations.

Keywords: BCL6; duplex sequencing; mutational signatures; somatic hypermutation; super-enhancer.

Fig. 1.

Mutation frequencies in various TSS-SE gene loci and in the Ig gene IgHV3-23 in B cells from 10 healthy individuals. Twelve aSHM targets as well as the Ig gene IgHV3-23 were captured from CD19⁺ B cells purified from 10 independent blood donors and sequenced by DS. The mutation frequency—the total number of mutations divided by total number of nucleotides sequenced—was calculated for each sample and plotted as shown. Note that the mutation frequency at the BCL6 SE (2.2 × 10⁻⁴) is as high as that of IgHV3-23 (1.3 × 10⁻⁴). Data are presented as mean ± SEM (n = 10). SEs with mutation frequencies >1.0 × 10⁻⁴, between 2.5 × 10⁻⁶ and 1.0 × 10⁻⁴, and <2.5 × 10⁻⁶ are highlighted in red, blue, and orange, respectively.

Fig. 2.

Landscape of aSHM at the BCL6 SE locus. (A) Average MAFs (number of mutations at a given nucleotide position/sequence read depth at that position) at each nucleotide position within the 1,001-bp region of the BCL6 SE. Average MAFs in the two 50-bp hotspot clusters and in a 50-bp cold spot are indicated. (B) Mutational profiles of the BCL6 SE locus are conserved in individuals of different ethnicities. Most of the mutations are present in the 600-bp segment at the 5′-end of the genomic DNA sequence, that is, between positions +400 and +1,000 from the TSS. Three mutational clusters are apparent. Whereas mutations in clusters 1 and 2 have higher mutant allele fractions, those in the TAA/WA sequence have lower fractions but higher density.

Fig. 3.

Order of AID and Pol η activities revealed by mutational frequencies and spectra. Cumulative mutations in BCL6 SE from 10 individuals were sorted into 5 bins based on the mutant allele fractions at each nucleotide position: bin 1, >1 × 10⁻³; bin 2, >5 × 10⁻⁴ to 1 × 10⁻³; bin 3, >1 × 10⁻⁴ to 5 × 10⁻⁴; bin 4, >5 × 10⁻⁵ to 1 × 10⁻⁴; and bin 5, >1.7 × 10⁻⁵ to 5 × 10⁻⁵. (A) Order of mutations as a function of the fraction of each base substitution type in each bin. C > T/G > A (AID-mediated) and C > G/G > C substitutions dominate bin 1 (∼80%), decrease in bins 2 and 3, and then gradually increase in bins 4 and 5. In contrast, A > G/T > C substitutions (Pol η-generated) constitute only a minor fraction of all substitutions in bin 1 but increase in bins 2 and 3 before declining. (B) Order of mutations at G:C vs. A:T pairs.

Fig. 4.

Deconstruction of signatures of unique mutations in BCL6 SE (A) and IgHV3-23 (B) from 10 merged B cell samples using deconstructSigs (24). Panels represent the original profiles (A-1 and B-1), the signature as computed using the best-fitting combination of COSMICv3 SBS signatures (A-2 and B-2), the difference between the preceding 2 figures (A-3 and B-3), and the breakdown of the original profile into COSMIC v3 SBS signatures (A-4 and B-4). Types of base substitutions and the array of trinucleotide motifs are displayed; motifs with the most frequent substitutions found in the original profile (panel 1) are highlighted with colors designated for each of the six base substitution types.