Position-Dependent Differential Targeting of Somatic Hypermutation

Abstract

Somatic hypermutation (SHM) generates much of the Ab diversity necessary for affinity maturation and effective humoral immunity. The activation-induced cytidine deaminase-induced DNA lesions and error-prone repair that underlie SHM are known to exhibit intrinsic biases when targeting the Ig sequences. Computational models for SHM targeting often model the targeting probability of a nucleotide in a motif-based fashion, assuming that the same DNA motif is equally likely to be targeted regardless of its position along the Ig sequence. The validity of this assumption, however, has not been rigorously studied in vivo. In this study, by analyzing a large collection of 956,157 human Ig sequences while controlling for the confounding influence of selection, we show that the likelihood of a DNA 5-mer motif being targeted by SHM is not the same at different positions in the same Ig sequence. We found position-dependent differential SHM targeting for about three quarters of the 38 and 269 unique motifs from more than half of the 292 and 1912 motif-allele pairs analyzed using productive and nonproductive Ig sequences, respectively. The direction of the differential SHM targeting was largely conserved across individuals with no allele-specific effect within an IgH variable gene family, but was not consistent with general decay of SHM targeting with increasing distance from the transcription start site. However, SHM targeting did correlate positively with the mutability of the wider sequence neighborhood surrounding the motif. These findings provide insights and future directions for computational efforts toward modeling SHM.

Figure 1

Motif-allele pairs (MAPs) in which the central nucleotide of the motif can only mutate synonymously. (A) MAPs found in an example set of germline IGHV alleles. The list of MAPs in IGHV2-5*01 is enumerated in the box. (B) Distribution of the number of MAPs harbored in a germline IGHV allele. (C) Distribution of the nucleotide distance between the central nucleotides at the 5’ and 3’ sites of a MAP.

Figure 2

Position-dependent differential SHM targeting within MAPs. (A) Examples of MAPs being tested for differential SHM targeting in individual-subject S5F analysis and aggregate RS5NF analysis pooling all non-productively rearranged sequences. Gray shaded regions correspond to CDR1 and CDR2. (B) Difference in likelihood of mutating at one site but not at the other between the 5’ and 3’ sites of MAPs showing significant differential SHM targeting in each individual-subject S5F analysis. Each point represents a MAP and is colored by the classical categorization of the motif in the MAP. Numbers at the top indicate the number of MAPs found significant. (C) Percentage of MAPs showing significant differential SHM targeting in observed sequences (solid) versus sequences from 500 simulated datasets (hollow) based on the S5F model.

Figure 3

Direction of differential SHM targeting is largely conserved across individuals. A meta-analysis identified 190 MAPs with significant differential SHM targeting, for which the directions observed in individual-subject S5F analyses, aggregate S5F analysis pooling all productively rearranged sequences, and aggregate RS5NF analysis pooling all non-productively rearranged sequences are also shown. Each column identifies a MAP. The identities of the MAPs are detailed in Supplemental Figure 6.

Figure 4

Direction of significant differential SHM targeting for MAPs found in multiple (A) IGHV1 alleles; (B) IGHV2 alleles; (C) IGHV3 alleles; (D) IGHV4 alleles; and (E) alleles across different IGHV families. Each point represents a MAP, with the x-axis indicating the allele in which the MAP is found and the y-axis indicating the 5-mer motif and the IMGT-numbered positions of the central nucleotides at the 5’ and 3’ sites.

Figure 5

Potential factors related to the direction of (A) Position-dependent differential SHM targeting in 190 MAPs that tested significant in the meta-analysis. Gray shaded regions correspond to CDR1 and CDR2. (B) Fold change in mutation frequency at 5’ site over that at 3’ site versus distance between the 5’ and 3’ sites of a MAP, with the linear regression line shown in red. Relationship between the mutation frequency of each MAP and (C) the percentage of classical hot spots, or (D, E) the average mutability, in a neighborhood extending 16 (C, D) or 23 (E) 5-mer motifs in the 5’ direction and 27 5-mer motifs in the 3’ direction. Each line represents a MAP. MAPs for which the slope was undefined due to 5’ and 3’ sites having equal percentages of surrounding hot spots or average mutability were excluded. One-sided binomial tests were performed for the proportions of positive slopes being >0.5.