Analysis of 6912 Unselected Somatic Hypermutations in Human VDJ Rearrangements Reveals Lack of Strand Specificity and Correlation between Phase II Substitution Rates and Distance to the Nearest 3′ Activation-Induced Cytidine Deaminase Target

Abstract

The initial event of somatic hypermutation (SHM) is the deamination of cytidine residues by activation-induced cytidine deaminase (AID). Deamination is followed by the replication over uracil and/or different error-prone repair events. We sequenced 659 nonproductive human IgH rearrangements (IGHV3-23*01) from blood B lymphocytes enriched for CD27-positive memory cells. Analyses of 6,912 unique, unselected substitutions showed that in vivo hot and cold spots for the SHM of C and G residues corresponded closely to the target preferences reported for AID in vitro. A detailed analysis of all possible four-nucleotide motifs present on both strands of the VH gene showed significant correlations between the substitution frequencies in reverse complementary motifs, suggesting that the SHM machinery targets both strands equally well. An analysis of individual JH and D gene segments showed that the substitution frequencies in the individual motifs were comparable to the frequencies found in the VH gene. Interestingly, JH6-carrying sequences were less likely to undergo SHM (average 15.2 substitutions per VH region) than sequences using JH4 (18.1 substitutions, p = 0.03). We also found that the substitution rates in G and T residues correlated inversely with the distance to the nearest 3′ WRC AID hot spot motif on both the nontranscribed and transcribed strands. This suggests that phase II SHM takes place 5′ of the initial AID deamination target and primarily targets T and G residues or, alternatively, the corresponding A and C residues on the opposite strand.

Overlapping hotspots in CDRs are critical sites for V region diversification

“…To assess the role of the local sequence context in recruiting AID-induced mutations in human heavy chain V regions, we first reexamined the relative frequency and distribution of 2,657 independent mutations in IGHV3-23*01 V regions from circulating memory B cells of 24 normal individuals reported by Ohm-Laursen and colleagues (45,46). These mutations were from 438 V regions that had premature stop codons or frame shifts that likely arose during the creation of the CDR3 and were extracted by Ohm-Laursen and colleagues (45,46) from a larger database of IGHV3-23*01 V region sequences. Because the mutations were at sites of variable, diversity, and joining (VDJ) region rearrangement, the V regions are presumed to have been nonproductive throughout the differentiation of the B-cell clone that contained them, so the subsequent mutations that arose in those V regions were not subjected to antigen selection (45,46).…”

“…To assess the role of the local sequence context in recruiting AID-induced mutations in human heavy chain V regions, we first reexamined the relative frequency and distribution of 2,657 independent mutations in IGHV3-23*01 V regions from circulating memory B cells of 24 normal individuals reported by Ohm-Laursen and colleagues (45,46). These mutations were from 438 V regions that had premature stop codons or frame shifts that likely arose during the creation of the CDR3 and were extracted by Ohm-Laursen and colleagues (45,46) from a larger database of IGHV3-23*01 V region sequences.…”

“…These mutations were from 438 V regions that had premature stop codons or frame shifts that likely arose during the creation of the CDR3 and were extracted by Ohm-Laursen and colleagues (45,46) from a larger database of IGHV3-23*01 V region sequences. Because the mutations were at sites of variable, diversity, and joining (VDJ) region rearrangement, the V regions are presumed to have been nonproductive throughout the differentiation of the B-cell clone that contained them, so the subsequent mutations that arose in those V regions were not subjected to antigen selection (45,46). Because none of the V regions shared the same CDR3, the observed mutations arose from different B-cell clones, and each of these nonproductive V regions reflects sets of independent mutational events (45,46).…”

“…The IGVH3 family represents about half of the functional genomic human V regions (40), and IGVH3-23 is particularly interesting because it is the most commonly used V region during normal immune responses (41)(42)(43), as well as in some B-cell malignancies (44). Fortunately, there is a large database available for bioinformatic analysis with independently mutated productive and nonproductive human IGHV3-23*01 sequences (45,46). By analyzing the patterns of mutation in the nonproductive genes in this memory B-cell database, we found that the overlapping AID hotspot (OHS), WGCW, especially AGCT, has a marked propensity to undergo AID deamination, especially in CDR2 of human IGHV3-23*01, and that if the OHS in CDRs do not undergo mutation, there is a decrease in the mutation frequency throughout the V region.…”

Coupling mammalian cell surface display with somatic hypermutation for the discovery and maturation of human antibodies

“…AID is essential for the initiation of SHM in B cells by the deamination of cytidine residues directly in Ig genes (4,5). To achieve this, AID is targeted to V-region DNA sequences, termed hotspots (e.g., WRCH) that result in mutations and amino acid substitutions, which are frequently in positions biased to modulate antigen binding (6). Expression of AID alone has been shown to be sufficient to reproduce the salient features of SHM in both B cells and other mammalian cells (4,7,8).…”