Tandem Substitutions in Somatic Hypermutation

Upon antigen recognition, activation-induced cytosine deaminase initiates affinity maturation of the B-cell receptor by somatic hypermutation (SHM) through error-prone DNA repair pathways. SHM typically creates single nucleotide substitutions, but tandem substitutions may also occur. While tandem substitutions have been described in mice and other species, the incidence of this phenomenon and its underlying mechanism in humans is currently unknown. We investigated incidence and sequence context of tandem substitutions by massive parallel sequencing of V(D)J repertoires in healthy human donors generated by unbiased ARTISAN PCR. Selection of unique, clonally unrelated, antigen-experienced sequences carrying up to 5% mutations yielded 13.532 VDJ, 7.952 VJ-kappa and 7.598 VJ-lambda. Comparison to the closest germline allele allowed for identification of a total of 122.878 single nucleotide substitutions (SNS), 10.735 tandem dinucleotide substitutions (TDNS) and 2.615 longer contiguous substitutions. After correcting for expected clusters of adjacent SNS, tandem substitutions comprised 5,7% of all AID-induced mutations. The mutation of more than one nucleotide in a single event, was shown to overcome amino acid codon redundancy and may therefore enhance the adaptive immune response. Clustering of such mutations around AID hotspots and their overall distribution indicates that tandem substitutions are an integral part of the SHM spectrum. In the majority of tandem substitutions, the mutated sequence may be identified in the directly adjacent reference sequence context. Tandem substitutions in humans therefore represent single nucleotide juxtalocations. Such juxtalocations appear to be favored in polydipyramidine stretches. These observations could be confirmed in patients with MSH2/6 deficiency, but were absent in a VDJ library from an UNG-deficient patient, indicating a strict dependence on abasic sites as an instigating mechanism. Together, these findings delineate a model where tandem substitutions are predominantly generated by translesion synthesis across an apyramidinic site that is typically created by UNG. During replication, apyrimidinic sites transiently adapt an extruded configuration, causing skipping of the extruded base. Consequent strand decontraction leads to the juxtalocation, after which exonucleases repair the apyramidinic site and any directly adjacent mismatched base pairs. The mismatch repair pathway appears to account for the remainder of tandem substitutions. Our study shows that a significant portion of mutations acquired during SHM are caused by tandem substitutions, and that this mechanism may enhance affinity maturation and expedite the adaptive immune response by overcoming amino acid codon degeneracies or mutating two adjacent amino acid residues simultaneously. Figure legend. Corrected incidence of tandem dinucleotide substitutions in healthy donors. (A) Dinucleotide substitutions from unique IGHV, IGKV and IGLV sequences and corrected after in silico predictions of dinucleotide substitutions that did not occur in tandem. Burgundy cells represent sequence inversions, light and dark purple cells represent juxtalocations of the 5' and 3' base in the pair (as seen from the non-transcribed strand), respectively. For unshaded cells, juxtalocation could not be assessed due to one or more nucleotides in the reference sequence matching the mutated sequence. (B) Relative contribution of sequence inversions and juxtalocations. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.