Characterization of Ig gene somatic hypermutation in the absence of activation-induced cytidine deaminase

Abstract

Somatic hypermutation (SHM) of Ig genes depends upon the deamination of C nucleotides in WRCY (W = A/T, R = A/G, Y = C/T) motifs by activation-induced cytidine deaminase (AICDA). Despite this, a large number of mutations occur in WA motifs that can be accounted for by the activity of polymerase eta (POL eta). To determine whether there are AICDA-independent mutations and to characterize the relationship between AICDA- and POL eta-mediated mutations, 1470 H chain and 1313 kappa- and lambda-chain rearrangements from three AICDA(-/-) patients were analyzed. The Ig mutation frequency of all V(H) genes from AICDA(-/-) patients was 40-fold less than that of normal donors, whereas the mutation frequency of mutated V(H) sequences from AICDA(-/-) patients was 6.8-fold less than that of normal donors. AICDA(-/-) B cells lack mutations in WRCY/RGYW motifs as well as replacement mutations and mutational targeting in complementarity-determining regions. A significantly reduced mutation frequency in WA motifs compared with normal donors and an increased percentage of transitions, which may relate to reduced uracil DNA-glycosylase activity, suggest a role for AICDA in regulating POL eta and uracil DNA-glycosylase activity. Similar results were observed in V(L) rearrangements. The residual mutations were predominantly G:C substitutions, indicating that AICDA-independent cytidine deamination was a likely, yet inefficient, mechanism for mutating Ig genes.

Figure 1. The nucleotide mutation pattern in productive and nonproductive heavy chain rearrangements in AICDA^−/− B cells

The individual nucleotide substitutions detected in the variable segment of (A) nonproductive V_HDJ_H and (B) productive V_HDJ_H rearrangements in peripheral blood B cells from 4 normal donors and 3 AICDA^−/− patients are presented. The percentages of all mutations (lower panels in each group) are scored after correction for base composition of the target sequence. Transitions appear in bold face type. *Significant (p=0.03) difference between AICDA^−/− and normal donors; †significant (p<0.0001) difference between transitions and transversions and significant (p<0.0008) difference in transitions between AICDA^−/− and normal donor.

Figure 2. The nucleotide mutation pattern in nonproductive light chain rearrangements in AICDA^−/− B cells

The light chain data is collected from AICDA^−/− patients P4 and P5 and consists of 18 mutations from 294 nonproductive kappa rearrangements and 16 mutations from 142 nonproductive lambda rearrangements. (A) Individual nucleotide substitutions in the light chain repertoires are presented in which the percentage of all mutations (lower panels) are scored after correction for base composition of the target sequence. (B) The mutation frequency of a specific nucleotide inside vs outside a hypermutable motif was calculated as shown in Table 3. *Significant (p=.02) difference between AICDA^−/− and normal donors; †significant (p<.0001) difference between transitions and transversions and significant (p=.0008) difference in transitions between AICDA^−/− and normal donor.