Characterization of Ig Gene Somatic Hypermutation in the Absence of Activation-Induced Cytidine Deaminase

This information is current as Nancy S. Longo, Colleen L. Satorius, Alessandro Plebani, of September 29, 2021. Anne Durandy and Peter E. Lipsky J Immunol 2008; 181:1299-1306; ; doi: 10.4049/jimmunol.181.2.1299 http://www.jimmunol.org/content/181/2/1299 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Characterization of Ig Gene Somatic Hypermutation in the Absence of Activation-Induced Cytidine Deaminase1

Nancy S. Longo,* Colleen L. Satorius,* Alessandro Plebani,† Anne Durandy,‡§¶ and Peter E. Lipsky2*

؍ A/G, Y ؍ A/T, R ؍ Somatic hypermutation (SHM) of Ig genes depends upon the deamination of C nucleotides in WRCY (W 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 ␩ (POL ␩). To determine whether there are AICDA-independent mutations and to characterize the relationship between AICDA- and POL ␩-mediated mutations, 1470 H chain and 1313 ␬- and ␭-chain ؊/؊ ؊/؊ rearrangements from three AICDA patients were analyzed. The Ig mutation frequency of all VH genes from AICDA ؊/؊ patients was 40-fold less than that of normal donors, whereas the mutation frequency of mutated VH 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 Downloaded from 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 ␩ and uracil DNA-glycosylase activity. Similar results were observed in VL 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. The Journal of Immunology, 2008, 181: 1299–1306. http://www.jimmunol.org/ xpression of B cell-specific activation-induced cytidine owing to the activity of other mechanisms, such as error-prone deaminase (AICDA)3 in germinal center (GC) B cells is polymerase (POL) ␩, are not known. E required for generating somatic hypermutation (SHM) of AICDA preferentially deaminates Cs in the WRCY motif (W ϭ Ig genes and affinity maturation of Abs during an immune response A/T, R ϭ A/G, Y ϭ C/T) in both DNA strands (6–9) and is (1, 2). AICDA-deficient B cells can undergo a GC reaction but fail directly associated with generating the G/C mutation spectrum. to accumulate mutations in the Ig variable regions following im- The targeting mechanism of AICDA and certain features of Ig H munization (3). The lack of mutation may contribute to shifts in the chain gene CDR, such as the presence of overlapping targeted H and L chain repertoires (4). Although AICDA is critical for motifs, are associated with favoring CDR as opposed to framework by guest on September 29, 2021 SHM, other mechanisms contribute to the overall mutational pat- region (FR) mutations and also replacement (R) substitutions that tern (5) and some mutations may occur independent of AICDA (1, impact the Ag binding site (8, 10) and enable affinity maturation. 3). However, the nature of the mutations in the absence of AICDA The A/T mutation spectrum is associated with the activity of and the relationship between AICDA-induced mutations and those error-prone POL ␩ that preferentially targets A residues in the WA motif. Linkage between AICDA and POL ␩ activity was suggested by a detailed analysis of mutation patterning showing that muta- *Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin tions of POL ␩-targeted WA motifs were increased when these † Diseases, Bethesda, MD 20892; Clinica Pediatrica and Istituto di Medicina, Mole- motifs overlapped AICDA-targeted RGYW motifs (N. S. Longo, colare “Angelo Nocivelli,” Universita`di Brescia, Brescia, Italy; ‡Institut National de la Sante´de la Recherche Me´dicale, Unite´768, Paris, France; §Universite´Paris-Des- P. L. Lugar, S. Yavuz, W. Zheng, P. H. L. Krijger, D. E. Russ, cartes, Faculte´deMe´dicine Rene´Descartes, Paris, France; and ¶Assistance Publique- A. C. Grammer, and P. E. Lipsky, manuscript submitted for pub- Hoˆpitaux de Paris, Hoˆpital Necker Enfants Malades, Service d’Immunologie et d’He´matologie Pe´diatrique, Paris, France lication). This pattern of mutations suggested that the AICDA- Received for publication February 11, 2008. Accepted for publication May 5, 2008. induced U:G lesions may foster additional mutation in adjacent residues through the recruitment of a WA/TW motif mutator, such The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance as POL ␩. Consistent with this mechanism, POL ␩ deficiency re- with 18 U.S.C. Section 1734 solely to indicate this fact. sults in a loss of mutations in WA motifs in AICDA-targeted 1 This work was supported in part by the National Institutes of Health, National WRCY/RGYW motifs (11). However, because POL ␩ deficiency Institute of Arthritis and Musculoskeletal and Skin Diseases, Intramural Research Program, grants from the Institut National de la Sante´et de la Recherche Me´dicale, also results in a loss of WA mutations remote from RGYW/ Association de la Recherche Contre le Cancer, the European Community No. PL WRCY motifs, it is possible that this error-prone POL might also 006411 (EUROPOLICY-PID)-6th Programme Cadre de Recherche De´veloppment, have an AICDA-independent function. the Association Nationale pour la Recherche, Institut National du Cancer, and a grant from the Fondazione C. Golgi and the Associazione Immunodeficienze Primitive, To characterize the mutational pattern that occurs in the absence Brescia, Italy. of AICDA and to explore the relationship between AICDA and 2 Address correspondence and reprint requests to Dr. Peter E. Lipsky, National In- POL ␩ in more detail as well to characterize the mutational pattern stitute of Arthritis and Musculoskeletal and Skin Diseases, 9000 Rockville Pike, Building 10, 6D47C, Bethesda, MD 20892. E-mail address: [email protected] that occurs in the absence of AICDA, we analyzed the H chain and L chain mutations from three AICDA-deficient patients. We fo- 3 Abbreviations used in this paper: AICDA, activation-induced cytidine deaminase; FR, framework region; MMR, mismatch repair; POL, polymerase; R, replacement; S, cused on mutations in nonproductive rearrangements that are not silent; SHM, somatic hypermutation; GC, germinal center; UNG, uracil-DNA influenced by selection to identify mutational mechanisms. There was glycosylase. a significant reduction in the frequency of mutations, but still signif- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 icantly more than could be anticipated in genes of non-B cells. Many www.jimmunol.org 1300 Ig MUTATIONS IN HUMAN AICDA DEFICIENCY

of the mutations could be attributed to non-targeted AICDA-indepen- [77], IGKV1-27*01 [94], IGKV1-12*01 [24], IGLV 13-21*01 [108], and dent cytidine deamination resulting in a loss of targeting of mutations IGLV 6-57*01 [49]. to CDRs. The mutation pattern in AICDA deficiency was biased to- ward transitions and lacked mutations in POL ␩-targeted WA motifs, Statistical analysis indicating that AICDA is necessary for the recruitment of POL ␩ and The ␹2 test and the p Ͻ 0.05 limit were used to determine statistically also uracil-DNA glycosylase (UNG) during SHM. significant differences. The ratio of nucleotide mutational targeting inside the motif vs mutation of the nucleotide outside the motif was used to compare differences between AICDA and normal donors (see Table III and Materials and Methods Fig. 2A). Patient samples and cell preparation Calculation of the PCR error rate Three AICDA-deficient patients donated peripheral blood samples after signing informed consent forms in accordance with institutional review The PCR error of this technique was initially demonstrated to be 1 ϫ 10Ϫ4 board-approved protocols. The control samples (12–14) and aicda muta- by amplifying an unmutated germline Ig sequence 96 times using the same tions and Ig levels for each patient (P4 age 11, P5 age 7, and P6 age 21) amplification and sequencing conditions used here (19). The low PCR error have been described previously (3). Blood mononuclear cells were isolated rate was confirmed by amplifying c-kit genes from B cells (0.8 ϫ 10Ϫ4,6

on Ficoll gradients and stained with mouse IgG1 anti-human CD19 PE (BD of 72,261) (20). Finally, unrearranged VH family gene segments were am- Biosciences), mouse IgG2 anti-human IgD FITC (BD Pharmingen), or goat plified and sequenced from CD19Ϫ peripheral blood cells. Analysis of 96

anti-human IgD FITC (Caltag Laboratories) and anti-CD27-PE and iso- VH4 segments from CD19-negative cells demonstrated a PCR error rate of type controls (BD Pharmingen). Patient mononuclear cells were sorted 0.76 ϫ 10Ϫ4 (2 of 25,986). into CD19ϩ or CD19ϩCD27Ϫ and CD19ϩCD27ϩ subsets using a BD

Biosciences FACSVantage SE DiVa or a MoFlo Cell Sorter (Dako Downloaded from Cytomation). Sorted populations were 94–98% pure. Results Decreased mutation frequency of B cells from AICDAϪ/Ϫ Single-cell PCR patients Aliquots of approximately one cell were diluted in buffer (10 mM NaCl, 5 B cell phenotyping and mutation analysis confirmed previous re- mM Tris-HCl (pH 8) at 25°C, and 0.1% Triton X-100) containing 0.4 ports (3) that AICDA-deficient patients have a normal percentage mg/ml proteinase K (Sigma-Aldrich), and genomic DNA from individual of CD19ϩCD27ϩ B cells but unlike normal donors in which cell lysates was linearly amplified using random 15-mer (Qiagen). Aliquots http://www.jimmunol.org/ ϩ of the preamplification product were used as template for internal and CD27 expression correlates with a mutated memory B cell pop- Ϫ/Ϫ ϩ external nested amplification of the transcribed strand using VH3, VH4, and ulation, the AICDA CD27 B cells are infrequently mutated. VH1 H chain-specific upstream primers and downstream JH-specific prim- Ϫ/Ϫ In this study, 20–28% of the peripheral blood B cells from ers as described previously (14). The AICDA L chains were analyzed Ϫ/Ϫ ϩ ␬ ␬ ␬ ␭ ␭ ␭ AICDA patients were CD27 (data not shown). Both subsets with V 1, V 2, and V 3, and V 1-, V 2-, and V 3-specific primers as ϩ ϩ ϩ Ϫ described previously (15, 16). of CD19 CD27 and CD19 CD27 B cells contained mutated sequences. However, there was no significant difference in the per- Sequencing analysis centage of mutated sequences or the mutation frequency between Ϫ ϩ PCR products were separated by electrophoresis on 1.2% agarose gels, the CD27 and CD27 subsets. Since CD27 expression did not Ϫ/Ϫ Ϫ purified (Edge Biosystems), and directly sequenced using the Applied Bio- correlate with mutation in AICDA B cells and since CD27 by guest on September 29, 2021 systems Prism Big Dye Terminator Cycle Sequencing Kit. PCR products human memory cells have been identified (21), the subsets were were directly sequenced using on an automated capillary sequencer (Ap- plied Biosystems Prism 3100 Genetic Analyzer). combined for the mutation analysis. The percentages of mutated Ϫ/Ϫ VHDJH sequences in AICDA nonproductive and productive Database repertoires (12 and 9%, respectively) were significantly ( p Ͻ The analysis included 105 nonproductive and 636 productive rearrange- 0.0001) less than those of normal donors (71–75%) (Table I). The ments from normal donors that included CD19ϩIgMϩ B cells from overall mutation frequency was 40-fold less than that of normal accession numbers X87006–X87082, CD19ϩIgMϩCD5ϩ and CD19ϩ controls, whereas the mutation frequency of mutated sequences IgMϩCD5Ϫ B cells from accession numbers Z80363–Z80770 (12–14), ϩ ϩ was only 6.8-fold less than normal controls. Most of the mutated IgD CD27 B cells from accession numbers EF542547–EF542687, Ϫ/Ϫ Ϫ ϩ nonproductive AICDA sequences had a unique, single-nucle- and IgD CD27 B cells from accession numbers EF542688– EF542796. The AICDA-deficient analysis included 320 nonproductive otide substitution, except four sequences that contained two mu- ϩ Ϫ ϩ ϩ and 1150 productive CD19 CD27 and CD19 CD27 VHDJH rear- tations. In the mutated productive repertoire, 100 (96%) of 104 rangements with accession numbers (EU237493–EU238970), 294 non- rearrangements had one to two mutations, three sequences had three ␬ ␬ productive and 482 productive rearrangements (EU788043-516) mutations, and one sequence had four mutations. These mutations (EU788791-919) (EU788982-9157), and 142 nonproductive and 395 productive ␭ rearrangements ␭ (EU788517-790) (EU788920-981) clearly were not polymorphisms and occurred at approximately a 10- (EU789158-362). fold higher frequency than the PCR error for this method (6 mutations of 75,182 bp; 8 ϫ 10Ϫ5) (20) or 2 mutations of 25,986 (0.76 ϫ 10Ϫ4) Mutation analysis determined for VH4 sequences in non-B cells.

Nonproductive and productive VHDJH rearrangements were determined by using the JOINSOLVER sequence and mutation analysis program (17). Ig-hypermutable motifs are not mutated in AICDAϪ/Ϫ B cells Enumeration of nucleotides used an algorithm that takes into consideration the finding that some bp positions in the germline genes reside within more Because mutations do occur in Ig genes in the absence of AICDA, than one motif and/or within ambiguous motifs (i.e., AGCT is a RGYW we next characterized the frequency of mutation in Ig-hypermut- and WRCY motif; TA is a WA and TW motif). This approach avoided a able motifs, particularly those in the nonproductive rearrangements subjective bias in motif nucleotide designation, but also was the source of that are not influenced by positive and negative selection. An in- noninteger values. creased focus of mutations in any of the four bases constituting Seven new polymorphisms within codons 25–94 in six VH genes were excluded from the mutation analysis (N. S. Longo, P. L. Lugar, S. Yavuz, WRCY (W ϭ A/T, R ϭ A/G, Y ϭ C/T) and the inverse comple- W. Zheng, P. H. L. Krijger, D. E. Russ, A. C. Grammer, and P. E. Lipsky, mentary motif RGYW are the hallmarks of AICDA-dependent manuscript submitted for publication). The following 12 substitutions in L SHM (22, 23). In the nonproductive repertoire, the percentage of chain genes (ImMuno GeneTics (IMGT) numbering for codons in brackets) ϭ appeared to be new polymorphisms and were excluded from the mutation mutations that occurred in WRCY motifs was significantly ( p analysis: IGKV1-16*01 [75], IGKV1D-17*01 [26], IGKV1D-17*01 [52], 0.004) reduced compared with normal controls (3% vs 16%; Table IGKV1-5*03 [57], IGKV1-9*01 [86], IGKV1-9*01 [90], IGKV1-8*01 II). Similarly, the frequency of mutations in RGYW (10%) was no The Journal of Immunology 1301

a Table I. Mutation frequency of VH segment of AICDA-deficient B cells

AICDAϪ/Ϫ Normal Donors

NP P NP P

Total no. of rearrangements 320 1150 105 636 Total no. of mutations 43 124 627 4864 Percentage of sequences mutated 39/320b (12%) 104/1,150b (9%) 76/105 (71%) 479/636 (75%) Mutation range 1–2 1–4 1–33 1–38 Mutation frequency overall 43/64,081b (0.07%) 124/230,514b (0.05%) 627/22,693 (2.8%) 4864/152,977 (3.2%) Mutation frequency of mutated 43/7,799b (0.6%) 124/19,938b (0.6%) 627/15,149 (4.1%) 4864/113,434 (4.3%) rearrangements

a NP, Nonproductive rearrangements; P, productive rearrangements. The AICDAϪ/Ϫ data include CD19ϩCD27Ϫ and CD19ϩCD27ϩ sequences from three patients. The control data come from previous reports of four individuals and three different experiments that collected IgMϩ, IgMϩCD5ϩ, ϩ Ϫ ϩ Ϫ Ϫ ϩ IgM CD5 , IgD CD27 , and IgD CD27 B cells which represent normal unmutated and mutated populations in peripheral blood. The mutation frequencies were calculated from the number of mutations/total number of bases analyzed or from the total number of bases analyzed from mutated sequences only. b Significant ( p Ͻ 0.0001) difference between AICDAϪ/Ϫ and normal donors. greater than that expected from random chance (12.5%) and sig- motifs suggested that POL ␩ may be recruited to the site of the Downloaded from nificantly less than in normal controls (24%, p ϭ 0.03). The fre- AICDA-induced lesion during mismatch repair in normal human B quency of mutations in either motif (13%) was also significantly cells. In support of the human data, transgenic mice with SHM ( p ϭ 0.01) less than in normal B cells (40%). These motifs be- substrates acquired the capacity to acquire A/T mutations only come hot spots for hypermutation primarily because of AICDA when the substrate contained an AICDA-targeted cytosine (28). To targeting C in WRCY on both strands of DNA (N. S. Longo, P. L. examine the role of POL ␩ in the absence of AICDA, Lugar, S. Yavuz, W. Zheng, P. H. L. Krijger, D. E. Russ, A. C. we examined mutations in WA motifs in the H chain genes of the http://www.jimmunol.org/ Grammer, and P. E. Lipsky, manuscript submitted for publication AICDAϪ/Ϫ B cells. WA motif mutations (10%) were no more and Refs. 7, 9, and 24). In the AICDA-deficient B cells, the hy- frequent than that expected from random mutation (12.5%) and permutable nucleotides (C in WRCY and G in RGYW, which were significantly less frequent than WA motif mutations found in represents an AICDA attack on the opposite strand) were not tar- normal B cells (Table II). Notably, mutations of the hypermutable geted for mutation more frequently than mutation of C or G res- A in WA motifs were no more frequent than A mutations else- idues anywhere else in the H chain variable segment (Table III), where in the variable segment, indicating that POL ␩ activity was consistent with a complete loss of AICDA activity in these sub- greatly reduced (Table III). Similar results were noted with L chain jects. By comparison, there was nearly 4-fold more targeting of rearrangements (Fig. 2A). these specific nucleotides inside WRCY/RGYW motifs in normal by guest on September 29, 2021 Despite reduced targeting in all hypermutable motifs, mutations B cells. A similar loss of G/C mutational targeting was noted in the Ϫ Ϫ in RGYW motifs appeared to be positively selected as indicated by AICDA / L chain rearrangements (Fig. 2A). These results indi- the increased mutation frequency in the V productive compared cate that the preferential targeting of the C in WRCY (and G in H with nonproductive repertoires (Table II). A similar trend was ob- RGYW) is completely dependent on AICDA activity and provides the opportunity to examine the AICDA dependence of other served with the other motifs, but significant differences were not known components of the SHM machinery. found. Therefore, some form of selection is intact, although this is not evident in normal controls in which a large number of muta- Loss of POL ␩ mutational targeting in AICDAϪ/Ϫ B cells tions have been analyzed, presumably because large numbers of There is substantial evidence that error-prone POL ␩ plays a sig- mutations of Ig genes may disable the Ig molecule and contribute nificant role in SHM (11, 25, 26). Analysis of Ig gene mutational to B cell loss. patterns from individuals deficient in POL ␩ has indicated that this polymerase specifically targets the A in WA motifs, (11, 27) and Mutations in AICDAϪ/Ϫ B cells are not associated with a new preferentially targets the TA dinucleotide (N. S. Longo, P. L. Lu- consensus mutation motif gar, S. Yavuz, W. Zheng, P. H. L. Krijger, D. E. Russ, A. C. Grammer, and P. E. Lipsky, manuscript submitted for publication). Although the known hypermutable motifs were not targeted in More recently, evidence of WA mutations overlapping RGYW AICDAϪ/Ϫ B cells, there was the possibility that another motif

Table II. Percentage of mutations in RGYW/WRCY and WA/TW motifsa

AICDAϪ/Ϫ Normal Donors

NP P NP P

WRCY 1.5/43b (3%) 13.8/124c (11%) 98.1/627c (16%) 756.7/4864c (16%) RGYW 4.5/43b (10%) 30.3/124 (24%) 152.4/627 (24%) 1285.9/4864 (26%) WA 4.5/43b (10%) 25.5/124d (21%) 140.4/627d (22%) 888.5/4864d (18%) TW 1.5/43 (4%) 9/124 (7%) 73.7/627 (12%) 490.5/4864 (10%)

a The enumeration of nucleotides was adjusted for nucleotides that occupy a position in more than one motif or ambiguous motifs. This calculation, therefore, can assign a fraction of a mutation for a given motif. NP, Nonproductive; P, productive. b Significant ( p Յ 0.05), difference between AICDAϪ/Ϫ and ND. c Significant ( p Ͻ 0.008) difference between WRCY and RGYW. d Significant ( p Ͻ 0.009) difference between WA and TW. 1302 Ig MUTATIONS IN HUMAN AICDA DEFICIENCY

Table III. Target nucleotide mutation frequency (%) in hypermutable a motifs in nonproductive VH rearrangements

Mutated Base Motif AICDAϪ/Ϫ Normal Donor

G Inside RGYW 2/2,374b (0.08%) 94/937c (10.0%) All other G 18/13,016b (0.14%) 99/6,093 (1.6%) C Inside WRCY 0/2,140b (0%) 62/832c (7.5%) All other C 7/13,596b (0.05%) 75/5,685 (1.3%) A Inside WA 4.5/8,273b (0.05%) 115.9/2,473c (4.7%) All other A 6.5/8,564b (0.08%) 79.1/3,615 (2.2%) T Inside TW 1.5/5,180b (0.03%) 56.5/1,550c (3.6%) All other T 3.5/8,288b (0.04%) 45.5/3,714 (1.2%)

a The targeted nucleotide refers to the base in the underlined position (or two positions in WA, where W ϭ A or T). The mutation frequency of the targeted nu- cleotide inside a motif was calculated from the number of target nucleotide mutations divided by the total number of the motif (or total number of A in WA motifs). To calculate the mutation frequency outside the motif, the total number of base mutations in the sequence minus the number of targeted nucleotide mutations was divided by the total number of the base in the sequence minus the number of target nucleotides inside the motif. The extent of targeting was determined by calculating the fold difference between the mutation frequency inside a motif vs all other positions. Downloaded from b Significant ( p Ͻ 0.0001) difference between AICDAϪ/Ϫ and normal controls. c Significant ( p Ͻ 0.01) difference between the nucleotide mutation frequency inside a motif vs all other positions. was targeted. To address this question, the Ig sequences were di- vided into oligomers, each of which contained a mutated nucleo- http://www.jimmunol.org/ tide from the AICDAϪ/Ϫ B cells in the context of five nucleotides flanking both sides of the mutation. The sequences were interro- gated for shared motifs by using the web-based Genio/logo pro- gram (http://www.biogenio.com/logo/logo.cgi). This procedure failed to identify a motif within which residual mutations occurred (data not shown). Other potential patterns of mutational activity FIGURE 1. The nucleotide mutation pattern in productive and nonpro- were investigated, such as UV-induced dipyrimidine substitutions ductive H chain rearrangements in AICDAϪ/Ϫ B cells. The individual nu- or oxidative damage generating mutations of guanine dinucleoti- cleotide substitutions detected in the variable segment of (A) nonproductive des, but no pattern with a mutation frequency greater than normal VHDJH and (B) productive VHDJH rearrangements in peripheral blood B by guest on September 29, 2021 was found (data not shown). We concluded that the major mutation cells from four normal donors and three AICDAϪ/Ϫ patients are presented. mechanism in AICDAϪ/Ϫ was a random event involving individ- The percentages of all mutations (lower panels in each group) are scored ual Ig gene nucleotides. after correction for base composition of the target sequence. Transitions Significant (p ϭ 0.03) difference between ,ء .appear in boldface Ϫ Ϫ Mutational targeting of CDRs is decreased in AICDA deficiency AICDA / and normal donors; †, significant (p Ͻ 0.0001) difference be- tween transitions and transversions and significant (p Ͻ 0.0008) difference Ϫ Ϫ In normal nonproductive VH rearrangements, the frequency of mu- in transitions between AICDA / and normal donor. tation in the CDRs in VH genes is 2-fold greater than the frequency of mutations in the FRs after normalization for the number of nucleotides analyzed in each region (Table IV and Ref. 8). By the AICDAϪ/Ϫ rearrangements, an increased frequency of CDR contrast, in the AICDA-deficient VH rearrangements, the prefer- mutations and a decrease in FR mutations was noted in the productive ential mutation targeting of CDRs was lost. These data indicate repertoire and, as a result, the ratio of mutations in CDRs vs FRs was that AICDA activity is essential for targeting of mutations to normalized, presumably because of positive selection of CDR muta- CDRs. Enrichment of mutations is normally observed in CDR re- tions and negative selection of FR mutations. gions in the productive repertoire when productive and nonpro- The ratio of replacement:silent mutations (R:S) is 5.3 in normal ductive VH rearrangements are compared (Table IV). However, in donor nonproductive CDRs compared with the 3.4 in FRs (Table IV).

Table IV. The effect of AICDA deficiency on amino acid and nucleotide substitutions in CDRs and FRs of H chain rearrangementsa

AICDAϪ/Ϫ Normal Donors

NP P NP P

CDR R:S 3/1b (3.0) 41/2c (20.5) 170/32b (5.3) 1383/391 (3.5) FR R:S 24/15c (1.6) 54/27 (2.0) 329/96b (3.4) 1,940/1,150 (1.7) CDR mutation frequency 4/11,393b,c (3.5 ϫ 10Ϫ4) 43/40,699c (1 ϫ 10Ϫ3) 198/4,826b (4.1 ϫ 10Ϫ2) 1741/29,250 (6.0 ϫ 10Ϫ2) FR mutation frequency 39/52,688b,c (7.4 ϫ 10Ϫ4) 81/189,815c (0.4 ϫ 10Ϫ3) 426/20,244b (2.1 ϫ 10Ϫ2) 3,092/120,907 (2.6 ϫ 10Ϫ2) Mutation frequency 3.5:7.4b,c 1:0.4 4.1:2.1 6.0:2.6 CDR:FR 0.5 2.5 2.0 2.3

a The FR:CDR mutation frequency ratio was normalized to correct for the number of nucleotides analyzed in FRs and CDRs. The analysis excludes the CDR3 and the first 25 codons of FR1. b Significant ( p Ͻ 0.007) difference between nonproductive (NP) and productive (P) rearrangements. c Significant ( p ϭ 0.03) difference between AICDAϪ/Ϫ and normal donors. The Journal of Immunology 1303

FIGURE 2. The nucleotide mutation pattern in nonproductive L chain rearrangements in AICDAϪ/Ϫ B cells. The L chain data were collected from AICDAϪ/Ϫ patients P4 and P5 and consists of 18 mutations from 294 nonproductive ␬ rearrange- ments and 16 mutations from 142 nonproductive ␭ rearrangements. A, Individual nucleotide substitu- tions in the L 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 Downloaded from specific nucleotide inside vs outside an hypermut- able motif was calculated as shown in Table III. Significant (p ϭ 0.02) difference between ,ء AICDAϪ/Ϫ and normal donors; †, significant (p Ͻ 0.0001) difference between transitions and transver- sions and significant (p ϭ 0.0008) difference in tran- Ϫ/Ϫ sitions between AICDA and normal donor. http://www.jimmunol.org/ by guest on September 29, 2021

By comparison, the R:S ratio anticipated by random chance is 4.5 in p ϭ 0.39), whereas the percentage of G mutations was significantly CDRs and 3.2 in FRs because of codon bias. In normal donors, the increased in AICDAϪ/Ϫ compared with normal (AICDAϪ/Ϫ, 41% vs R:S ratios of both the CDRs and the FRs in the nonproductive rep- normal, 27%; p ϭ 0.03). This suggested that a major source of mu- ertoire were 1.5- to 2-fold greater than those of the productive rear- tation in AICDA-deficient cells was a G:C-focused mutational mech- rangements, suggesting that large numbers of R mutations in both anism (Fig. 1). AICDA-independent cytidine deamination is the most regions result in the loss of cells, as previously reported (8). In the common form of DNA damage in non-B cells and could contribute to absence of AICDA, R substitutions in the nonproductive repertoire the mutation pattern in AICDAϪ/Ϫ B cells (31). Presumably, this were less common in both CDRs and FRs than in normal rearrange- could target both the transcribed and nontranscribed strands with a ments. Notably, R substitutions appeared to be positively selected bias toward the latter with little or no involvement of POL ␩.By when occurring in CDRs and not eliminated when occurring in FRs, comparing the mutation frequency of AICDAϪ/Ϫ B cells to our es- as evidenced by comparison of R:S ratios in nonproductive and pro- timated mutation frequency of non-B cells, it appears that AICDA- ductive rearrangements. Presumably, the very low mutational activity independent C deamination in B cells is subjected to less of the nor- in AICDAϪ/Ϫ B cells introduces fewer deleterious mutations so that mal repair mechanisms operative in non-B cells. elimination of rearrangements with R mutations is not greater than the increase resulting from positive selection. AICDA-independent mutations are biased toward transitions The individual nucleotide substitutions in the AICDAϪ/Ϫ nonpro- Mutation of individual nucleotides in AICDA deficiency ductive rearrangements were biased toward transitions (77%), In the nonproductive AICDA-deficient repertoire, G residues were compared with normal donors in which transitions and transver- the most frequently mutated (41%), followed by A (24%), C sions occur at comparable frequencies (Fig. 1). The most evident (17%), and T residues (14%) compared with AϾGϾCϾT in nor- reason for this mutation shift was the significant ( p ϭ 0.01) in- mal donors (Fig. 1). Similar to normal controls, a bias favoring A crease in GϾA transitions and to a lesser extent AϾG transitions. mutations more than T mutations was present in AICDAϪ/Ϫ B Similarly, mutations in nonproductive AICDA-deficient L chain cells. Although POL ␩ targets A residues more than T, the absence rearrangements were skewed significantly toward transitions (Fig. of targeting in WA motifs suggested that another polymerase, such 2B). One of the mechanisms known to contribute to a loss of trans- as POL ␨, POL␪,orPOL␫, each of which is expressed in human versions is UNG deficiency (32). Even though UNG expression is tonsilar B cells, may be contributing to the residual A:T mutations increased in tonsillar GC cells, UNG may be less active or not in AICDAϪ/Ϫ B cells (29, 30). accessible when AICDA is not available. A similar phenomenon Ϫ/Ϫ Of the few mutations in AICDA VH genes, the percentage of C was noted when mutations in X-HIgM B cells, which are also mutations was similar to normal (AICDAϪ/Ϫ, 17% vs normal, 21%; characterized by less functional AICDA activity, were analyzed. 1304 Ig MUTATIONS IN HUMAN AICDA DEFICIENCY

These data suggest that AICDA plays a role not only in the initi- targeting WA motifs was related to AICDA (35). The loss of POL ation and targeting of SHM, but also in the use of UNG and POL ␩ activity in xeroderma pigmentosa variant resulted in a reduction ␩ that are involved in processing the AICDA-induced lesion. in WA mutations, regardless of their position inside or outside RGYW/WRCY motifs, indicated that WA mutations in RGYW/ Discussion WRCY were related to POL ␩ targeting and not to the direct ac- The current study demonstrated that a small number of mutations tivity of AICDA (11, 36). Thus, the loss of POL ␩ targeting in WA can develop in B cell Ig genes of individuals who totally lack motifs in the AICDAϪ/Ϫ Ig genes may reflect the observation expression of AICDA. However, in the absence of AICDA, the given that pol ␩ is constitutively expressed in B cells and not mutational frequency is markedly reduced and there is a loss of up-regulated upon CD40 ligation to a level of expression that is RGYW/WRCY mutations as well as of mutations in POL ␩-tar- sustained in GCs that are present and accentuated in size in sub- geted WA motifs. In addition, UNG-dependent transversions were jects with AICDA deficiency (3). Based upon the current finding markedly reduced. These results suggest that AICDA induces a that there was a marked reduction of WA targeting in AICDAϪ/Ϫ process that involves POL ␩ and UNG to induce the normal spec- B cells, it is likely that AICDA may directly or indirectly recruit trum of SHM. The loss of AICDA results in the absence of a POL ␩ into a mutation complex. This conclusion is consistent with mutational focus on CDRs and a bias toward R mutations in CDRs recent findings that the induction of A/T mutations in a transgenic and, therefore, little ability of SHM to contribute to affinity mat- mutation substrate required a potentially AICDA-targeted G/C site uration of the Ab response. (28). However, the current data extend this finding to implicate Because the measured mutational frequency was relatively low, POL ␩ as the error-prone polymerase targeting WA motifs in an it was essential to be certain that PCR error did not confound AICDA-dependent manner. These findings also support the data Downloaded from interpretation of the data. PCR amplification of genomic DNA from X-HIgM studies suggesting a linkage between POL ␩ tar- from individual cells with direct sequencing of the PCR products geting WA within the AICDA-induced lesion. Notably, however, used here has been documented to have a very low PCR error rate the current data indicate that AICDA is also responsible for re- (0.8–1.0 ϫ 10Ϫ4). To confirm this, we sequenced unrearranged Ig cruiting POL ␩ activity to sites remote from the AICDA-targeted Ϫ VH gene segments from CD19 non-B cells and confirmed the lesion, possibly through long patch repair (37). Ϫ4 very low PCR error rate (0.76 ϫ 10 ). Thus, few of the detected The AICDA-deficient B cells demonstrated a marked skewing http://www.jimmunol.org/ bp changes could be accounted for by PCR error. Moreover, be- toward transitions, whereas normal B cells had equal numbers of cause the majority of the data related to the absence of expected transitions and transversions. A similar skewing toward transitions mutational targeting (G in RGYW, C in WRCY, A in WA), it was was attributed to the absence of UNG in a chicken cell line (32) unlikely that these few bp changes potentially introduced by the and mouse (38) and human B cells (39). Importantly, these results PCR amplification affected the interpretation of the results. suggest a role for AICDA in recruiting UNG2 to sites of Importantly, the Ig mutation frequency of AICDAϪ/Ϫ B cells AICDA-dependent uracil generation, perhaps by forming a was greater than that expected from DNA instability in non-B multiprotein structure with proliferating cell nuclear Ag and cells. We directly measured the mutational frequency of unrear- replication protein A. by guest on September 29, 2021 ranged VH4 genes in non-B cells and determined that it was no The lack of targeting to WRCY/RGYW motifs and the decrease more than the PCR error rate. Thus, the mutation frequency of in CDR mutations as well as R substitutions in the CDRs provide AICDAϪ/Ϫ B cells was nearly 10-fold that of Ig genes in non-B support for the proposal that two intrinsic features of Ig genes may cells. Other approaches have been used to estimate the mutation have coevolved with AICDA to focus the mutation mechanism on frequency of eukaryotic cells, which is generally reported to be the Ag binding site. First, WRCY/RGYW motifs are more con- Յ Ϫ10 ϫ Ϫ3 10 per bp per cell division (33), compared with 1 10 per centrated in CDRs than in the FRs of the VH genes (8). This may bp per cell division for Ig genes in B cells (34). Assuming a DNA bias the mutational machinery initiated by AICDA to induce mu- length of 240 bp, equivalent to that of VH gene sequences, and that tations preferentially into CDRs, since the current data clearly the cells undergo 25 cell divisions, which would yield a predicted show that in the absence of AICDA, the bias for replacement mu- Ig gene mutational frequency of 2.5% (similar to that measured for tations to localize into CDRs was markedly reduced. The second normal B cells), the AICDA-deficient B cells have as much as a feature of Ig genes noted in normal B cells to influence mutational 2.8 ϫ 105-fold increase in mutational frequency compared with patterning is the biased use of codons that favor replacement sub- that of DNA in non-B cells. This estimate does not take into ac- stitutions. This was highlighted by the increase in the predicted count the increased mutability of Ig genes within B cells, but it R:S ratio in CDRs (5.2) compared with FRs (3.4) in the current clearly is consistent with the current data that the frequency of analysis. It is notable that positive selection of R mutations in the mutations of Ig genes is significantly greater in AICDAϪ/Ϫ B cells CDRs was apparent with the low mutation frequency of than that of non-Ig genes in B cells or Ig genes in non-B cells. AICDAϪ/Ϫ B cells, but was absent from the normal mutated B Whether this reflects an increased likelihood of introducing bp cells that have a markedly higher mutation frequency compared changes or an altered tendency for error-prone repair is currently with AICDAϪ/Ϫ mutated B cells. This may be attributed to the unknown. increased loss of B cells that accumulate large numbers of muta- During the GC reaction, expression of AICDA and multiple tions either because the R mutations in the CDRs introduce auto- enzymes involved in DNA repair and translesion synthesis is reactivity or R mutations in the FRs alter the structural integrity of uniquely altered, resulting in high-frequency mutation with spe- the molecule (8). The very low mutation frequency in AICDAϪ/Ϫ cific complex patterns targeted to Ig genes. A greater understand- B cells may make it less likely that deleterious mutations are in- ing of how individual components contribute to SHM in humans troduced and, therefore, more probable that positive selection of has resulted from the analysis of the Ig gene mutational pattern in mutations can be observed. B cells from individuals with a deficiency in one of the enzymes The residual mutations in AICDAϪ/Ϫ Ig genes did not appear to involved in SHM or the GC reaction itself. In normal B cells, WA be targeted by a new mutator to an identifiable motif. Moreover, mutations occur both when motifs overlap RGYW/WRCY tetrads, the mutations did not exhibit a pattern of targeting within G–G or as well as when they are distant (11). This pattern has made it Y–Y dinucleotides compatible with oxidative damage to DNA. A difficult to distinguish whether the underlying mutation process likely source of the predominant G/C nucleotide mutations was The Journal of Immunology 1305 spontaneous cytidine deamination that is common to all cells but 2. Milstein, C. 1987. Diversity and the genesis of high affinity antibodies. Biochem. apparently occurs more frequently in Ig genes, possibly because of Soc. Trans. 15: 779–787. 3. Revy, P., T. Muto, Y. Levy, F. Geissmann, A. Plebani, O. Sanal, N. 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Analysis of the frequency and pattern of somatic muta- prone polymerase is currently unknown but the targeting to G/C tions within nonproductively rearranged human variable heavy chain genes. ␩ ␪ J. Immunol. 158: 2779–2789. nucleotides indicates that POL is not involved, leaving POL or 9. Xue, K., C. Rada, and M. S. Neuberger. 2006. The in vivo pattern of AID tar- POL ␨ as likely candidates. In mice it appears that all A/T muta- geting to immunoglobulin switch regions deduced from mutation spectra in Ϫ Ϫ Ϫ Ϫ tions are attributed to POL ␩ recruitment by ⌴S⌯2 (41). However, msh2 / ung / mice. J. Exp. Med. 203: 2085–2094. 10. Milstein, C., M. S. Neuberger, and R. Staden. 1998. Both DNA strands of anti- ␩ Downloaded from A/T mutations remain in the pol and msh2 single knockouts body genes are hypermutation targets. Proc. Natl. Acad. Sci. USA 95: when AICDA and UNG are available, suggesting that MSH2 may 8791–8794. be capable of recruiting other enzymes if POL ␩ is absent or, in the 11. Yavuz, S., A. S. Yavuz, K. H. Kraemer, and P. E. Lipsky. 2002. The role of polymerase ␩ in somatic hypermutation determined by analysis of mutations in absence of MSH2, error-prone enzymes other than POL ␩ may a patient with xeroderma pigmentosum variant. J. Immunol. 169: 3825–3830. leave a mild A/T footprint. Thus, the mechanism that accounts for 12. Brezinschek, H. P., R. I. Brezinschek, and P. E. Lipsky. 1995. Analysis of the the other 40% of mutations in AICDA-deficient B cells remains heavy chain repertoire of human peripheral B cells using single-cell polymerase chain reaction. J. Immunol. 155: 190–202. unknown. 13. Do¨rner, T., H. P. Brezinschek, S. J. Foster, R. I. Brezinschek, N. L. Farner, and http://www.jimmunol.org/ Overall, the mutations that occur in the absence of AICDA are P. E. Lipsky. 1998. Comparable impact of mutational and selective influences in shaping the expressed repertoire of peripheral IgMϩCD5Ϫ and IgMϩCD5ϩ B likely to have little impact on the conventional Ag binding site or cells. Eur. J. Immunol. 28: 657–668. the nonvariable FR regions that superantigens bind (42). Despite 14. Brezinschek, H. P., S. J. Foster, R. I. Brezinschek, T. Do¨rner, R. Domiati-Saad, the positive selection of mutated B cells that occurs in AICDAϪ/Ϫ and P. E. Lipsky. 1997. Analysis of the human VH gene repertoire: differential effects of selection and somatic hypermutation on human peripheral CD5ϩIgMϩ subjects, the data suggest that the process may not be sufficiently and CD5ϪIgMϩ B cells. J. Clin. Invest. 99: 2488–2501. robust to allow B cells to undergo affinity maturation. The lack of 15. Monson, N. L., T. Dorner, and P. E. Lipsky. 2000. Targeting and selection of affinity maturation and class switching is reflected in the absence mutations in human V␭ rearrangements. Eur. J. 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