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 References This article cites 41 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/181/2/1299.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 29, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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).