Activation-induced cytidine deaminase (AID) is a mutator enzyme essential for somatic hypermutation (SHM) and class switch recombination (CSR) during effective adaptive immune responses. Its aberrant expression and activity have been detected in lymphomas, leukemias, and solid tumors. In chronic lymphocytic leukemia (CLL) increased expression of alternatively spliced AID variants has been documented. We used real-time RT-PCR to quantify the expression of AID and its alternatively spliced transcripts (AIDΔE4a, AIDΔE4, AIDivs3, and AIDΔE3E4) in 149 CLL patients and correlated this expression to prognostic markers including recurrent chromosomal aberrations, the presence of complex karyotype, mutation status of the immunoglobulin heavy chain variable gene, and recurrent mutations. We report a previously unappreciated association between higher AID transcript levels and trisomy of chromosome 12. Functional analysis of AID splice variants revealed loss of their activity with respect to SHM, CSR, and induction of double-strand DNA breaks. In silico modeling provided insight into the molecular interactions and structural dynamics of wild-type AID and a shortened AID variant closely resembling AIDΔE4, confirming its loss-of-function phenotype.

Central European Institute of Technology Center of Molecular Medicine Masaryk University Kamenice 5 A35 625 00 Brno Czech Republic

Department of Biomedical Sciences School of Veterinary Medicine University of Pennsylvania Philadelphia PA USA

Department of Internal Medicine Hematology and Oncology Faculty of Medicine Masaryk University and University Hospital Brno Brno Czech Republic

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Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, Buchbinder A, Budman D, Dittmar K, Kolitz J, Lichtman SM, Schulman P, Vinciguerra VP, Rai KR, Ferrarini M, Chiorazzi N (1999) Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 94(6):1840–1847 PubMed

Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK (1999) Unmutated Ig V-H genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 94(6):1848–1854 PubMed

Dohner H, Stilgenbauer S, Benner A et al. (2000) Genomic aberrations and survival in chronic lymphocytic leukemia. New Engl J Med 343(26):1910–1916 PubMed

Landau DA, Carter SL, Stojanov P, McKenna A, Stevenson K, Lawrence MS, Sougnez C, Stewart C, Sivachenko A, Wang L, Wan Y, Zhang W, Shukla SA, Vartanov A, Fernandes SM, Saksena G, Cibulskis K, Tesar B, Gabriel S, Hacohen N, Meyerson M, Lander ES, Neuberg D, Brown JR, Getz G, Wu CJ (2013) Evolution and impact of subclonal mutations in chronic lymphocytic leukemia. Cell 152(4):714–726 PubMed PMC

Baliakas P, Iskas M, Gardiner A, Davis Z, Plevova K, Nguyen-Khac F, Malcikova J, Anagnostopoulos A, Glide S, Mould S, Stepanovska K, Brejcha M, Belessi C, Davi F, Pospisilova S, Athanasiadou A, Stamatopoulos K, Oscier D (2014) Chromosomal translocations and karyotype complexity in chronic lymphocytic leukemia: a systematic reappraisal of classic cytogenetic data. Am J Hematol 89(3):249–255 PubMed

Rigolin GM, del Giudice I, Formigaro L, Saccenti E, Martinelli S, Cavallari M, Lista E, Tammiso E, Volta E, Lupini L, Bassi C, Bardi A, Sofritti O, Daghia G, Cavazzini F, Marinelli M, Tavolaro S, Guarini A, Negrini M, Foà R, Cuneo A (2015) Chromosome aberrations detected by conventional karyotyping using novel mitogens in chronic lymphocytic leukemia: clinical and biologic correlations. Gene Chromosome Canc 54:818–826 PubMed

Muramatsu M, Kinoshita K, Fagarasan S, Yamada S, Shinkai Y, Honjo T (2000) Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102(5):553–563 PubMed

Revy P, Muto T, Levy Y, Geissmann F, Plebani A, Sanal O, Catalan N, Forveille M, Dufourcq-Lagelouse R, Gennery A, Tezcan I, Ersoy F, Kayserili H, Ugazio AG, Brousse N, Muramatsu M, Notarangelo LD, Kinoshita K, Honjo T, Fischer A, Durandy A (2000) Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the hyper-IgM syndrome (HIGM2). Cell 102(5):565–575 PubMed

Bransteitter R, Pham P, Calabrese P, Goodman MF (2004) Biochemical analysis of hypermutational targeting by wild type and mutant activation-induced cytidine deaminase. J Biol Chem 279(49) :51612–51621 PubMed

Pham P, Bransteitter R, Petruska J, Goodman MF (2003) Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation. Nature 424(6944):103–107 PubMed

Iacobucci I, Lonetti A, Messa F et al. (2010) Different isoforms of the B-cell mutator activation-induced cytidine deaminase are aberrantly expressed in BCR-ABL1-positive acute lymphoblastic leukemia patients. Leukemia 24(1):66–73 PubMed

Klemm L, Duy C, Iacobucci I, Kuchen S, von Levetzow G, Feldhahn N, Henke N, Li Z, Hoffmann TK, Kim YM, Hofmann WK, Jumaa H, Groffen J, Heisterkamp N, Martinelli G, Lieber MR, Casellas R, Müschen M (2009) The B cell mutator AID promotes B lymphoid blast crisis and drug resistance in chronic myeloid leukemia. Cancer Cell 16(3):232–245 PubMed PMC

Okazaki IM, Kotani A, Honjo T (2007) Role of AID in tumorigenesis. Adv Immunol 94:245–273 PubMed

Pasqualucci L, Bhagat G, Jankovic M, Compagno M, Smith P, Muramatsu M, Honjo T, Morse HC, Nussenzweig MC, Dalla- Favera R (2008) AID is required for germinal center-derived lymphomagenesis. Nature Genet 40(1):108–112 PubMed

Pasqualucci L, Guglielmino R, Houldsworth J, Mohr J, Aoufouchi S, Polakiewicz R, Chaganti RS, Dalla-Favera R (2004) Expression of the AID protein in normal and neoplastic B cells. Blood 104(10): 3318–3325 PubMed

Ramiro AR, Jankovic M, Eisenreich T, Difilippantonio S, Chen-Kiang S, Muramatsu M, Honjo T, Nussenzweig A, Nussenzweig MC (2004) AID is required for c-myc/IgH chromosome translocations in vivo. Cell 118(4):431–438 PubMed

Robbiani DF, Bunting S, Feldhahn N, Bothmer A, Camps J, Deroubaix S, McBride KM, Klein IA, Stone G, Eisenreich TR, Ried T, Nussenzweig A, Nussenzweig MC (2009) AID produces DNA double-strand breaks in non-Ig genes and mature B cell lymphomas with reciprocal chromosome translocations. Mol Cell 36(4):631–641 PubMed PMC

Kumar R, DiMenna LJ, Chaudhuri J et al. (2014) Biological function of activation-induced cytidine deaminase (AID). Biom J 37: 269–283 PubMed

Cattoretti G, Buttner M, Shaknovich R et al. (2006) Nuclear and cytoplasmic AID in extrafollicular and germinal center B cells. Blood 107(10):3967–3975 PubMed

Gonda H, Sugai M, Nambu Y, Katakai T, Agata Y, Mori KJ, Yokota Y, Shimizu A (2003) The balance between Pax5 and Id2 activities is the key to AID gene expression. J Exp Med 198(9):1427–1437 PubMed PMC

Park SR, Zan H, Pal Z, Zhang J, al-Qahtani A, Pone EJ, Xu Z, Mai T, Casali P (2009) HoxC4 binds to the promoter of the cytidine deaminase AID gene to induce AID expression, class-switch DNA recombination and somatic hypermutation. Nat Immunol 10(5):540–550 PubMed PMC

Sayegh CE, Quong MW, Agata Y, Murre C (2003) E-proteins directly regulate expression of activation-induced deaminase in mature B cells. Nat Immunol 4(6):586–593 PubMed

Tran TH, Nakata M, Suzuki K, Begum NA, Shinkura R, Fagarasan S, Honjo T, Nagaoka H (2010) B cell-specific and stimulation-responsive enhancers derepress Aicda by overcoming the effects of silencers. Nat Immunol 11(2):148–U67 PubMed

Patten PEM, Chu CC, Albesiano E, Damle RN, Yan XJ, Kim D, Zhang L, Magli AR, Barrientos J, Kolitz JE, Allen SL, Rai KR, Roa S, Mongini PK, MacCarthy T, Scharff MD, Chiorazzi N (2012) IGHV-unmutated and IGHV-mutated chronic lymphocytic leukemia cells produce activation-induced deaminase protein with a full range of biologic functions. Blood 120(24):4802–4811 PubMed PMC

Rush JS, Liu M, Odegard VH, Unniraman S, Schatz DG (2005) Expression of activation-induced cytidine deaminase is regulated by cell division, providing a mechanistic basis for division-linked class switch recombination. Proc Natl Acad Sci U S A 102(37): 13242–13247 PubMed PMC

Albesiano E, Messmer BT, Damle RN, Allen SL, Rai KR, Chiorazzi N (2003) Activation-induced cytidine deaminase in chronic lymphocytic leukemia B cells: expression as multiple forms in a dynamic, variably sized fraction of the clone. Blood 102(9):3333–3339 PubMed

de Yebenes VG, Belver L, Pisano DG et al. (2008) miR-181b negatively regulates activation-induced cytidine deaminase in B cells. J Exp Med 205(10):2199–2206 PubMed PMC

Sernandez IV, de Yebenes VG, Dorsett Y et al. (2008) Haploinsufficiency of activation-induced deaminase for antibody diversification and chromosome translocations both in vitro and in vivo. PLoS One 3(12):e3927. PubMed PMC

Teng G, Hakimpour P, Landgraf P, Rice A, Tuschl T, Casellas R, Papavasiliou FN (2008) MicroRNA-155 is a negative regulator of activation-induced cytidine deaminase. Immunity 28(5):621–629 PubMed PMC

Marantidou F, Dagklis A, Stalika E, Korkolopoulou P, Saetta A, Anagnostopoulos A, Laoutaris N, Stamatopoulos K, Belessi C, Scouras Z, Patsouris E (2010) Activation-induced cytidine deaminase splicing patterns in chronic lymphocytic leukemia. Blood Cells Mol Dis 44(4):262–267 PubMed

van Maldegem F, Jibodh RA, van Dijk R, Bende RJ, van Noesel CJM (2010) Activation-induced cytidine deaminase splice variants are defective because of the lack of structural support for the catalytic site. J Immunol 184(5):2487–2491 PubMed

Wu XS, Darce JR, Chang SK, Nowakowski GS, Jelinek DF (2008) Alternative splicing regulates activation-induced cytidine deaminase (AID): implications for suppression of AID mutagenic activity in normal and malignant B cells. Blood 112(12):4675–4682 PubMed PMC

van Maldegem F, Scheeren FA, Aarti Jibodh R, Bende RJ, Jacobs H, van Noesel CJM (2009) AID splice variants lack deaminase activity. Blood 113:1862–1864 PubMed

Rebhandl S, Huemer M, Zaborsky N, Gassner FJ, Catakovic K, Felder TK, Greil R, Geisberger R (2014) Alternative splice variants of AID are not stoichiometrically present at the protein level in chronic lymphocytic leukemia. Eur J Immunol 44:2175–2187 PubMed PMC

Sali A, Blundell TL (1993) Comparative protein modeling by satisfaction of spatial restraints. J Mol Biol 234(3):779–815 PubMed

Case DA, Babin V, Berryman JT et al. (2014) AMBER 14. University of California, San Francisco

Le Bris Y, Struski S, Guièze R et al. (2016) Major prognostic value of complex karyotype in addition to TP53 and IGHV mutational status in first-line chronic lymphocytic leukemia. Hematol Oncol 35:664–670. 10.1002/hon.2349 PubMed DOI

Huemer M, Rebhandl S, Zaborsky N, Gassner FJ, Hainzl S, Weiss L, Hebenstreit D, Greil R, Geisberger R (2014) AID induces intraclonal diversity and genomic damage in CD86(+) chronic lymphocytic leukemia cells. Eur J Immunol 44(12):3747–3757 PubMed PMC

Jiang YW, Soong TD, Wang L et al. (2012) Genome-wide detection of genes targeted by non-Ig somatic hypermutation in lymphoma. PLoS One 7(7):e40332. PubMed PMC

Liu M, Duke JL, Richter DJ, Vinuesa CG, Goodnow CC, Kleinstein SH, Schatz DG (2008) Two levels of protection for the B cell genome during somatic hypermutation. Nature 451(7180): 841–845 PubMed

Pasqualucci L, Neumeister P, Goossens T, Nanjangud G, Chaganti RSK, Küppers R, Dalla-Favera R (2001) Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas. Nature 412(6844):341–346 PubMed

Aoufouchi S, Faili A, Zober C, D’Orlando O, Weller S, Weill JC, Reynaud CA (2008) Proteasomal degradation restricts the nuclear lifespan of AID. J Exp Med 205(6):1357–1368 PubMed PMC

Geisberger R, Rada C, Neuberger MS (2009) The stability of AID and its function in class-switching are critically sensitive to the identity of its nuclear-export sequence. Proc Natl Acad Sci U S A 106(16):6736–6741 PubMed PMC

McBride KM, Barreto V, Ramiro AR et al. (2004) Somatic hypermutation is limited by CRM1-dependent nuclear export of activation-induced deaminase. J Exp Med 199(9):1235–1244 PubMed PMC

Zahn A, Eranki AK, Patenaude AM, Methot SP, Fifield H, Cortizas EM, Foster P, Imai K, Durandy A, Larijani M, Verdun RE, di Noia JM (2014) Activation induced deaminase C-terminal domain links DNA breaks to end protection and repair during class switch recombination. Proc Natl Acad Sci U S A 111(11):E988–E997 PubMed PMC

Betts L, Xiang SB, Short SA, Wolfenden R, Carter CW Jr (1994) Cytidine deaminase. The 2.3 A crystal structure of an enzyme: transition-state analog complex. J Mol Biol 235(2):635–656 PubMed

Heintel D, Kroemer E, Kienle D et al. (2004) High expression of activation-induced cytidine deaminase (AID) mRNA is associated with unmutated IGVH gene status and unfavourable cytogenetic aberrations in patients with chronic lymphocytic leukaemia. Leukemia 18:756–762 PubMed

Leuenberger M, Frigerio S, Wild PJ, Noetzli F, Korol D, Zimmermann DR, Gengler C, Probst-Hensch NM, Moch H, Tinguely M (2010) AID protein expression in chronic lymphocytic leukemia/small lymphocytic lymphoma is associated with poor prognosis and complex genetic alterations. Mod Pathol 23(2): 177–186 PubMed

McCarthy H, Wierda WG, Barron LL, Cromwell CC, Wang J, Coombes KR, Rangel R, Elenitoba-Johnson KS, Keating MJ, Abruzzo LV (2003) High expression of activation-induced cytidine deaminase (AID) and splice variants is a distinctive feature of poor-prognosis chronic lymphocytic leukemia. Blood 101(12):4903–4908 PubMed

Palacios F, Moreno P, Morande P, Abreu C, Correa A, Porro V, Landoni AI, Gabus R, Giordano M, Dighiero G, Pritsch O, Oppezzo P (2010) High expression of AID and active class switch recombination might account for a more aggressive disease in unmutated CLL patients: link with an activated microenvironment in CLL disease. Blood 115(22):4488–4496 PubMed

Oppezzo P, Vuillier F, Vasconcelos Y, Dumas G, Magnac C, Payelle-Brogard B, Pritsch O, Dighiero G (2003) Chronic lymphocytic leukemia B cells expressing AID display dissociation between class switch recombination and somatic hypermutation. Blood 101(10):4029–4032 PubMed

Eagle GL, Zhuang JG, Jenkins RE, Till KJ, Jithesh PV, Lin K, Johnson GG, Oates M, Park K, Kitteringham NR, Pettitt AR (2015) Total proteome analysis identifies migration defects as a major Pathogenetic factor in immunoglobulin heavy chain variable region (IGHV)-unmutated chronic lymphocytic leukemia. Mol Cell Proteomics 14(4):933–945 PubMed PMC

Balatti V, Bottoni A, Palamarchuk A, Alder H, Rassenti LZ, Kipps TJ, Pekarsky Y, Croce CM (2012) NOTCH1 mutations in CLL associated with trisomy 12. Blood 119(2):329–331 PubMed PMC

Chigrinova E, Rinaldi A, Kwee I, Rossi D, Rancoita PMV, Strefford JC, Oscier D, Stamatopoulos K, Papadaki T, Berger F, Young KH, Murray F, Rosenquist R, Greiner TC, Chan WC, Orlandi EM, Lucioni M, Marasca R, Inghirami G, Ladetto M, Forconi F, Cogliatti S, Votavova H, Swerdlow SH, Stilgenbauer S, Piris MA, Matolcsy A, Spagnolo D, Nikitin E, Zamo A, Gattei V, Bhagat G, Ott G, Zucca E, Gaidano G, Bertoni F (2013) Two main genetic pathways lead to the transformation of chronic lymphocytic leukemia to Richter syndrome. Blood 122(15):2673–2682 PubMed

Liso V, Capalbo S, Lapietra A, Pavone V, Guarini A, Specchia G (1999) Evaluation of trisomy 12 by fluorescence in situ hybridization in peripheral blood, bone marrow and lymph nodes of patients with B-cell chronic lymphocytic leukemia. Haematologica 84(3): 212–217 PubMed

Burger JA, Montserrat E (2013) Coming full circle: 70 years of chronic lymphocytic leukemia cell redistribution, from glucocorticoids to inhibitors of B-cell receptor signaling. Blood 121(9):1501–1509 PubMed PMC

Riches JC, O’Donovan CJ, Kingdon SJ, McClanahan F, Clear AJ, Neuberg DS, Werner L, Croce CM, Ramsay AG, Rassenti LZ, Kipps TJ, Gribben JG (2014) Trisomy 12 chronic lymphocytic leukemia cells exhibit upregulation of integrin signaling that is modulated by NOTCH1 mutations. Blood 123(26):4101–4110 PubMed PMC

Reiniger L, Bodor C, Bognar A et al. (2006) Richter’s and prolymphocytic transformation of chronic lymphocytic leukemia are associated with high mRNA expression of activation-induced cytidine deaminase and aberrant somatic hypermutation. Leukemia 20(6):1089–1095 PubMed

Smit LA, van Maldegem F, Langerak AW, van der Schoot C, de Wit MJ, Bea S, Campo E, Bende RJ, van Noesel C (2006) Antigen receptors and somatic hypermutation in B-cell chronic lymphocytic leukemia with Richter’s transformation. Haematologica 91(7):903–911 PubMed

Ito S, Nagaoka H, Shinkura R, Begum N, Muramatsu M, Nakata M, Honjo T (2004) Activation-induced cytidine deaminase shuttles between nucleus and cytoplasm like apolipoprotein B mRNA editing catalytic polypeptide 1. Proc Natl Acad Sci USA 101(7): 1975–1980 PubMed PMC

Ta VT, Nagaoka H, Catalan N, Durandy A, Fischer A, Imai K, Nonoyama S, Tashiro J, Ikegawa M, Ito S, Kinoshita K, Muramatsu M, Honjo T (2003) AID mutant analyses indicate requirement for class-switch-specific cofactors. Nat Immunol 4(9): 843–848 PubMed