Background. BCR-ABL1-positive ALL is the most frequent and prognostically unfavorable subtype of adult ALL, mainly because of genetic instability. Activation-induced cytidine deaminase (AID) introduces single-strand breaks into target DNA and produces immunediversity by inducing somatic hypermutations and class-switch recombinations (CSR) in human immunoglobulin genes (Ig). Aim. We investigated the expression of the AID in BCR-ABL1-positive ALL. Patients and Methods. 61 adult de novo BCR-ABL1-positive ALL patients (pts) were analyzed. AID cDNA, obtained from bone marrow or peripheral blood, was amplified with two pairs of oligonucleotides, the forward primer of each couple conjugated with a fluorescent dye (fluorescein) at its 5’ end. PCR products were then loaded on the ABI Prism 3730 DNA Analyzer and the results were plotted with the AbiPrism GeneMapper v3.5 software (Applied Biosystems). Results. On the 61 de novo adult BCR-ABL1-positive ALL pts, AID mRNA and protein were detected in 36 (59%); their expression correlated with BCR-ABL1 transcript levels and disappeared after treatment with tyrosine kinase inhibitors. Different isoforms of AID were identified: 13/61 (21%) pts expressed the full-length isoform (AID-FL); 19/61 (31%) co-expressed the wild-type and different splice variants. In particular, we found an isoform with a 30 bp deletion of exon 4 (AID-deltaE4a); an isoform characterized by the deletion of the entire exon 4 (AID-deltaE4) which led to a C-terminal truncation due a frameshift. In 2 patients, AIDins3, characterized by retention of intron 3, was expressed. This variant maintained the cytidine domain but lacked the class switch recombination (CSR) domain due a frameshift. Four out 61 Ph+ ALL patients (7%) expressed the AID deltaE3-E4 isoform without deaminase activity but retaining intact the CSR domain. Since splicing induced alterations in the nuclear export signal (NES) at the Cterminus we found that. AID-FL exhibited predominant cytoplasmic localization, as did the AID-deltaE4a and AID-deltaE3E4 variants whereas the C-terminal truncated AID-deltaE4 showed a slightly increased nuclear localization. Conclusions. Our findings show that BCR-ABL1-positive ALL cells aberrantly express different isoforms of AID that may act as mutator outside the Ig gene loci in promoting genetic instability in leukemia cells. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, Strategico di Ateneo, GIMEMA Onlus.
THE B-CELL MUTATOR ACTIVATION-INDUCED CYTIDINE DEAMINASE IS ALTERNATIVELY SPLICED IN BCR-ABL1-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIAPATIENTS
LONETTI, ANNALISA;IACOBUCCI, ILARIA;SOVERINI, SIMONA;OTTAVIANI, EMANUELA;PAPAYANNIDIS, CRISTINA;PICCALUGA, PIER PAOLO;PAOLINI, STEFANIA;MARTINELLI, GIOVANNI
2009
Abstract
Background. BCR-ABL1-positive ALL is the most frequent and prognostically unfavorable subtype of adult ALL, mainly because of genetic instability. Activation-induced cytidine deaminase (AID) introduces single-strand breaks into target DNA and produces immunediversity by inducing somatic hypermutations and class-switch recombinations (CSR) in human immunoglobulin genes (Ig). Aim. We investigated the expression of the AID in BCR-ABL1-positive ALL. Patients and Methods. 61 adult de novo BCR-ABL1-positive ALL patients (pts) were analyzed. AID cDNA, obtained from bone marrow or peripheral blood, was amplified with two pairs of oligonucleotides, the forward primer of each couple conjugated with a fluorescent dye (fluorescein) at its 5’ end. PCR products were then loaded on the ABI Prism 3730 DNA Analyzer and the results were plotted with the AbiPrism GeneMapper v3.5 software (Applied Biosystems). Results. On the 61 de novo adult BCR-ABL1-positive ALL pts, AID mRNA and protein were detected in 36 (59%); their expression correlated with BCR-ABL1 transcript levels and disappeared after treatment with tyrosine kinase inhibitors. Different isoforms of AID were identified: 13/61 (21%) pts expressed the full-length isoform (AID-FL); 19/61 (31%) co-expressed the wild-type and different splice variants. In particular, we found an isoform with a 30 bp deletion of exon 4 (AID-deltaE4a); an isoform characterized by the deletion of the entire exon 4 (AID-deltaE4) which led to a C-terminal truncation due a frameshift. In 2 patients, AIDins3, characterized by retention of intron 3, was expressed. This variant maintained the cytidine domain but lacked the class switch recombination (CSR) domain due a frameshift. Four out 61 Ph+ ALL patients (7%) expressed the AID deltaE3-E4 isoform without deaminase activity but retaining intact the CSR domain. Since splicing induced alterations in the nuclear export signal (NES) at the Cterminus we found that. AID-FL exhibited predominant cytoplasmic localization, as did the AID-deltaE4a and AID-deltaE3E4 variants whereas the C-terminal truncated AID-deltaE4 showed a slightly increased nuclear localization. Conclusions. Our findings show that BCR-ABL1-positive ALL cells aberrantly express different isoforms of AID that may act as mutator outside the Ig gene loci in promoting genetic instability in leukemia cells. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, Strategico di Ateneo, GIMEMA Onlus.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
