Background. Activation-induced cytidine deaminase (AID) initiates somatic hypermutation and class switch recombination of immunoglobulin (Ig) genes by deaminating deoxycytidine residues in mature germinal center B cells. AID can also induce mutations in non-Ig genes. In addition to the full-length isoform, five splice variants have been reported in normal B cells and in mature B-cell disorders. Furthermore, aberrant expression of AID has been detected in B-lineage acute lymphoblastic leukemia (B-ALL) and a strong correlation between AID expression and BCR/ABL-positivity has been found; intriguingly, BCR/ABL+ ALL, the most frequent genetically-defined ALL in adults, is characterized by a high genetic instability. Aims. To deepen the role of AID in BCR/ABL+ ALL, AID expression was studied in these patients and then its association with gene expression profiling was evaluated. Methods. We analyzed 16 adult BCR/ABL+ ALL patients at the onset of disease for AID expression: after cDNA amplification, PCR products were loaded on the ABI Prism 3730 DNA Analyzer for automated capillary gel electrophoresis and the results were plotted with the AbiPrism GeneMapper v3.5 software (Applied Biosystems). Subsequently, gene expression profiling experiments were performed on the same set of patients using the HGU133 Plus 2.0 arrays (Affymetrix); statistical analysis was carried out using the dChip software. Unsupervised and supervised analyses (Analysis of Variance, ANOVA) were used to evaluate the presence of potential subsets and to compare the BCR/ABL+ ALL subgroup based on AID results. Results. AID mRNA was detected in 7 patients: 4 expressed only the full-lenght isoform, 2 displayed the splice variant that retains exons 1, 2 and 5, while 1 patient co-expressed the full-lenght isoform and two splice variants. The remaining cases proved negative for AID expression. By unsupervised clustering, gene expression profiling data analysis revealed that AID-fulllenght and AID-splice variants patients display a similar profile. This finding was confirmed by supervised analysis: in fact, comparison on the 3 AID subsets (i.e. AID-full-lenght, AID-splice variants and AID-negative) grouped AID-full-lenght and AID-splice variants in the same cluster, as opposed to AID-negative cases. Among the 1151 differentially expressed genes, 328 resulted specifically and homogeneously upregulated in AIDfull- lenght patients. Of interest, this group of patients was characterized by the overexpression of a large set of genes involved in i) DNA repair (PCNA, RPA3, RAD50, XRCC4, TP53), ii) DNA replication (ORC5L, MCM10, CETN3, ANAPC7), iii) cell cycle regulation (CDK6, MKI67, MYC), iv) nucleotide metabolism (NT5C3, PPAT, PRPS2). Functional annotation analysis, performed using the DAVID software, corroborated these findings. Conclusions. These results indicate that AID-full-lenght and splice variants exert a similar genomic profile, distinct from AIDnegative patients, suggesting that gene expression profiling is mainly affected by AID positivity or negativity, rather than by the presence of AID-splice variants. Furthermore, AID positive patients are characterized by a peculiar signature in which genes involved in DNA repair and replication are overrepresented, indicating a possible deregulation of these mechanisms: it is intriguing to speculate that this phenomenon might be implicated in the genetic instability observed in these patients. Functional studies are ongoing to confirm this hypothesis.

AID EXPRESSION IN BCR/ABL POSITIVE ALL IS ASSOCIATED WITH A PECULIAR GENE EXPRESSION PROFILE AND DEREGULATION OF DNA REPAIR AND REPLICATION GENES

IACOBUCCI, ILARIA;LONETTI, ANNALISA;PAPAYANNIDIS, CRISTINA;SOVERINI, SIMONA;MARTINELLI, GIOVANNI;
2009

Abstract

Background. Activation-induced cytidine deaminase (AID) initiates somatic hypermutation and class switch recombination of immunoglobulin (Ig) genes by deaminating deoxycytidine residues in mature germinal center B cells. AID can also induce mutations in non-Ig genes. In addition to the full-length isoform, five splice variants have been reported in normal B cells and in mature B-cell disorders. Furthermore, aberrant expression of AID has been detected in B-lineage acute lymphoblastic leukemia (B-ALL) and a strong correlation between AID expression and BCR/ABL-positivity has been found; intriguingly, BCR/ABL+ ALL, the most frequent genetically-defined ALL in adults, is characterized by a high genetic instability. Aims. To deepen the role of AID in BCR/ABL+ ALL, AID expression was studied in these patients and then its association with gene expression profiling was evaluated. Methods. We analyzed 16 adult BCR/ABL+ ALL patients at the onset of disease for AID expression: after cDNA amplification, PCR products were loaded on the ABI Prism 3730 DNA Analyzer for automated capillary gel electrophoresis and the results were plotted with the AbiPrism GeneMapper v3.5 software (Applied Biosystems). Subsequently, gene expression profiling experiments were performed on the same set of patients using the HGU133 Plus 2.0 arrays (Affymetrix); statistical analysis was carried out using the dChip software. Unsupervised and supervised analyses (Analysis of Variance, ANOVA) were used to evaluate the presence of potential subsets and to compare the BCR/ABL+ ALL subgroup based on AID results. Results. AID mRNA was detected in 7 patients: 4 expressed only the full-lenght isoform, 2 displayed the splice variant that retains exons 1, 2 and 5, while 1 patient co-expressed the full-lenght isoform and two splice variants. The remaining cases proved negative for AID expression. By unsupervised clustering, gene expression profiling data analysis revealed that AID-fulllenght and AID-splice variants patients display a similar profile. This finding was confirmed by supervised analysis: in fact, comparison on the 3 AID subsets (i.e. AID-full-lenght, AID-splice variants and AID-negative) grouped AID-full-lenght and AID-splice variants in the same cluster, as opposed to AID-negative cases. Among the 1151 differentially expressed genes, 328 resulted specifically and homogeneously upregulated in AIDfull- lenght patients. Of interest, this group of patients was characterized by the overexpression of a large set of genes involved in i) DNA repair (PCNA, RPA3, RAD50, XRCC4, TP53), ii) DNA replication (ORC5L, MCM10, CETN3, ANAPC7), iii) cell cycle regulation (CDK6, MKI67, MYC), iv) nucleotide metabolism (NT5C3, PPAT, PRPS2). Functional annotation analysis, performed using the DAVID software, corroborated these findings. Conclusions. These results indicate that AID-full-lenght and splice variants exert a similar genomic profile, distinct from AIDnegative patients, suggesting that gene expression profiling is mainly affected by AID positivity or negativity, rather than by the presence of AID-splice variants. Furthermore, AID positive patients are characterized by a peculiar signature in which genes involved in DNA repair and replication are overrepresented, indicating a possible deregulation of these mechanisms: it is intriguing to speculate that this phenomenon might be implicated in the genetic instability observed in these patients. Functional studies are ongoing to confirm this hypothesis.
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M. Messina; S. Chiaretti; I. Iacobucci; S. Tavolaro; A. Vitale; I. Della Starza; A. Lonetti; C. Papayannidis; A.D. Negulici; S. Soverini; D. Cilloni; A. Guarini; G. Martinelli; R. Foà
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/154559
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