Introduction.Pax-5 gene codifies for a transcription factor central to B-cell differentiation and function, expressed during the early stage of differentiation and alternatively spliced during B-cell development. In addition to the full-length isoform (Pax-5a), isoforms arising from the inclusion or exclusion of exons 2, 7, 8, and/or 9, and lacking the DNA-binding and the transactivating domains, have been described. These isoforms and their levels of expression increase during B-cell maturation. In particular, Pax-5b isoform (deleted of exon 2), resulting in protein with partial DNA-binding domain, is related with the differentiation stage. Recently, mutations of Pax-5 gene were reported in 31.7% of 242 children (Mullighan, Nature 2007) and 30% of 119 adults (Familiades, ASH 2007 abs. 2806) with B-cell precursor acute lymphoblastic leukemia (ALL). We investigated Pax-5 gene mutations and isoforms expression in patients with ALL at the diagnosis and in selected cell populations from control subjects.

Methods.Pax-5 mutations and mRNA isoforms were investigated by sequence analysis. Pax-5a and Pax-5b expression levels were evaluated by quantitative PCR in leukemic cells. Total RNA was obtained from leukemic blasts of 95 patients with B-cell precursor ALL at diagnosis, 45 adults and 50 children. These last had been selected according to the presence of adverse translocations: BCR/ABL p190 (n=10), E2A-PBX1 (n=10), TEL/AML (n=10), MLL/AF4 (n=10), or none (n=10). mRNA was obtained from purified peripheral blood CD19+ lymphocytes (6 samples), from bone marrow CD34+ hematopoietic progenitor cells (6 samples), and pro-B CD34+CD19+ cells (3 samples) from a total of 8 healthy bone marrow donors.

Results. Pax-5 gene point mutations were found in 8/45 adults (18%) and 14/50 children (28%) (p=n.s.), resulting in a total of 20 different mutations. Mutations were located as follows: exon 2 (53G>C, 76delG, 101C>G, 113G>A, 197G>A), exon 3 (223A>G, 247delA, 296T>C), exon 4 (446A>T, 526G>A), exon 5 (580A>C, 601G>A), exon 6 (625C>T, 716G>A), exon 7 (836C>A), exon 8 (955G>A, 964insC, 971A>G), exon 9 (1058C>T), exon 10 (1133G>A). There was no correlation between the presence of mutations and the listed genetic subtypes. Pax-5 alternative splicing was observed in 47/95 cases (49%): 29/45 adults (64%) and 18/50 children (36%) (p=n.s.). Alternative splicing resulted in increased frequencies of the Pax-5b isoform and the deletion of exons 8 and/or 9; furthermore, a novel isoform resulting from the skipping of exon 5 was documented. On the contrary, only the fulllenght isoform was present in CD34+ progenitors and CD34+CD19+ cells from healthy donors, in which deleted isoforms were detected only at the stage of mature B-lymphocytes.

Conclusion: Pax-5 mutations are frequent in childhood and adult ALL and are scattered all over the gene. We extend the current knowledge on their pathogenic role by demonstrating that alternative splicing is a common event in these patients. Imbalance between the fulllength and the Pax-5b isoforms is expected to affect the maturation of the B-cell and its propensity to apoptosis (Robichaud Nucleic Acids Res 2008), thus contributing to the process of leukemogenesis. Based on our findings, this mechanism acts broadly and is independent of the most common ALL genetic translocations.

Disclosures: No relevant conflicts of interest to declare.

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