Frequent Occurrence of Aberrant Pax5 Splice Variants in Philadelphia Chromosome-Positive Lymphoblastic Leukemia.

Pax5 is a key regulator of B cell commitment and is indispensable throughout the early stages of B cell differentiation. B cell development is blocked at the pro-B cell stage in Pax5-null mice. On the other hand, several investigators have reported that aberrant Pax5 transcripts as well as forced expression of Pax5 result from genetic and/or epigenetic mechanisms especially in B cell malignancies, suggesting the involvement of Pax5 abnormality in development and progression of B cell malignancies. In the present study, we examined Pax5 expression in Philadelphia chromosome (Ph)-positive lymphoid malignancies including 17 cases of Ph-positive acute lymphoblastic leukemia (Ph-ALL) and 3 cases of lymphoid blast crisis of chronic myeloid leukemia (CML-BC). By the reverse transcription-polymerase chain reaction (RT-PCR) method, we detected 4 types of Pax5 isoforms among those. In 16 samples, full-length as well as truncated forms of Pax5 were expressed. Expression of Pax5 could not be detected in 3 cases, and was only faintly found in the remaining one. These 4 cases did not express or very weakly expressed EBF, early B cell transcription factor upstream of Pax5. Sequencing analysis revealed that all 4 truncated isoforms were derived from exon skipping as follows. Type I is lacking in exon 2 and exon 3 (D2/3), type II shows deletion of exons 2–6 (D2-6), type III; D2-7, and type IV; D2-8. Two isoforms resulted in frame-shift, and two isoforms were in-frame splice variants without exons coding DNA binding domain. Transactivation potential of these two in-frame variants was examined by transfection of 293T cells with a reporter plasmid containing high-affinity Pax5 binding sites from the CD19 gene linked to the minimal TATA-like promoter, indicating much lower transcriptional activity in comparison to wild-type Pax5. These results suggest the frequent occurrence in adult Ph-positive lymphoid malignancies of aberrant Pax5 splice variants with reduced transcriptional activity, leading to impairment of normal B cell differentiation.