Molecular Cloning And Leukemogenesis Studies Of A Novel Fusion Gene Involving The PAX5Gene, PAX5-UBE2D4

Objective:1. To explore the incidence of PAX5gene rearrangement in B-cell acute lympholasticleukemia (B-ALL) and patients harboring9p abnormalities and to analyze its clinical andother cytogenetic features.2. To provide considerable insight into the mechanisms of PAX5-related fusion genesin leukemia through molecular cloning and functional study of the fusion gene by thechromosome translocation dic (7;9)(p11-13; p13) in a mixed-phenotype acute leukemiapatient.3. To investigate the incidence of the BTG1deletions in BCR/ABL1positive acuteleukemia and to analyze its clinical and other cytogenetic features.Furthermore, todetermine the relationship between BTG1deletions and leukemogenesis and relapse inBCR/ABL1positive leukemia.Methods:1.84cases were studied by R-band karyotypic analysis and bacterial artificialchromosomes RPl1-344823and RPl1-652D9encompassing the PAX5gene wereselected.DNA was extracted with conventional method and labeled with fluorescein bynicking transition．Fiuorescence in situ hybridization (FISH) was used to determine therearrangement or deletion of the PAX5gene in B-ALL and patients harboring chromosome9p abnormalities. Clinical and laboratory features of patients were analyzed．2. Array-based comparative genomic hybridization (Array-CGH) analysis wasperformed in4hematologic malignancies patients for whom at least1.5ug genomic DNAwas available. Agilent human CGH microarrays (Agilent Technologies，Santa Clara，CA，USA) containing244,000probes (244k) with an average spatial resolution of6.4Kb were used for the Array-CGH experiments according to protocols provided by themanufacturer．Image analysis，normalization and annotation were based on FeatureExtraction9.1(Agilent)，while data were visualized with Agilent Genomic Workbench LiteEdition6.5software. RT-PCR wsa used to detect the fusion transcript and to clone thefull-lenth coding sequence of the fusion transcript which was afterwards inserted to atransfer plasmid-LV5.The fusion gene was transfected into NIH-3T3and BaF3cells byDNA-calucium phosphate.Western Blot was used to determine the fusion protein. Growthrate of the above cells was detected with the cell counting kit8(CCK8) and growth curvewas made according to it. Growth ability of such cells in the methylcellulose and real-timequantitative PCR were also investigated to detect the malignant transformation ability ofthe above genes.3. A cohort of44patients following admission to Jiangsu Institute of Hematologyenrolled in this study from December2008to August2013was recruited in this research toassess the BTG1deletion. Array-CGH analysis was performed in16BCR/ABL1positiveBCP-ALL patients,5MPAL patients and1CML-BC (B-lineage) patient.To further screenand map BTG1deletion breakpoints, RT-PCR analysis was performed on44samples andthe REH cell line followed by direct bidirectional DNA sequencing. Clinical andlaboratory features of patients were analyzed．Results:1. Clinical and cytogenetic features of patients with PAX5alterationsPAX5gene alterations in B-ALL and patients harboring9p abnormalities were foundin23patients. PAX5deletions were observed in20patients(23.8%), rearragements wereobserved in2patients(2.4%),and addition was detected only in1case(1.2%).The totalfrequence of abnormalities was27.4%(23/84).The incidence of PAX5alterations in thosecases with t(9;22) karyotype was higher than that in cases without t(9;22). There were nosignificant differences in median white blood cell count, hemoglobin concentration,platelet count, bone marrow blast count, sex, age, or overall complete remission ratebetween patients with and without PAX5alterations. 2. Molecular cloning and leukemogenesis studies of a novel fusion gene involving thePAX5gene, PAX5-UBE2D4In this study, we identified7patients with leukemia harboring dic (7;9)(p11-13; p13)rearrangements by screening patients with leukemia. Moreover, we identified a novelfusion gene involving PAX5gene in a patients with MPAL, which results in an in-framefusion of PAX5to ubiquitin-conjugating enzyme E2D4(UBE2D4).RT-PCR wsa used to detect the fusion transcript and to clone the full-lenth codingsequence of the fusion transcript which was afterwards inserted to a transferplasmid-LV5.The fusion gene was transfected into NIH-3T3and BaF3cells byDNA-calucium phosphate. PAX5-UBE2D4fusion protein could confer the NIH-3T3andBaF3cells with growth advantage, and the combination with BCR/ABL could inhibite theadhesion and apoptosis.Moreover, it would make BaF3cells grow without IL-3.Localization of the fusion proteins was also confirmed by Western Blot, which detected thefusion protein predominantly in the nucleus, but also in the cytoplasm.The overexpressionof PAX5was detected in infected cells by real-time PCR.3. The frequence and function of BTG1deletions in patients with BCR/ABL1positiveacute leukemiaBTG1deletions were presented in31.8%of BCR/ABL1positive acute leukemiapatients, including31.3%of BCP-ALL (10/32),33.3%of MPAL (2/6) and33.3%ofCML-BC (B-lineage)(2/6). Of note, the intragenic deletion breakpoints, mapping to5different positions, clustered tightly within exon2of BTG1, which located within a stretchof20base pairs from nucleotide284to304and led to truncated BTG1transcripts. Therewere no significant differences in median white blood cell count, hemoglobinconcentration, platelet count, bone marrow blast count, sex, age, or overall completeremission rate between patients with and without BTG1deletions.Conclusion:1. In this study, PAX5alterations is a recurrent cytogenetics abnormality in B-ALL,but also in AML-M2. PAX5alterations were associated with common recurrent translocations. FISH analysis plays an important role in identifying the rearrangement ofPAX5.2. We identified a novel fusion gene involving PAX5gene in a patients with precursorB-ALL, which results in an in-frame fusion of PAX5-UBE2D4. Molecular analysis showedthat exon7of PAX5was fused in·frame to exon2of UBE2D4in this case. The fusionprotein was predominantly localized in the nucleus, but also was detected in the cytoplasm,which might play an important role in transcription of PAX5. PAX5-UBE2D4fusionprotein could confer the NIH-3T3and BaF3cells with growth advantage, and thecombination with BCR/ABL could inhibite the adhesion and apoptosis.Moreover, it wouldmake BaF3cells grow without IL-3.3. In our study, the frequency of BTG1deletions in BCP-ALL (10/32,31.3%)harboring BCR/ABL1transcript is higher than expected based on the literature. Moreover,we first detected the deletion of BTG1in2CML-BC (B-lineage) patients. Wedemonstrated BTG1deletions in a significant proportion of BCR/ABL1positive acuteleukemia, not only in BCP-ALL, but also in MPAL and CML-BC (B-lineage). Therefore,BTG1deletions may play an important role in leukemogenesis in patients with BCR/ABL1transcript.