Genome-Wide Analysis of Alternative Splicing Points to Novel Leukemia Relevant Genes in Acute Myeloid Leukemia.

Abstract 2391

Poster Board II-368

Alternative mRNA splicing represents an effective mechanism of regulating gene function as well as a key element to increase the coding capacity of the human genome. Today, an increasing number of reports illustrates that aberrant splicing events can contribute to human disease and that alterations in the splicing machinery are common and functionally important for cancer development. Aberrant splice forms can for example have genome-wide effects by deregulating key signaling pathways. However, for most of the aberrant mRNA transcripts detected it remains unclear whether they directly contribute to the malignant phenotype or just represent a by-product of cellular transformation. Thus, more comprehensive analyses of the transcriptome splicing are warranted in order to get novel insights into the biology underlying malignancies like, e.g., acute myeloid leukemia (AML).

Here, we performed a genome-wide screening of splicing events in AML using the Exon microarray platform GeneChip Human Exon 1.0 ST (Affymetrix). We analyzed forty AML cases with complex karyotypes and twenty Core Binding Factor (CBF) AML cases (entered on a multicenter trial for patients <60 years, AMLSG 07-04) using this microarray approach allowing the detection of splice variants. In order to detect alternative splicing events distinguishing different leukemia subgroups we applied a commercial and an open source software tool: XRAY version 3.9 (Biotique Systems) and the OneChannelGUI package for R (version 1.10.7 available at http://www.bioinformatica.unito.it/oneChannelGUI/). Using XRAY supervised analysis comparing subgroups of CBF and complex karyotype AML we identified 1120 transcripts to be potentially alternatively spliced. In parallel, the analysis of the same AML subgroups using the OneChannelGUI package in R revealed 1439 candidates with an overlap of only 211 genes. Of these transcripts, that have been indicated by both programs as potentially alternatively spliced, selected candidates were further investigated by RT-PCR, quantitative RT-PCR and sequence analysis for the presence of splice-variants. Of 26 candidate genes studied, we could confirm alternative splice forms for 5 genes that might potentially be involved in driving leukemogenesis, such as the protein coding gene arginine methyltransferase 1 (PRMT1), which regulates transcription through histone methylation and participates in DNA damage response. Furthermore, we could confirm differential exon usage in the protein tyrosine phosphatase non-receptor type (PTPN6) transcript, which encodes for a negative regulator of numerous signaling pathways involved in cell cycle control and apoptosis. Similarly, the mRNA of the protein Rho GTPase activating protein 4 (ARHGAP4), which has been shown to regulate cell motility, was alternatively spliced between CBF and complex karyotype subgroups.

In summary, these first gene expression data demonstrate that the attempt to elucidate the splicing of transcriptome in AML by applying Exon microarray technology is challenging in particular with regard to the currently available software solutions. Nevertheless, our results show that this approach offers the ability to detect novel alternatively spliced candidate genes. Being involved in cell cycle control, regulation of transcription or remodeling of the cytoskeleton, alternative splicing of these genes might play a potential role in the pathomechanism of distinct AML subgroups. Thus, in the future more extensive Exon array profiling with more sophisticated software solutions at hand is likely to provide additional insights into the molecular mechanisms of leukemogenesis and might reveal novel targets for refined therapeutic strategies in AML.