Identification of Intronic RNA Expression in CD34+ Cells of Patients with Myelodysplatic Syndrome by RNA Microarray Analysis.

Myelodysplatic syndromes (MDS) are a group of clonal hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. RNAs transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and in the regulation of exon-skipping and intron retention. The characterization of intronic transcripts in progenitor cells of MDS patients could be an important strategic to understand the gene expression regulation in this disease. We conducted a pilot study in CD34+ cells of 4 MDS-RARS patients and 4 healthy individuals. Gene expression analysis was performed using a 44k intron-exon oligoarray custom-designed by Verjorvski-Almeida et al. and printed by Agilent Technologies. This oligoarray included protein-coding genes, sense and antisense strands of totally intronic noncoding (TIN) and partially intronic nonconding (PIN) RNAs. CD34+ cells were isolated from bone marrow samples using MACS magnetic columns. The quality of total extracted RNA was confirmed with the Agilent Bioanalyzer 2100. We amplified 300ng of each total RNA using the Agilent Low RNA Input Fluorescent Linear Amplification Kit PLUS, two-Color and samples were hybridized using the Gene Expression Hybridization Kit (Agilent) and then scanned on a GenePIX 4000B Scanner (Molecular Devices). The extraction analysis was performed using Agilent Feature Extration Software 9.5. Considering only the fluorescent spots in samples, the data were normalized by quantil using Spotfire DecisionSite®. To identify genes differentially expressed between MDS-RARS and healthy samples, we applied the SAM (Significance Analysis of microarray) approach using as parameters: two-class unpaired response, t-statistic, 1,000 permutations, fold > 2 and FDR = 0%. We identified 139 genes differentially expressed (67 up-regulated and 72 down-regulated), of which 33 were TIN and PIN transcripts (21 up-regulated and 12 down-regulated). These transcripts were grouped according to the main role: gene transcription (ZNF76, CC2D1A, ASXL1, TOP2B, NR4A3 and NR4A2); immune response (CTSH, CTSS, IFI30, NPY, SERPINA1 and PAG1); growth factor and receptor (RP5-1022P6.2, PRG4 and FGFR1OP2); adhesion (PPP1R15A and FN1); cell differentiation (B3GNT5, RALGPS1, C16orf67 and C5orf13); cell cycle and apoptosis (CYFIP2, PPP1R15A, DDX3X and NASP) and cellular trafficking (WIPI1, ICA1 and SLC11A2). These results demonstrated that 22% of all the genes differentially expressed correspond to TIN and PIN transcripts in CD34+ cells of MDS-RARS patients, suggesting that intronic transcripts can play an important role during the development of myelodysplastic syndrome.