ALBERT

Description: Recent work (Ebert et al, Nature 2008, Jan 17:335-9) has shown that ribosomal protein S14 (RPS14) is underexpressed in myelodysplasia (MDS) with deletion of chromosome 5q and plays a role in its pathogenesis. The role of ribosomal proteins in other more common subtypes of MDS is unknown. We conducted a meta-analytical comparison of gene expression profiles of 60 cases of non 5q- MDS CD34+ cells with 52 normal CD34+ profiles. These datasets were obtained from seven independent studies from NCBI’s GEO database. The data was integrated based on UniGene IDs and were quantile normalized to ensure cross-study comparability. (Based on our previous approach; Sohal et al PLOS One, 2008, Zhou et al, Blood, 2008). Using significance analysis of microarrays (SAM) and a false discovery rate (FDR) of just 0.04%, we found that ribosomal protein were the class of genes that were most significantly altered in MDS. We observed that RPL35a, RPS9, RPL10, RPL22, RPS14, RPS10, RPS15a, RPS24, RPL24, RPL36, RPL21, RPL23 were strikingly downregulated in non-5q- MDS CD34+ cells. To determine if these alterations were a result of changes in DNA copy numbers of these genes, we examined 20 MDS samples by high resolution array comparative genome hybridization (aCGH) performed on Nimblegen whole genome tiling arrays. aCGH at 6kb resolution revealed deletions in RPL14, RPL22, RPL36, RPS10, RPS5 and even RPS14 in distinct selected cases of non 5q- MDS. These small deletions, which were not identifiable by traditional karyotyping methods, may be putative mechanisms of ribosomal protein downregulation and ultimately, in MDS development and/or progression. Since MDS is also characterized by aberrant epigenetic silencing of genes, we next examined the methylation status of ribosomal gene promoters by high resolution global DNA methylation profiling by using the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)). This assay uses differential methylation-specific digestion by HpaII and MspI followed by amplification, two color labeling and hybridization to quantitatively determine individual promoter CpG island methylation. While there were sporadic changes in some patients, we did not observe any consistently significant changes in methylation of these ribosomal gene promoters on comparison with normal anemic controls. In summary, we show novel widespread alterations in ribosomal protein expression in MDS, at least some of which are associated with genomic deletions. These findings illustrate the applicability of meta-analytical genomic approaches in a heterogeneous disease such as MDS. Most importantly, our data points to the dramatic role of alteration in the protein translational machinery in pathogenesis of MDS.

Description: Acute Myeloid Leukemia (AML) and Myelodysplastic syndrome (MDS) arise from accumulation of multiple stepwise genetic and epigenetic changes in hematopoietic stem cells (HSC) and/or committed progenitors. A series of transforming events can initially give rise to pre-leukemia stem cells (pre-LSC) as well as fully transformed leukemia stem cells (LSC), both of which need to be targeted in strategies aimed at curing these diseases. We conducted parallel transcriptional and epigenetic analysis of highly fractionated stem and progenitor populations in individual patients of MDS and identified STAT3 upregulation in MDS HSCs. qRTPCR in an independent set of sorted MDS/AML HSCs (Lineage-negative, CD34+, CD38-) confirmed the significant increase in STAT3 expression in 60% of cases when compared to healthy controls. We further analyzed gene expression profiles of CD34+ cells from 183 MDS patients and found significant increased expression of STAT3 in MDS when compared to healthy controls (FDR