GeneSpring GX Software Research Articles

Exploring the Mechanism of ETO2-Dependent Transcriptional Regulation in Erythroid Cells

Abstract 1307 Background:Developmental control mechanisms often utilize multimeric complexes containing transcription factors, coregulators, and additional non-DNA binding components. It is challenging to ascertain how such components contribute to complex function at endogenous loci. We recently analyzed the function of components of a complex containing master regulators of hematopoiesis (GATA-1 and Scl/TAL1) and the non-DNA binding components ETO2, the LIM domain protein LMO2, and the chromatin looping factor LDB1. We revealed that ETO2 and LMO2 regulate distinct target gene ensembles in erythroid cells. Furthermore, it was found that ETO2 commonly represses GATA-1 function via suppressing histone H3 acetylation, and also regulates methylation of histone H3 at lysine 27 (H3-trimeK27) at select loci, which suggested that ETO2 might be an important determinant of the erythroblast epigenome (Fujiwara et al. PNAS. 2010). Here, we investigated the role of ETO2 in the epigenetic regulation of erythroid genes. Methods:CBFA2T3 mRNA (which encodes ETO2 protein) was cloned into pcDNA3.1 (Clontech) and Flexi HaloTag vector (Promega), and ETO2 was transiently overexpressed in K562 cells using Amaxa nucleofection technology™ (Amaxa Inc.). For ETO2 knockdown, pGIPZ lentiviral shRNAmir (Open Biosystems) was used. Quantitative ChIP analysis was performed using antibodies for acetylated H3K9 (abcam), trimethyl H3K27 (Millipore), ETO2, c-Myc (Santa Cruz), and HaloCHIP™ system (Promega). To obtain human primary erythroblasts, CD34-positive cells isolated from cord blood were induced in liquid suspension culture. For transcription profiling, human whole expression array was used (Agilent), and the data was analyzed with GeneSpring GX software. Results:First, we conducted microarray analysis to characterize ETO2 target gene ensemble using erythroid cell line (K562 cells). The analysis demonstrated that 598 genes were downregulated in the ETO2-overexpressed cells (> 2 fold). To test what percentages of ETO2-repressed genes could be direct target genes of GATA-1 or GATA-2 in K562 cells, we merged the microarray results with ChIP-seq profile (n= 5,749 and n=21,167 for GATA-1 and GATA-2 ChIP-seq, respectively) (Fujiwara et al. Mol Cell. 2009), and demonstrated that 23.1% and 40.5% of ETO2-repressed genes contained significant GATA-1 and GATA-2 peaks in their loci, respectively. Gene Ontology analysis among ETO2-repressed genes revealed significant enrichment of genes related to “oxygen transporter” and “hemoglobin complex” (p=0.00128), corresponding to HBG, HBB, HBE, HBA, HBQ, HBM and HBZ. We also confirmed that shRNA-mediated knockdown of ETO2 de-repressed globin genes in K562 cells. Quantitative ChIP analysis confirmed endogeneous and exogeneous ETO2 protein occupancy at beta-globin locus control region (LCR) and alpha-globin HS-40 in K562 cells. Furthermore, the overexpression significantly increased H3-trimeK27 and reduced acetylated H3K9 at these loci. Co-immunoprecipitation analysis revealed the interaction of ETO2 with EZH2/SUZ12, known as components of histone H3K27 methyltransferase complex, polycomb repressor complex 2 (PRC2), implying that the complex might be involved in ETO2-mediated transcriptional repression. To test if ETO2-mediated repression of globin genes is also observed in primary erythroblasts, we conducted shRNA-mediated knockdown of ETO2 in cord blood cell-derived primary erythroblasts, and demonstrated that ETO2 knockdown significantly de-repressed HBB and HBA expression. We are currently analyzing the mechanism of ETO2-dependent transcriptional repression and how ETO2-dependent histone marks are established in erythroid cells. Conclusion:In conjunction with the evidence that ETO2 binds histone deacetylases and associates with GATA-Scl/TAL1 complex that binds epigenetic modifiers, our results suggest that ETO2 appears to have important roles in establishing the erythroblast epigenome. Disclosures:No relevant conflicts of interest to declare.

Abstract 5011: Difference in expression of STAT3 and STAT5b in Swedish and Egyptian patients with activated b-cell subtype of diffuse large b-cell lymphoma

Abstract Diffuse Large B-cell lymphoma (DLBCL), the most common type of lymphoma in adults, accounts for approximately 30 to 40% of newly diagnosed lymphomas. Using gene expression profiling, DLBCL can be divided into three immunologically indistinguishable subtypes: the germinal center B-cell like (GBC), the activated B-cell like (ABC) and the primary mediastinal B-cell lymphoma (PMBL) subtype. The aim of the study was to compare gene expression profiles of Swedish and Egyptian ABC DLBCL patients. Formalin fixed and paraffin embedded material of 67 Egyptian and 15 Swedish ABC DLBCL patients were used in the study. Affymetrix gene expression platform was used for profiling and the analysis was carried out using GeneSpring GX software. Quantitative PCR (qPCR) and confocal microscopy was used to verify the findings. The GeneSpring GX analysis showed that 3247 genes were at least 2 fold up- or downregulated comparing the two patient groups. Among these were 1827 genes upregulated and 1420 genes downregulated. Gene expression analysis was verified using qPCR showing that STAT3 was upregulated with a fold change of 3.1 in the Swedish patients and that STAT5b was upregulated with a fold change of 1.3 in the Egyptian patients. Confocal microscopy showed protein activity of STAT3 and 5b in the nucleus of Swedish and Egyptian patients respectively using monoclonal antibodies. This is the first study that reveals diversity in expression of STATs in different populations of ABC DLBCL patients. STAT3 and 5b are known to have different pathways of activation and targeted disruption has shown their functional differences. Using gene expression profiling, we revealed different expression levels of STAT3 and STAT5b in Swedish and Egyptian ABC DLBCL patients. Since the populations of Sweden and Egypt are exposed to different infectious agents, the results of the present study further raise the question concerning viral etiology in ABC-DLBCL possibly due to repeated infections and chronic inflammation. In general, these findings give additional evidence to the importance of signaling pathways involving STATs in ABC DLBCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5011. doi:10.1158/1538-7445.AM2011-5011

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

BackgroundDiet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life.ResultsThe expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR.ConclusionsCtgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.

A20 Gene Deregulation In Waldenstrom's Macroglobulinemia.

AbstractAbstract 3628 Background.Waldenstrom's macroglobulinemia (WM) is a rare lymphoproliferative disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells and monoclonal IgM gammopathy. The deletion 6q is the most frequent cytogenetic aberration in WM and it has been suspected that this region harbours a tumour suppressor gene of pathogenic significance for WM. Comparative genomic hybridization (CGH) array delineated several minimal deleted regions (MDR) on 6q and pointed out key regulator genes of the NFKB pathway involved in these deletions, including A20 gene. The zinc finger protein A20 has been characterized as dual inhibitor of NFKB activation and was recently described as a tumor suppressor gene in lymphoma. However, the mechanisms of A20 deregulation are not fully understood in WM. We aimed to study the mechanisms of A20 deregulation in WM using gene expression profiling (GEP) and single nucleotide polymorphism (SNP) based arrays, a powerful high resolution method allowing both the detection of loss of heterozygosity (LOH) and copy number alteration (CNA) analysis in the same experiment. Methods.We have studied BM samples from 42 patients (pts) with WM (31 males, mean age: 67 years). Conventional karyotype study was analyzed following stimulation with IL2 and DSP30 of BM cells. FISH analysis was performed to detect deletion 6q21 (MYB probe). DNA and RNA were extracted following BM CD19 B cell negative selection. Quantification of copy number of A20 gene was performed using real time PCR, RNAse P gene was used as reference gene. Genome-Wide Human SNP Array 6.0 (Affymetrix chips) was used to detect both LOH and CNA in 31 pts. Paired samples (B cells/normal T lymphocytes) were used as an intra-individual reference to distinguish germline polymorphisms from somatic abnormalities in 29 patients. Size, position and location of genes were referenced using UCSC HG18 assembly, LOH and CNA were identified using genotyping console 3.02 software (Affymetrix) and Partek genomic suite. Gene expression profiling (GEP) was performed in 11 pts using Affymetrix U133A arrays. Gene-expression intensity values were log transformed, normalized and analyzed using GeneSpring GX software. Results.SNP array identified deletion 6q in 9/31 (29%) patients, confirmed by FISH analysis. All cases were monoallelic deletion. In 90% of cases, deletion 6q was associated with others genetic abnormalities. In one case, we observed a loss of 6q combined with a gain of 6p. Deletion 6q was associated with gain of chromosome 4 in 2 cases, and with deletion of 13q in 3 cases. The MDR was located on chromosome 6q21-6q25. This MDR contained several candidate genes included always deletion of A20 gene, located on 6q23, and was confirmed by real time DNA PCR quantification in all cases. Overall, the frequency of A20 gene deletion in our entire WM cohort (N=42 pts) was 29.2% (12/42), determined by real time DNA PCR of the copy number of A20 gene.In patient without deletion 6q, we looked at potential deregulated mechanisms of A20 by LOH with no CNA, e.g. UPD (uniparental disomy). We have not seen any UPD targeting the A20 gene.We then looked at A20 gene expression by GEP, and found no significant variation of A20 gene expression related to A20 monoallelic deletion as compared to pts with two copies, reflecting probably the existence of other mechanisms of A20 gene deregulation. This result was consistent with the absence of clear difference in genome-wide expression profiling in pts with 6q deletion and those without the deletion. As A20 is a key player in the negative feedback loop regulation of NFKB, we also looked at the gene expression of a set of genes involved in NFKB pathway. The presence or absence of A20 deletion did not influence the gene expression profiling of this NFKB pathway gene set. Conclusion.We described a high frequency of deletion of A20 gene. In most cases, deletion of A20 was associated with other genetic abnormalities. A20 deletion was not associated with a significant signature by GEP. Additional studies are needed to understand the cellular consequences of A20 deletions in the pathogenesis of WM. Disclosures:Leleu:Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Overexpression of S100A4 in human cancer cell lines resistant to methotrexate

BackgroundMethotrexate is a chemotherapeutic drug that is used in therapy of a wide variety of cancers. The efficiency of treatment with this drug is compromised by the appearance of resistance. Combination treatments of MTX with other drugs that could modulate the expression of genes involved in MTX resistance would be an adequate strategy to prevent the development of this resistance.MethodsThe differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. A global comparison of all the studied cell lines was performed in order to find out differentially expressed genes in the majority of the MTX-resistant cells. S100A4 mRNA and protein levels were determined by RT-Real-Time PCR and Western blot, respectively. Functional validations of S100A4 were performed either by transfection of an expression vector for S100A4 or a siRNA against S100A4. Transfection of an expression vector encoding for β-catenin was used to inquire for the possible transcriptional regulation of S100A4 through the Wnt pathway.ResultsS100A4 is overexpressed in five out of the seven MTX-resistant cell lines studied. Ectopic overexpression of this gene in HT29 sensitive cells augmented both the intracellular and extracellular S100A4 protein levels and caused desensitization toward MTX. siRNA against S100A4 decreased the levels of this protein and caused a chemosensitization in combined treatments with MTX. β-catenin overexpression experiments support a possible involvement of the Wnt signaling pathway in S100A4 transcriptional regulation in HT29 cells.ConclusionsS100A4 is overexpressed in many MTX-resistant cells. S100A4 overexpression decreases the sensitivity of HT29 colon cancer human cells to MTX, whereas its knockdown causes chemosensitization toward MTX. Both approaches highlight a role for S100A4 in MTX resistance.

CXCR3 Binding Chemokine and TNFSF14 Over Expression in Bladder Urothelium of Patients With Ulcerative Interstitial Cystitis

We investigated the genes responsible for ulcerative interstitial cystitis by DNA microarray analysis and quantitative real-time polymerase chain reaction. Bladder urothelial tissues were taken from a site apart from the ulcerative lesion in 9 patients with ulcerative interstitial cystitis and from a normal-looking area in 9 controls, including 7 with bladder carcinoma and 2 with benign prostatic hyperplasia. Total RNA was extracted from bladder samples and gene expression was compared between these 2 groups using Whole Human Genome DNA microarray 44K (Agilent Technologies, Santa Clara, California). Microarray data were analyzed by GeneSpring GX software and Ingenuity Pathway Analysis. Chosen genes were confirmed for altered transcription by quantitative real-time polymerase chain reaction. We identified 564 probes that were significantly expressed in mRNA more than 4-fold vs those in controls using volcano plot analysis (p <0.001). Further network Ingenuity Pathway Analysis of these genes showed the top 3 functions, including 1) cell-to-cell signaling and interaction, and hematological system development and function, 2) inflammatory disease and 3) cellular development. Quantitative real-time polymerase chain reaction confirmed increased mRNA expression of several genes in the bladder samples of patients with ulcerative interstitial cystitis, including CXCR3 binding chemokines (CXCL9, 10 and 11) and TNFSF14 (LIGHT). Our study using DNA microarray analysis followed by quantitative real-time polymerase chain reaction reveals over expression of genes related to immune and inflammatory responses, including T-helper type 1 related chemokines, and cytokines such as CXCR3 binding chemokines and TNFSF14. These genes may be potential interstitial cystitis biomarkers.

Expression profiling of formalin-fixed paraffin embedded primary human uveal melanomas using DASL matrices

Fresh biopsied ocular tumor tissues are difficult to obtain for the purpose of performing microarray experiments on extracted nucleic acids. Present technology allows for extraction of total RNA from formalin-fixed paraffin embedded (FFPE) tissue analyzed by the cDNA mediated Annealing Sectioning and Ligation (DASL) method. We aimed to correlate gene transcript differences between two uveal melanoma (UM) clinical-histopathological parameters (metastasis, cell type). A total of 43 FFPE UM were used. The expression of RPL13a, a ribosomal protein gene, for each sample was used to evaluate the quality of RNA extracted from FFPE tissue. Gene expression values generated from the array were analyzed using the GeneSpring GX software (Agilent). Immunohistochemistry was used in order to validate transcriptional findings at the protein level. A total of 106 genes were identified with (P < 0.05, Welch ANOVA test) a difference in transcript abundance for the metastasis clinical parameter. Furthermore, we identified 64 genes with a statistically significant (P < 0.05) difference in transcript abundance between the spindle and epithelioid cell types. Each individual sample for both groups (metastasis, cell type) exhibited distinct transcriptional profiles that were separated on a PCA. Positive nuclear immunostaining for LIG4-metastasis, ErbB3-cell type was found to be associated with better patient prognosis and outcome. To the best of our knowledge, this is the first time that a successful retrospective analysis has been done with UM FFPE RNA. This data may lead to future customized therapeutic targets, which may improve the now unchanged mortality rate of this particular malignancy.

Use of Gene Expression Profiles of Peripheral Blood Lymphocytes to Distinguish BRCA1 Mutation Carriers in High Risk Breast Cancer Families

Mutations in two major genes, BRCA1 and BRCA2, account for up to 30% of families with hereditary breast cancer. Unfortunately, in most families there is little to indicate which gene should be targeted first for mutation screening, which is labor intensive, time consuming and often prohibitively expensive. As BRCA1 is a tumor suppressor gene involved in various cellular processes, heterozygous mutations could deregulate dependent pathways, such as DNA damage response, and disturb transcriptional activity of genes involved in the downstream signaling cascade. We investigated gene expression profiling in peripheral blood lymphocytes to evaluate this strategy for distinguishing BRCA1 mutation carriers from non-carriers. RNA from whole blood samples of 15 BRCA1 mutation carriers and 15 non-carriers from BRCA1 or BRCA2 families were hybridized to Agilent Technologies Whole Human Genome OligoMicroarrays (4 x 44 K multiplex format) containing 41,000 unique human genes and transcripts. Gene expression data were analyzed with Welch's t-tests and submitted to hierarchical clustering (GeneSpring GX software, Agilent Technologies). Statistical analysis revealed a slight tendency for 133 genes to be differentially expressed between BRCA1 mutation carriers and non-carriers. However, hierarchical clustering of these genes did not accurately discriminate BRCA1 mutation carriers from non-carriers. Expression variation for these genes according to BRCA1 mutation status was weak. In summary, microarray profiling of untreated whole blood does not appear to be informative in identifying breast cancer risk due to BRCA1 mutation.

Networking of differentially expressed genes in human cancer cells resistant to methotrexate

BackgroundThe need for an integrated view of data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance, we constructed biological association networks of genes differentially expressed in cell lines resistant to methotrexate (MTX).MethodsSeven cell lines representative of different types of cancer, including colon cancer (HT29 and Caco2), breast cancer (MCF-7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. Genes deregulated in common between the different cancer cell lines served to generate biological association networks using the Pathway Architect software.ResultsDikkopf homolog-1 (DKK1) is a highly interconnected node in the network generated with genes in common between the two colon cancer cell lines, and functional validations of this target using small interfering RNAs (siRNAs) showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of genes differentially expressed in the two breast cancer cell lines. siRNA treatment against UGT1A also showed an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was overexpressed among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX.ConclusionsBiological association networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using siRNA technology against these three genes showed chemosensitization toward MTX.

Hypoxia alters gene expression in the gonads of zebrafish ( Danio rerio)

The objectives of this study were to characterize gene expression responses to hypoxia in gonads of mature zebrafish ( Danio rerio), and to start characterizing modes of action by which hypoxia could potentially alter reproduction. Adult male and female zebrafish were maintained under normoxia (7 mg O 2/L), moderate hypoxia (3 mg O 2/L), and severe hypoxia (1 mg O 2/L) for 4 and 14 days and changes in gene expression in gonadal tissues ( n = 5 per sex per treatment) were evaluated using a commercial 21,000 gene zebrafish oligonucleotide microarray. Differentially expressed genes were determined using ANOVA ( p < 0.05), and enriched gene ontology (GO) categories ( p < 0.01) identified using GeneSpring GX software. Short-term (4 d) exposure to hypoxia affected expression of genes associated with the initial adaptive responses such as: metabolism of carbohydrates and proteins, nucleotide metabolism, haemoglobin synthesis, reactive oxygen species metabolism, and locomotion. Prolonged (14 d) hypoxia affected a suite of genes belonging to different GO categories: lipid metabolism, reproduction (e.g., steroid hormone synthesis), and immune responses. Results of the present study demonstrate that reproduction likely would be affected by hypoxia via multiple modes of action. These include previously hypothesized mechanisms such as modulation of expression of steroidogenic genes, and downregulation of serotonergic pathway. In addition, we propose that there are multiple other points of disruption of reproductive system function linked, for example, to reorganization of lipid transport and other mechanisms involved in responding to hypoxia (e.g., hydroxysteroid dehydrogenase alterations, downregulation of contractile elements, etc.).

Gene Expression Profiling of Human Pancreatic Islets Undergoing a Simulated Process of Instant Blood-Mediated Inflammatory Reaction

Objective Islet cell transplantation, as a treatment for type 1 diabetes mellitus, has historically required islet infusions from more than one donor organ to achieve insulin independence. Significant islet mass may be destroyed upon infusion due to a yet undefined process known as instant blood-mediated inflammatory reaction (IBMIR). Our objective was to identify gene expression changes in islets undergoing a simulated process of IBMIR. Materials and Methods Human pancreatic islets were isolated from 2 cadaveric donors and divided into 3 groups each for a total of 18 samples. Group one (n = 3) was treated with autologous sera, group two (n = 3) with allogeneic sera, and group three (n = 3) with type 1 diabetic sera (T1DM). Each group was treated for 3 hours at 37°C. Islets were washed, lysed using TRI reagent, and mRNA was isolated using the Total Prep mRNA isolation kit. Isolated cRNA was used for microarray analysis using Illumina Gene Chips Hu6_v2. GeneSpring GX software was used for statistical analysis. Results were significant at P < .05. Results One-way ANOVA statistical analysis of the microarray data revealed that interleukin-11 (IL-11), interleukin-12A (IL-12A), and Ras related associated with diabetes (RRAD) were overexpressed in islets exposed to diabetic sera when normalized to autologous control ( P < .01). Under the same conditions, islet cells exposed to T1DM serum had down-regulation of IL-1 receptor antagonist (IL-1RN). Conclusion These findings suggested that T1DM serum elicited an adaptive and innate immune response to the transplanted islet mass making them more susceptible to cytokine-mediated destruction.

Endocrine effects on endometrium in human ectopic gestation

Endocrine effects on endometrium in human ectopic gestation

Kinesin Family Member 20A (KIF20A) Is Specifically down Regulated in 5q- CD34+ and CD61+ Cells and Uniparental Disomy Is Not a Feature of 5q- Syndrome.

The specificity of the clinicomorphological characteristics in 5q- syndrome has led to a search for putative tumour suppressor genes localised to the critical deleted region (CDR) of 5q-. To date, microarray expression studies in 5q- syndrome patients have been carried out on CD34+ cells, neutrophils or total bone marrow (BM) cells. We have used a Affymetrix microarray approach to investigate allelic imbalance (AI) and the transcription profile of CD61+ and CD34+ cells from patients (n=17) presenting with 5q- of which 10 have an “isolated 5q-“. the rest having additional cytogenetic abnormalities. BM CD34+ and CD61+ cells from normals (n=10) were controls in expression profiling. DNA or total RNA from CD61+ or CD34+ cells and constitutional DNA was prepared and hybridised to the 500K mapping or U113 plus 2.0 arrays. GC-RMA normalized expression data was analysed using non-parametric testing with Genespring GX software to generate lists of differentially regulated genes. DChip was used to analyse SNP data. From a list of changing genes 48/720 occur in both the CD61+ and CD34+ cell profiles. 16/48 of these genes map to 5q of which 10/16 map to 5q31. Of these genes KIF20A showed the most marked change (5 and 3.8 fold down) in CD34+ and CD61+ cells respectively. KIF20A is a microtubule-associated protein that is upregulated in mitosis and localizes to the spindle midzone during anaphase. Knock out of KIF20A protein results in binucleate nuclei indicating its involvement in both cytokinesis and nucleokinesis. Haploinsufficiency of this gene, may account for mononuclear megakaryocytes that are so characteristic of 5q- syndrome. Interestingly other members of the kinesin family expressed in 5q- CD34+ cells are down regulated in 5q- CD61+ cells (KIF21B, KIF2, KIF3A and KIF1B) indicating that in CD34+ cells, a compensatory mechanism for KIF20A haploinsufficiency may exist which is absent in megakaryocytes. The 5q- deletion (q14.2–q33.3) was detected in 6/10 CD61+ and 11/17 CD34+ cell populations by SNP analysis. The disparity reflects the admixture of normal and MDS CD61+ and CD34+ cells. Three patients presented with additional cytogenetic abnormalities, two had −7 or del 7(q22–q36), both being detected with the SNP array. The third patient had trisomy 8 that was not detected. SNP microarray identified 5q31.1–5q33.2 as the minimal deleted segment, which includes previously described CDR. Paired analysis of CD61+ and CD34+ with constitutional DNA revealed only 2/17 patients had additional abnormalities in CD34+ cells. One with a del12(q21.1–22) (21Mb). The 2nd had −13(q12.3–34;83Mb),+ 13(q11–12.3; 12.5Mb) and +11(q22.3-qterm; 30Mb) These changes were not detected by cytogenetic analysis. 2/6 patients shared 13Mb of UPD on chromosome 3(p12.2–q11.2) and chromosome 9(q33.1–q34.1) in CD34+ cells as well as constitutive DNA. We conclude that unlike AML, 5q- Syndrome does not have significant rate of UPD. Only a small number of genes are under expressed in both CD61+ and CD34+ cells and amongst these KIF20A is the most significant change and may be responsible for some of the clinicomorphological features of 5q- abnormality.