- Email: [email protected]
8 Gene expression profiling in patients with myelodysplastic syndromes, including patients with the del(5q)
Invited Lectures In order to find out the mechanism of slow proliferation with differentiation in D171N cells, BMI-1 mRNA of BM CD34+ cells from MDS/AML patients were measured. The amount of mRNA in D171N type patients was found to be higher than that in S291fs type patients, suggesting that BMI-1 expression is needed for the intensive proliferation and self renewal of D171N cells in vitro. In fact, by introducing BMI1 gene with stepwise manner, D171N cells were able to be stimulated to renew and proliferate with significant CD34+ cell fraction. The expression of BMI-1 in vivo may be induced in cooperation with the mutated gene in the RTKRAS pathway, being frequently observed in this type of MDS/AML. Taken together, we have clearly demonstrated that the in vitro characteristics of each D171N cells and S291fs cells reflect the clinical characteristics of each type of MDS/AML.
7 Molecular changes as detected by high-throughput genomic techniques in MDS W.-K. Hofmann1 ° , D. Nowak2 , F. Nolte3 . 1 University Hospital, Mannheim, Germany; 2 UCLA School of Medicine, Los Angeles, CA, USA; 3 University Hospital Benjamin Franklin, Berlin, Germany Myelodysplastic syndromes are characterized by ineffective hematopoiesis presenting with peripheral cytopenias in combination with a hyperplastic bone marrow and an increased risk of evolution to acute myeloid leukemia. Development and progression of MDS is suggested to be a multistep alteration to hematopoietic stem cells. Different molecular alterations have been described, affecting genes involved in cell cycle control, mitotic checkpoints as well as growth factor receptors. Secondary signal proteins and transcription factors which gives the cell a growth advantage over its normal counterpart, may be affected as well. The accumulation of such defects may finally cause the leukemic transformation of MDS. Identification of genomic lesions in progenitor cells of patients with myelodysplastic syndrome could lead to the discovery of new disease specific genes and may be of prognostic value. We and others have carried out a genomewide mapping of DNA from CD34+ cells of MDS patients with high-resolution 500K single nucleotide polymorphism (SNP) arrays and a concomitant integration with global gene expression analysis. Copy number and loss of heterozygosity (LOH) analyses detected heterozygous deletions on chromosomes 2, 9, 13, 16, 17 and 20 ranging in size from 0.1 Mba to 2.1 Mba. Additionally, numerous regions with significant uniparental disomy (UPD) were detected. Integration of the genomic data with gene expression analysis showed that genes, which were downregulated at least 1.5 fold in regions of significant deletion and UPD were exclusively downregulated in those samples displaying the aberration. Genomics and
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gene expression data were confirmed by real time PCR and variable number tandem repeat (VNTR) analysis. In summary, high-density genomic mapping of CD34+ bone marrow cells from patients with MDS identifies cryptic genetic lesions and offers new opportunities for the discovery of target genes in MDS by integration with gene expression analysis.
8 Gene expression profiling in patients with myelodysplastic syndromes, including patients with the del(5q) J. Boultwood ° . LRF Molecular Haematology Unit, NDCLS, John Radcliffe Hospital, Oxford, UK We have studied the transcriptome of the CD34+ cells of a large group of patients with MDS to gain insight into the pathophysiology of MDS, to discover molecular markers for diagnosis and prognosis, and to determine whether distinct gene expression profiles could be identified for specific groups of MDS as defined by either the FAB classification or karyotype. The CD34+ cells have been obtained from 180 MDS patients and 17 healthy individuals and were analysed using Affymetrix U133 Plus2.0 arrays. Pathophysiology: Pathway analysis using the most commonly up-regulated genes in our patient cohort showed that the interferon signalling pathway was one of the most significantly deregulated pathways in MDS. Indeed IFIT1, the most up-regulated gene in MDS (up-regulated in approximately two thirds of the patients by >2-fold), is an interferon-stimulated gene (ISG). Other ISGs, which mediate growth inhibitory effects of interferon, such as IFITM1, IFI44L and IFIT3, were markedly up-regulated in the majority of MDS patients [1]. Up-regulation of ISGs is a major feature of MDS and may be responsible for some of the hematological characteristics of this disorder, such as peripheral blood cytopenias. The DLK1 gene, encoding a transmembrane protein belonging to the epidermal growth factor-like superfamily was another commonly up-regulated gene, in agreement with previous studies [2]. FAB classification: Hierarchical clustering performed using the significantly differentially expressed genes identified showed that MDS patients with RARS constitute the most homogeneous group, while MDS patients with RA and RAEB show more overlap. RARS gene expression profile was characterized by up-regulation of mitochondrial-related genes and by down-regulation of ABCB7 [3], a gene mutated in the rare inherited X-linked sideroblastic anemia with ataxia (XLSA/A). Moreover, a good separation between patients with RARS and patients with RCMD-RS was obtained by hierarchical clustering using the significantly differentially expressed genes between these two WHO subgroups. One of the most significant genes was MFN1, which is essential for mitochondrial fusion and maintenance of mitochondrial morphology.
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Invited Lectures
Karyotype: Chen et al have shown the deregulation of different groups of genes in MDS patients with monosomy 7 and trisomy 8, respectively, using gene expression profiling [4]. The association of distinct gene expression profiles with specific cytogenetic groups was examined in our study, and by hierarchical clustering we were able to separate MDS patients with del(5q), patients with −7/del(7q) and patients with trisomy 8. The expression profile of patients with the del(5q) was characterised by the downregulation of genes mapping to chromosome 5q and other groups of genes (see below). Genes differentially expressed in patients with −7/del(7q) include LOX and UBE2H , while genes differentially expressed in patients with trisomy 8 include HRSP12 and TPM4. These findings suggest distinct molecular pathogenetic pathways for MDS patients with del(5q), −7/del(7q) and trisomy 8. Disease progression: The application of microarray technology to the comprehensive analysis of gene expression in MDS during disease evolution offers a realistic possibility of identifying new molecular pathways of key importance in disease progression. In order to identify differences in gene expression associated with MDS disease progression, we compared patients with early MDS (RA) and patients with advanced MDS (RAEB2). Hierarchical clustering performed using the significantly differentially expressed probe sets resulted in a good separation between MDS patients with RA and patients with RAEB2. LEF1, a regulator of neutrophilic granulopoiesis, was the most significant differentially expressed gene with higher expression levels in patients with RA and decreasing in patients with RAEB2. Intriguingly, reduced or absent LEF1 expression is believed to be critical to pathogenesis in some patients with congenital neutropenia. Other genes showing higher expression levels in patients with RA than RAEB2 include CASC5, a cancer susceptibility candidate gene, and RBBP8, a gene that plays a role in DNA-damageinduced cell cycle checkpoint control. We have identified a gene signature for poor survival in MDS although this requires confirmation in a larger patient cohort. 5q− syndrome: Our research group has a particular interest in the study of MDS with the del(5q), the most commonly reported deletion in MDS and found in 10−15% of all patients. We demonstrated haploinsufficiency of the ribosomal gene RPS14, which is required for the maturation of 40S ribosomal subunits and maps to the commonly deleted region in patients with the 5q− syndrome [5]. Haploinsufficiency of RPS14 has since been shown to be the mechanism underlying the erythroid defect in this disorder by Ebert and colleagues [6]. We have shown that haploinsufficiency of RPS14 in patients with the 5q− syndrome is associated with deregulated expression of multiple ribosomal- and translation-related genes [7] and we have recently shown that other MDS patients with the del(5q) also show haploinsufficiency of RPS14 and deregulation of a group ribosomal and translation-related genes. An exciting possibility is that defective ribosomal
biogenesis also plays a role in the pathogenesis of other MDS with the del(5q). References [1] Pellagatti, A, Cazzola M, Giagounidis A, Malcovati L, Dalla Porta MG, Killick S, Campbell LJ, Wang L, Langford CF, Fidler C, Oscier D, Aul, C, Wainscoat, JS, Boultwood, J. (2006) Gene expression profiles of CD34+ cells in myelodysplastic syndromes: involvement of interferon-stimulated genes and correlation to FAB subtype and karyotype. Blood, 108:337−45. [2] Miyazato A, Ueno S, Ohmine K, et al. Identification of myelodysplastic syndrome-specific genes by DNA microarray analysis with purified hematopoietic stem cell fraction. Blood. 2001;98: 422–427 [3] Boultwood J, Pellagatti A, Nikpour M, Pushkaran B, Fidler C, Cattan H, Littlewood TJ, Malcovati, L, Dalla Porta, MG, J¨adersten M, Killick S, Giagounidis A, Bowen D, Hellstr¨om-Lindberg, E, Cazzola, M, Wainscoat JS. (2008) The role of the iron transporter ABCB7 in refractory anemia with ringed sideroblasts. PLoS ONE 4(4):e1970. [4] Chen, G, Zeng, W, Miyazato, A, Billings, E, Maciejewski, JP, Kajigaya, S, Sloand, EM, Young, NS (2004). Distinctive gene expression profiles of CD34 cells from patients with myelodysplastic syndrome characterized by specific chromosomal abnormalities. Blood 104(13): 4210−8. [5] Boultwood J, Pellagatti A, Cattan H, Lawrie CH, Giagounidis A, Malcovati, L, Dalla Porta, MG, J¨adersten M, Killick, S, TJ, Fidler C, Cazzola, M, Hellstr¨om-Lindberg, E, Wainscoat JS. (2007) Gene expression profiling of CD34+ cells in patients with the 5q− syndrome. British Journal of Haematology, 139: 578−89. [6] Ebert, BL; Pretz, J; Bosco, J; Chang, CY; Tamayo, P; Galili, N; Raza, A; Root, DE; Attar, E; Ellis, SR; Golub, TR. Identification of RPS14 as a 5q- syndrome gene by RNA interference screen. Nature. 2008;451:335–339. [7] Pellagatti P, Hellstr¨om-Lindberg E, Giagounidis A, Perry J, Malcovati L, Della Porta MG, J¨adersten M, Killick S, Fidler C, Cazzola M, Wainscoat JS, Boultwood J. (2008) Haploinsufficiency of RPS14 in 5q− syndrome causes marked deregulation of ribosomal- and translation-related genes. British Journal of Haematology, 142: 57−64.
7 Molecular changes as detected by high-throughput genomic techniques in MDS W.-K. Hofmann1 ° , D. Nowak2 , F. Nolte3 . 1 University Hospital, Mannheim, Germany; 2 UCLA School of Medicine, Los Angeles, CA, USA; 3 University Hospital Benjamin Franklin, Berlin, Germany Myelodysplastic syndromes are characterized by ineffective hematopoiesis presenting with peripheral cytopenias in combination with a hyperplastic bone marrow and an increased risk of evolution to acute myeloid leukemia. Development and progression of MDS is suggested to be a multistep alteration to hematopoietic stem cells. Different molecular alterations have been described, affecting genes involved in cell cycle control, mitotic checkpoints as well as growth factor receptors. Secondary signal proteins and transcription factors which gives the cell a growth advantage over its normal counterpart, may be affected as well. The accumulation of such defects may finally cause the leukemic transformation of MDS. Identification of genomic lesions in progenitor cells of patients with myelodysplastic syndrome could lead to the discovery of new disease specific genes and may be of prognostic value. We and others have carried out a genomewide mapping of DNA from CD34+ cells of MDS patients with high-resolution 500K single nucleotide polymorphism (SNP) arrays and a concomitant integration with global gene expression analysis. Copy number and loss of heterozygosity (LOH) analyses detected heterozygous deletions on chromosomes 2, 9, 13, 16, 17 and 20 ranging in size from 0.1 Mba to 2.1 Mba. Additionally, numerous regions with significant uniparental disomy (UPD) were detected. Integration of the genomic data with gene expression analysis showed that genes, which were downregulated at least 1.5 fold in regions of significant deletion and UPD were exclusively downregulated in those samples displaying the aberration. Genomics and
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gene expression data were confirmed by real time PCR and variable number tandem repeat (VNTR) analysis. In summary, high-density genomic mapping of CD34+ bone marrow cells from patients with MDS identifies cryptic genetic lesions and offers new opportunities for the discovery of target genes in MDS by integration with gene expression analysis.
8 Gene expression profiling in patients with myelodysplastic syndromes, including patients with the del(5q) J. Boultwood ° . LRF Molecular Haematology Unit, NDCLS, John Radcliffe Hospital, Oxford, UK We have studied the transcriptome of the CD34+ cells of a large group of patients with MDS to gain insight into the pathophysiology of MDS, to discover molecular markers for diagnosis and prognosis, and to determine whether distinct gene expression profiles could be identified for specific groups of MDS as defined by either the FAB classification or karyotype. The CD34+ cells have been obtained from 180 MDS patients and 17 healthy individuals and were analysed using Affymetrix U133 Plus2.0 arrays. Pathophysiology: Pathway analysis using the most commonly up-regulated genes in our patient cohort showed that the interferon signalling pathway was one of the most significantly deregulated pathways in MDS. Indeed IFIT1, the most up-regulated gene in MDS (up-regulated in approximately two thirds of the patients by >2-fold), is an interferon-stimulated gene (ISG). Other ISGs, which mediate growth inhibitory effects of interferon, such as IFITM1, IFI44L and IFIT3, were markedly up-regulated in the majority of MDS patients [1]. Up-regulation of ISGs is a major feature of MDS and may be responsible for some of the hematological characteristics of this disorder, such as peripheral blood cytopenias. The DLK1 gene, encoding a transmembrane protein belonging to the epidermal growth factor-like superfamily was another commonly up-regulated gene, in agreement with previous studies [2]. FAB classification: Hierarchical clustering performed using the significantly differentially expressed genes identified showed that MDS patients with RARS constitute the most homogeneous group, while MDS patients with RA and RAEB show more overlap. RARS gene expression profile was characterized by up-regulation of mitochondrial-related genes and by down-regulation of ABCB7 [3], a gene mutated in the rare inherited X-linked sideroblastic anemia with ataxia (XLSA/A). Moreover, a good separation between patients with RARS and patients with RCMD-RS was obtained by hierarchical clustering using the significantly differentially expressed genes between these two WHO subgroups. One of the most significant genes was MFN1, which is essential for mitochondrial fusion and maintenance of mitochondrial morphology.
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Invited Lectures
Karyotype: Chen et al have shown the deregulation of different groups of genes in MDS patients with monosomy 7 and trisomy 8, respectively, using gene expression profiling [4]. The association of distinct gene expression profiles with specific cytogenetic groups was examined in our study, and by hierarchical clustering we were able to separate MDS patients with del(5q), patients with −7/del(7q) and patients with trisomy 8. The expression profile of patients with the del(5q) was characterised by the downregulation of genes mapping to chromosome 5q and other groups of genes (see below). Genes differentially expressed in patients with −7/del(7q) include LOX and UBE2H , while genes differentially expressed in patients with trisomy 8 include HRSP12 and TPM4. These findings suggest distinct molecular pathogenetic pathways for MDS patients with del(5q), −7/del(7q) and trisomy 8. Disease progression: The application of microarray technology to the comprehensive analysis of gene expression in MDS during disease evolution offers a realistic possibility of identifying new molecular pathways of key importance in disease progression. In order to identify differences in gene expression associated with MDS disease progression, we compared patients with early MDS (RA) and patients with advanced MDS (RAEB2). Hierarchical clustering performed using the significantly differentially expressed probe sets resulted in a good separation between MDS patients with RA and patients with RAEB2. LEF1, a regulator of neutrophilic granulopoiesis, was the most significant differentially expressed gene with higher expression levels in patients with RA and decreasing in patients with RAEB2. Intriguingly, reduced or absent LEF1 expression is believed to be critical to pathogenesis in some patients with congenital neutropenia. Other genes showing higher expression levels in patients with RA than RAEB2 include CASC5, a cancer susceptibility candidate gene, and RBBP8, a gene that plays a role in DNA-damageinduced cell cycle checkpoint control. We have identified a gene signature for poor survival in MDS although this requires confirmation in a larger patient cohort. 5q− syndrome: Our research group has a particular interest in the study of MDS with the del(5q), the most commonly reported deletion in MDS and found in 10−15% of all patients. We demonstrated haploinsufficiency of the ribosomal gene RPS14, which is required for the maturation of 40S ribosomal subunits and maps to the commonly deleted region in patients with the 5q− syndrome [5]. Haploinsufficiency of RPS14 has since been shown to be the mechanism underlying the erythroid defect in this disorder by Ebert and colleagues [6]. We have shown that haploinsufficiency of RPS14 in patients with the 5q− syndrome is associated with deregulated expression of multiple ribosomal- and translation-related genes [7] and we have recently shown that other MDS patients with the del(5q) also show haploinsufficiency of RPS14 and deregulation of a group ribosomal and translation-related genes. An exciting possibility is that defective ribosomal
biogenesis also plays a role in the pathogenesis of other MDS with the del(5q). References [1] Pellagatti, A, Cazzola M, Giagounidis A, Malcovati L, Dalla Porta MG, Killick S, Campbell LJ, Wang L, Langford CF, Fidler C, Oscier D, Aul, C, Wainscoat, JS, Boultwood, J. (2006) Gene expression profiles of CD34+ cells in myelodysplastic syndromes: involvement of interferon-stimulated genes and correlation to FAB subtype and karyotype. Blood, 108:337−45. [2] Miyazato A, Ueno S, Ohmine K, et al. Identification of myelodysplastic syndrome-specific genes by DNA microarray analysis with purified hematopoietic stem cell fraction. Blood. 2001;98: 422–427 [3] Boultwood J, Pellagatti A, Nikpour M, Pushkaran B, Fidler C, Cattan H, Littlewood TJ, Malcovati, L, Dalla Porta, MG, J¨adersten M, Killick S, Giagounidis A, Bowen D, Hellstr¨om-Lindberg, E, Cazzola, M, Wainscoat JS. (2008) The role of the iron transporter ABCB7 in refractory anemia with ringed sideroblasts. PLoS ONE 4(4):e1970. [4] Chen, G, Zeng, W, Miyazato, A, Billings, E, Maciejewski, JP, Kajigaya, S, Sloand, EM, Young, NS (2004). Distinctive gene expression profiles of CD34 cells from patients with myelodysplastic syndrome characterized by specific chromosomal abnormalities. Blood 104(13): 4210−8. [5] Boultwood J, Pellagatti A, Cattan H, Lawrie CH, Giagounidis A, Malcovati, L, Dalla Porta, MG, J¨adersten M, Killick, S, TJ, Fidler C, Cazzola, M, Hellstr¨om-Lindberg, E, Wainscoat JS. (2007) Gene expression profiling of CD34+ cells in patients with the 5q− syndrome. British Journal of Haematology, 139: 578−89. [6] Ebert, BL; Pretz, J; Bosco, J; Chang, CY; Tamayo, P; Galili, N; Raza, A; Root, DE; Attar, E; Ellis, SR; Golub, TR. Identification of RPS14 as a 5q- syndrome gene by RNA interference screen. Nature. 2008;451:335–339. [7] Pellagatti P, Hellstr¨om-Lindberg E, Giagounidis A, Perry J, Malcovati L, Della Porta MG, J¨adersten M, Killick S, Fidler C, Cazzola M, Wainscoat JS, Boultwood J. (2008) Haploinsufficiency of RPS14 in 5q− syndrome causes marked deregulation of ribosomal- and translation-related genes. British Journal of Haematology, 142: 57−64.