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Functional Evidence Implicating Chromosome 7q22 Haploinsufficiency in Myelodysplastic Syndrome Pathogenesis

ABSTRACT: Purpose: 5A3+/del mice, which have a heterozygous germ line deletion of a 2-Mb interval of chromosome band 5A3 syntenic to a commonly deleted segment of human 7q22, exhibit hematopoietic stem cell (HSC) abnormalities that may contribute to myelodysplastic syndrome (MDS) pathogenesis. These defects are cell autonomous. The goal of this study is to compare transcriptome profiling (RNA-seq) data obtained from HSC and multipotent progenitors (MPP) isolated from 5A3+/del mice and their wildtype littermates, in order to identify differentially expressed genes and pathways that may contribute to the phenotype. Methods: Total RNA was isolated from CD150hi-HSC, CD150lo-HSC and CD150neg-MPP from wildtype and 5A3+/del mice. 10ng of total RNA was converted into double-stranded cDNA using the Ovation RNA Amplification System V2 (NuGen, CA), and the amplified cDNA products were then used to generate RNA-seq libraries using the TruSeq RNA Sample Preparation Kit v2 reagents (Illumina, CA) with 10 PCR amplification cycles. Library quality and quantity were assessed by the Agilent DNA1000 Chip (Agilent, CA) and qPCR (Kappa Biosystems Inc, MA). 10 pM of each library was sequenced using Illumina SBS chemistry at 2 x 100 bp reads on the HiSeq2000 (Illumina®, CA). The RNA-Seq paired-end reads were mapped to the mouse mm9 genome using an in-house mapping and quality assessment pipeline. The expression of each gene was estimated by the mean coverage of the highest-covered coding exon. Genes with low expression level (<10) across all samples were filtered out, followed by quantile normalization. Differential expression analysis was performed using limma with estimation of false-discovery rate. Gene Set Enrichment Analysis was used to assess pathway enrichment. Results:Transcriptome (RNA-Seq) and Taqman quantitative real-time PCR analyses revealed a ~50% reduction in the expression of multiple genes and of the long intergenic non-coding RNA 503142E22Rik within the 5A3 interval in mutant HSC and MPP. Gene Set Enrichment Analysis (GSEA) of the RNA-Seq data from 5A3+/del HSCs further demonstrated reduced expression of multiple gene sets related to oxidative phosphorylation (OXPHOS) that are similarly down-regulated in the early stages of human therapy-induced MDS and AML. Conclusions: Our study revealed that genes involved in OXPHOS were down-regulated in 5A3+/del HSC, and this finding provided novel insights into the impact of chromosome 7 deletions on the pathogenesis of MDS.

ORGANISM(S): Mus musculus

PROVIDER: GSE72811 | GEO | 2015/09/09

SECONDARY ACCESSION(S): PRJNA295098

REPOSITORIES: GEO

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Project description:Purpose: 5A3+/del mice, which have a heterozygous germ line deletion of a 2-Mb interval of chromosome band 5A3 syntenic to a commonly deleted segment of human 7q22, exhibit hematopoietic stem cell (HSC) abnormalities that may contribute to myelodysplastic syndrome (MDS) pathogenesis. These defects are cell autonomous. The goal of this study is to compare transcriptome profiling (RNA-seq) data obtained from HSC and multipotent progenitors (MPP) isolated from 5A3+/del mice and their wildtype littermates, in order to identify differentially expressed genes and pathways that may contribute to the phenotype. Methods: Total RNA was isolated from CD150hi-HSC, CD150lo-HSC and CD150neg-MPP from wildtype and 5A3+/del mice. 10ng of total RNA was converted into double-stranded cDNA using the Ovation RNA Amplification System V2 (NuGen, CA), and the amplified cDNA products were then used to generate RNA-seq libraries using the TruSeq RNA Sample Preparation Kit v2 reagents (Illumina, CA) with 10 PCR amplification cycles. Library quality and quantity were assessed by the Agilent DNA1000 Chip (Agilent, CA) and qPCR (Kappa Biosystems Inc, MA). 10 pM of each library was sequenced using Illumina SBS chemistry at 2 x 100 bp reads on the HiSeq2000 (Illumina®, CA). The RNA-Seq paired-end reads were mapped to the mouse mm9 genome using an in-house mapping and quality assessment pipeline. The expression of each gene was estimated by the mean coverage of the highest-covered coding exon. Genes with low expression level (<10) across all samples were filtered out, followed by quantile normalization. Differential expression analysis was performed using limma with estimation of false-discovery rate. Gene Set Enrichment Analysis was used to assess pathway enrichment. Results:Transcriptome (RNA-Seq) and Taqman quantitative real-time PCR analyses revealed a ~50% reduction in the expression of multiple genes and of the long intergenic non-coding RNA 503142E22Rik within the 5A3 interval in mutant HSC and MPP. Gene Set Enrichment Analysis (GSEA) of the RNA-Seq data from 5A3+/del HSCs further demonstrated reduced expression of multiple gene sets related to oxidative phosphorylation (OXPHOS) that are similarly down-regulated in the early stages of human therapy-induced MDS and AML. Conclusions: Our study revealed that genes involved in OXPHOS were down-regulated in 5A3+/del HSC, and this finding provided novel insights into the impact of chromosome 7 deletions on the pathogenesis of MDS. Transcriptome profiling (RNA-seq) data of HSC and MPP isolated from 5A3+/del mice and their wildtype littermates were generated by sequencing, in quintuplicate, using Illumina SBS chemistry at 2 x 100 bp reads on the HiSeq2000 (Illumina®, CA)

Project description:Disrupting mutations of the RUNX1 gene are found in 10% of patients with myelodysplasia (MDS) and 30% of patients with acute myeloid leukemia (AML). Previous studies have revealed an increase in hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells in conditional Runx1-knockout (KO) mice, but the molecular mechanism is unresolved. We investigated the myeloid progenitor (MP) compartment in KO mice, arguing that disruptions at the HSC/MPP level may be amplified in downstream cells. We demonstrate that the MP compartment is increased more than fivefold in Runx1 KO mice, with a prominent skewing toward megakaryocyte (Meg) progenitors. Runx1- deficient granulocyte-macrophage progenitors are characterized by increased cloning capacity, impaired development into mature cells, and HSC and Meg transcription signatures. An HSC/MPP subpopulation expressing Meg markers was also increased in Runx1-deficient mice. Rescue experiments coupled with transcriptome analysis and Runx1 DNA-binding assays demonstrated that commitment is marked by Runx1 suppression of genes encoding adherence and motility proteins (Tek, Jam3, Plxnc1, Pcdh7, and Selp) that support HSCâMeg interactions with the BM niche. In vitro assays confirmed that enforced Tek expression in HSCs/MPPs increases Meg output. Interestingly, besides this key repressor function of Runx1 to control lineage decisions and cell numbers in progenitors, our study also revealed a critical activating function in erythroblast differentiation, in addition to its known importance in Meg and granulocyte/monocyte (G/M) maturation. Thus both repressor and activator functions of Runx1 at multiple hematopoietic stages and lineages likely contribute to the tumor suppressor activity in MDS and AML. GMP were isolated either from Runx1+/+-Tg(vav-Cre) and Runx1fl/fl-Tg(vav-Cre) mice or from mice transplanted with Runx1fl/fl-Tg(vav-Cre) progenitors engineered to express GFP with RUNX1-ERt2 or ERt2. RUNX1-ERt2 activity was induced by i.p. injection of tamoxifen on 3 consecutive days prior to GMP isolation.

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Functional Evidence Implicating Chromosome 7q22 Haploinsufficiency in Myelodysplastic Syndrome Pathogenesis

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