Functional Evidence Implicating Chromosome 7q22 Haploinsufficiency in Myelodysplastic Syndrome Pathogenesis

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)研究背景：5A3+/del小鼠的生殖系为杂合型缺失，缺失区域为小鼠5号染色体带5A3上一段2 Mb的区间，该区间与人类7号染色体q22区段的常见缺失区域同源。该小鼠会出现造血干细胞（hematopoietic stem cell, HSC）异常，这可能参与骨髓增生异常综合征（myelodysplastic syndrome, MDS）的发病进程，且此类缺陷具有细胞自主性。本研究旨在对比从5A3+/del小鼠及其野生型同窝对照中分离得到的造血干细胞（HSC）与多能祖细胞（multipotent progenitors, MPP）的转录组测序（RNA-seq）数据，以鉴定可能与该表型相关的差异表达基因及信号通路。 实验方法：从野生型及5A3+/del小鼠的CD150高表达造血干细胞（CD150hi-HSC）、CD150低表达造血干细胞（CD150lo-HSC）以及CD150阴性多能祖细胞（CD150neg-MPP）中分离总RNA。取10 ng总RNA，使用Ovation RNA扩增系统V2（加利福尼亚州NuGen公司）反转录为双链cDNA；随后以扩增得到的cDNA产物为模板，使用TruSeq RNA样本制备试剂盒v2（加利福尼亚州Illumina公司），经10轮聚合酶链式反应（PCR）扩增构建RNA-seq文库。采用安捷伦DNA1000芯片（加利福尼亚州安捷伦公司）与实时定量PCR（qPCR，马萨诸塞州Kappa生物系统公司）评估文库的质量与浓度。取10 pM的各文库，在HiSeq2000测序仪（加利福尼亚州Illumina公司）上采用Illumina边合成边测序（SBS）化学技术进行2×100 bp读长的双端测序。使用自研的比对与质量评估流程，将RNA-seq双端读段比对至小鼠mm9参考基因组。以每个基因覆盖度最高的编码外显子的平均覆盖度估算其表达量；过滤所有样本中表达量低于10的基因，随后进行分位数标准化。采用limma软件包进行差异表达分析，并估算错误发现率（false-discovery rate, FDR）；采用基因集富集分析（Gene Set Enrichment Analysis, GSEA）评估信号通路的富集情况。 实验结果：转录组测序（RNA-seq）与Taqman实时定量PCR分析显示，突变型HSC与MPP中，5A3缺失区间内的多个基因以及长链基因间非编码RNA 503142E22Rik的表达量均下降约50%。对5A3+/del小鼠HSC的RNA-seq数据进行基因集富集分析（GSEA）进一步发现，多个与氧化磷酸化（oxidative phosphorylation, OXPHOS）相关的基因集表达下调，而此类基因集在人类治疗相关性骨髓增生异常综合征与急性髓系白血病（acute myeloid leukemia, AML）的早期阶段同样呈现表达下调。 研究结论：本研究发现，5A3+/del小鼠的HSC中参与氧化磷酸化（OXPHOS）的基因表达下调，该发现为7号染色体缺失对骨髓增生异常综合征（MDS）发病进程的影响提供了全新的研究视角。本研究通过HiSeq2000测序仪（加利福尼亚州Illumina公司）采用Illumina边合成边测序（SBS）化学技术，以2×100 bp读长对从5A3+/del小鼠及其野生型同窝对照中分离得到的HSC与MPP的转录组测序（RNA-seq）数据进行了5次生物学重复测序。