Characterization of Genetic Abnormalities in MDS and Their Clinical Impact

MDS 遗传异常的特征及其临床影响

• 批准号: 8242386
• 负责人: Rafael Bejar
• 金额: $ 3.67万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-02 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8242386
• 关键词: Actins; Acute Myelocytic Leukemia; Acute leukemia; Advisory Committees; Affect; Area; Asses; Biological Assay; Biometry; Blast Cell; Blood; Blood Cells; Bone Marrow; CD34 gene; Cell physiology; Cells; Chromosome Deletion; Chromosome abnormality; Chromosomes; Clinic; Clinical; Clinical Oncology; Clonal Expansion; Coculture Techniques; Collaborations; Complementary DNA; DNA Primers; DNA Sequence; Development; Development Plans; Differentiation and Growth; Disease; Doctor of Philosophy; Dysmyelopoietic Syndromes; EZH2 gene; Ensure; Epigenetic Process; Erythroid; Functional RNA; Gene Mutation; Gene Targeting; Genes; Genetic; Genomics; Genotype; Goals; HL60; Head; Hematology; Hematopoiesis; Hematopoietic; Histones; Hospitals; Human; In Vitro; Indium-111; Institutes; Journals; Lead; Lesion; Mass Spectrum Analysis; Medicine; Mentors; Methods; Methylation; Minority; Modeling; Modification; Molecular Abnormality; Mutate; Mutation; Mutation Detection; Mutation Spectra; Myelogenous; Neutrophil Activation; New England; Normal Cell; North America; Oncogenes; Oncogenic; Pathogenesis; Patients; Peripheral; Phagocytosis; Phenotype; Physicians; Plant Roots; Play; Point Mutation; Primer Extension; Production; Public Health Schools; Publications; Publishing; Qualifying; RNA Interference; Recruitment Activity; Recurrence; Research; Research Personnel; Risk; Risk Factors; Role; Sampling; Scientist; Surveys; Syndrome; System; Techniques; Testing; Time; Tumor Suppressor Genes; Woman; Work; base; career; career development; chromosome 5q loss; clinical phenotype; clinically significant; cohort; cytopenia; design; experience; high throughput technology; histone modification; knock-down; medical schools; meetings; member; migration; molecular marker; neutrophil; next generation; novel; oncology; outcome forecast; polymerization; prognostic; programs; small hairpin RNA; tumor

DESCRIPTION (provided by applicant): The candidate, Dr. Rafael Bejar, presents a 5-year career development plan that seeks to characterize the broad range of mutations present in patients with myelodysplastic syndromes while establishing an academic career as a physician scientist in the field of hematology. The specific aims of this proposal are to: (1) study a clinically annotated cohort of 439 bone marrow samples from patients with MDS to determine if the mutations they carry are associated with common clinical features of MDS and overall patient survival, (2) perform a pooled shRNA screen targeting each of the genes from the commonly deleted regions of chromosome 5q to determine which genes, when knocked down in human CD34+ hematopoietic cells, lead to clonal expansion in a stromal co-culture system, and (3) functionally characterize EZH2 mutations identified in MDS patient samples to determine their effects on hematopoietic differentiation, clonal expansion, and mature cell function. Myelodysplastic syndromes are clonal disorders of hematopoiesis that cause inefficient blood cell production, low blood counts, and risk of progression to acute leukemia. The prognosis for patients with MDS is highly variable, ranging from weeks to years. Chromosomal abnormalities present in some cases help stratify patients into risk groups, but prognosis is largely determined by clinical parameters. MDS patients are known to have acquired DNA mutations in their diseased cells, however, these genetic lesions have yet to be incorporated into prognostic scoring systems. Molecular markers are needed to better classify subtypes of MDS, define prognostic risk, and identify pathogenic mechanisms associated with the development and progression of these disorders. There is abundant evidence that genes that are somatically mutated and functional in one tumor type can play a role in other tumor types. A comprehensive analysis of known cancer genes in MDS has not been performed. In order to identify oncogenic mutations in MDS, a set of DNA primer extension/mass spectroscopic assays will be used to determine which of 439 patient samples contain any of 953 mutations in 111 known cancer genes. Tumor suppressor genes known to be mutated in MDS and other myeloid disorders will be sequenced in this cohort using the 454 quantitative next-generation sequencing platform. These highly sensitive techniques allow for detection of mutations even if samples contain a large fraction of normal cells or mutations are present in only a small subclone of diseased cells. With the help of collaborator Donna Neuberg and her team, a statistical analysis will be performed to determine if mutations are correlated with MDS phenotypes and the overall survival of patients even after known risk factors are considered. In collaboration with Dr. David Root from the RNAi Consortium at the Broad Institute, a pooled short hairpin RNA interference screen in human CD34+ hematopoietic cells will be performed. Genes from regions of chromosome 5q that are commonly deleted in patients with MDS will be targeted to determine which confer a clonal advantage when knocked down. Finally, the functional role of MDS associated EZH2 mutations will be examined in human hematopoietic assays. These include tests of clonal expansion, differentiation, histone methylation, and mature cell functions such as neutrophil activation, migration, phagocytosis, and actin polymerization. Dr. Bejar is well qualified to carry out the research outlined in this proposal. He has successfully completed a project of comparable complexity as part of his PhD thesis. His mentor, Dr. Benjamin Ebert, has experience studying the genetic basis of myelodysplastic syndromes and has published the result of short hairpin RNA interference screen that discovered RPS14 as a critical gene lost in patients with the 5q-minus syndrome subtype of MDS. Along with Dr. Ebert, Dr. Bejar has published an invited review on the genetic basis of MDS in Hematology/Oncology Clinics of North America and a review on pathogenic mechanisms in MDS in the Journal of Clinical Oncology. The preliminary work described in this proposal has been accepted for publication in the New England Journal of Medicine. Dr. Bejar has recruited a team of outstanding mentors that that in addition to Dr. Ebert, include Dr. David Williams as a co-mentor, Dr. David Steensma as a clinical mentor, and Dr. Nancy Berliner, head of the Hematology division at Brigham and Women's Hospital, as a fourth member of his advisory committee. The advisory committee will meet, at minimum, every six months to ensure a successful scientific research program. Included in the career development plan are courses on biostatistics and genomic methods at the Harvard Medical School and School of Public Health. Successful completion the specific aims and career development plan outlined in this proposal will allow the candidate to advance his academic career as an independent investigator in the field of hematology. PUBLIC HEALTH RELEVANCE: The goals of this project are to identify genetic mutations associated with the development and clinical manifestations of myelodysplastic syndromes (MDS) and to model their effects on hematopoietic growth and differentiation in vitro. To accomplish these goals, we will perform: (1) a survey of mutations in known cancer genes in a large cohort of MDS patient samples with detailed clinical annotation, (2) a pooled shRNA screen to identify genes from the commonly deleted portion of chromosome 5q, that when knocked down, provide a clonal advantage to hematopoietic cells, and (3) functionally characterize the mechanisms through which mutations in the EZH2 gene contribute to the development and progression of MDS. We anticipate that the results of these studies will identify novel genetic lesions and pathogenic mechanisms that can molecularly classify subtypes of MDS, helping to predict survival of patients with these disorders.

描述（由申请人提供）：候选人Rafael Bejar博士提出了一项为期5年的职业发展计划，旨在描述骨髓增生异常综合征患者中存在的广泛突变，同时建立血液学领域医生科学家的学术生涯。这项建议的具体目标是：（1）研究来自MDS患者的439个骨髓样品的临床注释组群，以确定它们携带的突变是否与MDS的常见临床特征和总体患者存活率相关，（2）进行靶向来自染色体5 q的通常缺失区域的每个基因的汇集的shRNA筛选，以确定哪些基因，当在人CD 34+造血细胞中被敲低时，导致基质共培养系统中的克隆扩增，和（3）功能性表征在MDS患者样品中鉴定的EZH 2突变，以确定它们对造血分化、克隆扩增和成熟细胞功能的影响。骨髓增生异常综合征是造血系统的克隆性疾病，其导致血细胞生成效率低下、血细胞计数低和进展为急性白血病的风险。MDS患者的预后是高度可变的，从数周到数年不等。在某些情况下，染色体异常有助于将患者分为风险组，但预后主要取决于临床参数。已知MDS患者在其病变细胞中具有获得性DNA突变，然而，这些遗传病变尚未纳入预后评分系统。需要分子标记物来更好地分类MDS亚型，确定预后风险，并确定与这些疾病的发展和进展相关的致病机制。大量证据表明，在一种肿瘤类型中发生体细胞突变并发挥功能的基因可以在其他肿瘤类型中发挥作用。尚未对MDS中已知的癌症基因进行全面分析。为了鉴定MDS中的致癌突变，将使用一组DNA引物延伸/质谱分析来确定439个患者样本中的哪些包含111个已知癌症基因中的953个突变中的任何一个。将使用454定量下一代测序平台对该队列中已知在MDS和其他骨髓疾病中突变的肿瘤抑制基因进行测序。这些高度敏感的技术允许检测突变，即使样品含有大部分正常细胞或突变仅存在于患病细胞的一小部分亚克隆中。在合作者Donna Neuberg和她的团队的帮助下，将进行统计分析，以确定突变是否与MDS表型和患者的总生存率相关，即使在考虑了已知的风险因素后。与Broad Institute RNAi Consortium的大卫·鲁特博士合作，将在人CD 34+造血细胞中进行合并短发夹RNA干扰筛选。来自MDS患者中通常缺失的染色体5 q区域的基因将被靶向，以确定当敲除时赋予克隆优势。最后，MDS相关EZH 2突变的功能作用将在人类造血测定中进行检查。这些包括克隆扩增、分化、组蛋白甲基化和成熟细胞功能（如中性粒细胞活化、迁移、吞噬作用和肌动蛋白聚合）的测试。Bejar博士完全有资格开展本提案中概述的研究。他成功地完成了一个相当复杂的项目，作为他博士论文的一部分。他的导师Benjamin Ebert博士具有研究骨髓增生异常综合征遗传基础的经验，并发表了短发夹RNA干扰筛选的结果，发现RPS 14是MDS 5 q-减综合征亚型患者中丢失的关键基因。Bejar博士沿着Ebert博士在《北美血液学/肿瘤学临床》上发表了关于MDS遗传基础的特邀综述，并在《临床肿瘤学杂志》上发表了关于MDS致病机制的综述。本提案中描述的初步工作已被接受，将在《新英格兰医学杂志》上发表。Bejar博士招募了一个由杰出导师组成的团队，除了Ebert博士之外，还包括大卫威廉姆斯博士作为共同导师，大卫斯汀斯玛博士作为临床导师，以及布莱根妇女医院血液科主任南希柏林博士作为他的咨询委员会的第四名成员。咨询委员会将至少每六个月举行一次会议，以确保科学研究计划的成功。职业发展计划中包括在哈佛医学院和公共卫生学院学习生物统计学和基因组方法课程。成功完成本提案中概述的具体目标和职业发展计划将使候选人能够作为血液学领域的独立研究者推进其学术生涯。 公共卫生相关性：该项目的目标是确定与骨髓增生异常综合征（MDS）的发展和临床表现相关的基因突变，并模拟其对体外造血生长和分化的影响。为了实现这些目标，我们将：（1）在具有详细临床注释的一大群MDS患者样品中调查已知癌症基因的突变，（2）合并的shRNA筛选以鉴定来自染色体5 q的通常缺失部分的基因，当敲除时，其为造血细胞提供克隆优势，和（3）功能性地表征EZH 2基因中的突变有助于MDS的发生和进展的机制。我们预计，这些研究的结果将确定新的遗传病变和致病机制，可以对MDS亚型进行分子分类，有助于预测这些疾病患者的生存率。