Characterization of Genetic Abnormalities in MDS and Their Clinical Impact

描述（由申请人提供）：候选人Rafael Bejar博士提出了一个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的内科科学家的学术生涯。这项建议的具体目的是：(1)对来自MDS患者的439份骨髓样本进行临床注释队列研究，以确定其携带的突变是否与MDS的常见临床特征和总体患者生存率相关；(2)对5q染色体常见缺失区域的每个基因进行shRNA筛选，以确定在人CD34+造血细胞中敲低哪些基因会导致基质共培养系统中的克隆扩增。(3)对MDS患者样本中发现的EZH2突变进行功能表征，确定其对造血分化、克隆扩增和成熟细胞功能的影响。骨髓增生异常综合征是一种克隆性造血障碍，可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患者的预后变化很大，从几周到几年不等。在某些情况下，染色体异常有助于将患者分为危险组，但预后在很大程度上取决于临床参数。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生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引物扩展/质谱分析来确定439例患者样本中哪些包含111个已知癌症基因中的953个突变。已知在MDS和其他髓系疾病中发生突变的肿瘤抑制基因将在该队列中使用454下一代定量测序平台进行测序。这些高度敏感的技术允许检测突变，即使样品中含有大部分正常细胞或突变仅存在于一小部分病变细胞亚克隆中。在合作者Donna Neuberg和她的团队的帮助下，即使考虑了已知的危险因素，也将进行统计分析，以确定突变是否与MDS表型和患者的总体生存率相关。我们将与Broad研究所RNAi联盟的David Root博士合作，对人类CD34+造血细胞进行短发夹RNA干扰筛选。MDS患者中通常缺失的染色体5q区域的基因将被定位，以确定哪些基因在被敲除时赋予克隆优势。最后，MDS相关的EZH2突变的功能作用将在人类造血分析中进行检查。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、吞噬和肌动蛋白聚合）的测试。Bejar博士完全有资格进行本建议中概述的研究。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，并发表了短发卡RNA干扰筛查的结果，发现RPS14是MDS患者5q-综合征亚型中缺失的关键基因。Bejar博士与Ebert博士一起在北美血液学/肿瘤学诊所发表了一篇关于MDS遗传基础的特邀综述，并在《临床肿瘤学杂志》上发表了一篇关于MDS致病机制的综述。本提案中描述的初步工作已被接受发表在《新英格兰医学杂志》上。Bejar博士已经招募了一个杰出的导师团队，除了Ebert博士之外，还包括David Williams博士作为联合导师，David Steensma博士作为临床导师，以及Nancy Berliner博士（Brigham and Women's Hospital血液学部门负责人）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召开一次会议，以确保科学研究项目的成功。职业发展计划包括哈佛医学院和公共卫生学院的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允许候选人作为血液学领域的独立研究者推进他的学术生涯。

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.