Molecular Pathogenesis of MDS and CMML

描述(申请人提供)：在骨髓增生异常综合征(MDS)中，染色体异常起着重要的诊断和预后作用，指向可能的致病途径。然而，使用传统的中期细胞遗传学(MC)，只有大约50%的患者可以发现染色体病变；更高分辨率的方法可能允许在MC正常和异常的患者中检测到隐蔽的染色体病变。检测这些缺陷有助于识别新的治疗靶点，辅助诊断，并通过解释已建立的MDS亚型的表型异质性来改善当前的预后方案。单核苷酸多态阵列(SNP-A)是一种用于高分辨率核型分析和检测包括体细胞单亲二体(UPD)在内的不平衡DNA缺陷的新方法。我们已经证明了躯体UPD在MDS中非常常见，并假设UPD的共同区域可能指向参与MDS发展的基因的突变。体细胞UPD的特定区域与双等位基因突变的存在有关，例如JAK2(UPD9p)和Ftl3(UPD13q)。基于这一理论，我们在一大群MDS患者中定位了不变的UPD和缺失，发现UPD11q在慢性粒单核细胞白血病患者中尤其常见。通过对该染色体区域的E3泛素连接酶c-Cbl基因进行测序，发现了涉及该基因关键部分的突变。在这项建议中，我们计划测试SNP-A核型分析的临床实用性，并确定其对预后、临床表型和MDS新分子靶点发现的影响。在SA1中，我们将使用Affymetrix 6.0 SNP阵列分析来自MDS患者的配对样本(骨髓和分选的CD3+细胞作为克隆对照)。新的不变克隆性病变，特别是躯体UPD，将被识别、定位并分析它们对临床表型、生存和进展的影响。新发现的病变的影响将在IPSS的背景下进行评估，以提高预后准确性。在SA2中，作为复发的躯体UPD指向重要突变的概念的证据，我们将详细分析UPD11q和c-Cbl突变的患者，并确定与这种病变相关的这种突变的频率和临床结果。我们将进行体外研究，以阐明c-Cbl突变对酪氨酸激酶受体和Src家族激酶的影响，并解释c-Cbl突变是否介导了病理机制。例如，我们将比较环指突变敲入与c-Cbl蛋白敲除的功能后果。使用SNP-A核型技术改进的细胞遗传学诊断方法可能有助于更好地了解MDS的分子发病机制，并有可能识别与MDS发病相关的基因，可能为分子治疗的发展指明治疗靶点。临床上，基于SNP-A的核型分析可以补充细胞遗传学诊断，并通过检测以前隐蔽的染色体病变(包括UPD)，可能允许更好的预后分配。

DESCRIPTION (provided by applicant): In myelodysplastic syndrome (MDS) chromosomal abnormalities play an important diagnostic and prognostic role, pointing towards possible disease-causing pathways. However, using traditional metaphase cytogenetics (MC), chromosomal lesions can be found in only about 50% of patients; higher resolution methods may allow for detection of cryptic chromosomal lesions in patients with normal and abnormal MC. Detection of such defects could facilitate identification of novel therapeutic targets, aid diagnosis and improve current prognostic schemes by explaining phenotypic heterogeneity within established MDS subtypes. Single nucleotide polymorphism arrays (SNP-A) are a new method for high resolution karyotyping and detection of unbalanced DNA defects including somatic uniparental disomy (UPD). We have demonstrated that somatic UPD is very frequent in MDS and hypothesized that shared regions of UPD may point towards mutations of genes involved in the development of MDS. In particular areas of somatic UPD have been linked to the presence of biallelic mutations, such as Jak2 (UPD9p) and Ftl3 (UPD13q). Based on this theory we mapped invariant UPD and deletions in a large group of patients with MDS and found that UPD11q was particularly frequent in patients with chronic myelomonocytic leukemia. By sequencing the c-Cbl gene, an E3 ubiquitin ligase in this chromosomal region, a mutation involving a critical part of this gene was identified. In this proposal we plan to test the clinical utility of SNP-A karyotyping and establish its impact on prognosis, clinical phenotype and the discovery of new molecular targets in MDS. In SA1 we will analyze paired samples (bone marrow and sorted CD3+ cells as a clonal control) from patients with MDS using the Affymetrix 6.0 SNP array. New invariant clonal lesions, in particular somatic UPD, will be identified, mapped and their impact on clinical phenotypes, survival and progression analyzed. The influence of newly detected lesions will be assessed in the context of IPSS to improve prognostic accuracy. In SA2, as a proof of concept that recurrent somatic UPD points towards important mutations, we will analyze in detail patients with UPD11q and c-Cbl mutations and determine the frequency and clinical outcomes of this mutation associated with this lesion. We will perform in vitro studies to delineate the effect of c-Cbl mutations on tyrosine kinase receptors and Src family kinases and explain whether pathologic mechanisms are mediated by mutant c-Cbl. For example, we will compare functional consequences of ring finger mutant knock-in vs. c-Cbl protein knockdown. Improved cytogenetic diagnostics using SNP-A karyotyping may contribute to a better understanding of the molecular pathogenesis of MDS and has the potential of identification of genes involved in the pathogenesis of MDS, perhaps pointing towards therapeutic targets for development of molecular therapies. Clinically, SNP-A-based karyotyping may complement cytogenetic diagnosis and, through the detection of previously cryptic chromosomal lesions (including UPD), may allow for better prognostic assignment.