Molecular Pathogenesis of MDS and CMML

描述（由申请人提供）：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），染色体病变可以发现只有约50%的患者，更高的分辨率的方法可以允许检测隐匿性染色体病变的正常和异常MC的患者。这些缺陷的检测可以促进新的治疗靶点的鉴定，通过解释已建立的MDS亚型内的表型异质性来辅助诊断和改善当前的预后方案。 单核苷酸多态性阵列（SNP-A）是一种高分辨率染色体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，体细胞UPD是非常常见的MDS和假设，共享区域的UPD可能指向突变的基因参与MDS的发展。特别是体细胞UPD的区域已经与双等位基因突变的存在相关联，例如Jak 2（α 9 p）和Ftl 3（α 13 q）。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，并发现在慢性粒单核细胞白血病患者中，UPD 11 q尤其常见。通过对c-Cbl基因（该染色体区域中的E3泛素连接酶）进行测序，鉴定了涉及该基因关键部分的突变。 在这个提议中，我们计划测试SNP-A核型分析的临床效用，并确定其对MDS预后、临床表型和新分子靶点发现的影响。在SA 1中，我们将使用Affyandroid 6.0 SNP阵列分析MDS患者的配对样本（骨髓和分选的CD 3+细胞作为克隆对照）。新的不变克隆病变，特别是体细胞UPD，将被确定，映射和其对临床表型，生存和进展的影响进行分析。将在IPSS的背景下评估新检测到的病变的影响，以提高预后准确性。在SA 2中，作为复发性体细胞UPD指向重要突变的概念证明，我们将详细分析具有c-Cbl和c-11 q突变的患者，并确定与该病变相关的该突变的频率和临床结局。我们将进行体外研究，以描述c-Cbl突变对酪氨酸激酶受体和Src家族激酶的影响，并解释病理机制是否由突变c-Cbl介导。例如，我们将比较环指突变体敲入与c-Cbl蛋白敲低的功能后果。 使用SNP-A核型分析改进的细胞遗传学诊断可能有助于更好地理解MDS的分子发病机制，并具有鉴定MDS发病机制中涉及的基因的潜力，可能为分子疗法的发展指明治疗靶点。在临床上，SNP-A为基础的核型分析可以补充细胞遗传学诊断，并通过检测以前隐藏的染色体病变（包括UPD），可以更好地预后分配。

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.