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，指出可能的致病途径。然而，使用传统的中期细胞遗传学（MC），染色体病变只能在约50%的患者中发现；更高分辨率的方法可以检测正常和异常MC患者的隐性染色体病变。检测这些缺陷可以通过解释已建立的MDS亚型的表型异质性，促进识别新的治疗靶点，帮助诊断和改善当前的预后方案。单核苷酸多态性阵列（SNP-A）是一种高分辨率核型和检测不平衡DNA缺陷（包括体细胞单亲二体）的新方法。我们已经证明体细胞UPD在MDS中非常常见，并假设UPD的共享区域可能指向参与MDS发展的基因突变。体细胞UPD的特定区域与双等位基因突变的存在有关，如Jak2 （UPD9p）和Ftl3 （UPD13q）。基于这一理论，我们在一大群MDS患者中绘制了不变的UPD和缺失图谱，发现UPD11q在慢性髓细胞白血病患者中尤为常见。通过对c-Cbl基因（该染色体区域的E3泛素连接酶）进行测序，发现了涉及该基因关键部分的突变。在本课题中，我们计划测试SNP-A核型的临床应用，并确定其对MDS预后、临床表型和新分子靶点发现的影响。在SA1中，我们将使用Affymetrix 6.0 SNP阵列分析MDS患者的成对样本（骨髓和作为克隆对照的CD3+细胞）。新的不变克隆病变，特别是体细胞UPD，将被识别、绘制并分析其对临床表型、生存和进展的影响。新发现病变的影响将在IPSS的背景下进行评估，以提高预后的准确性。在SA2中，作为复发性体细胞UPD指向重要突变的概念的证明，我们将详细分析UPD11q和c-Cbl突变患者，并确定与该病变相关的该突变的频率和临床结果。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c-Cbl突变是否介导了病理机制。例如，我们将比较无名指突变敲入与c-Cbl蛋白敲入的功能后果。利用SNP-A染色体组型改进细胞遗传学诊断有助于更好地了解MDS的分子发病机制，并具有鉴定MDS发病机制相关基因的潜力，可能为开发分子疗法指明治疗靶点。临床上，基于snp的核型可以补充细胞遗传学诊断，并且通过检测先前的隐性染色体病变（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.