Long-read sequencing of SF3B1 mutated and wildtype chronic lymphocytic leukemia and myelodysplastic syndrome to identify mechanisms of genomic instability and therapy resistance and their impact on prognosis.

慢性淋巴细胞白血病（CLL）是西方成人中最常见的白血病。CLL中最常见的突变基因之一是剪接因子SF3B1，它与CLL的不良预后相关。SF3B1的突变已被证明可诱导剪接改变，并与DNA损伤反应的改变相关，进而影响染色体的稳定性。有趣的是，SF3B1也在骨髓增生异常综合征（MDS）中反复突变，MDS是一种造血干细胞疾病。然而，与CLL相比，MDS中的SF3B1突变与良好的预后相关。本研究的总体目标是分析SF3B1突变对CLL中RNA剪接、重复扩增和基因组稳定性的影响。为了回答这些问题，我们将使用PacBio的单分子实时测序（SMRT）进行长读测序。我们将对SF3B1和健康b细胞突变和野生型cll样本的RNA进行isoseq测序。结合我们在CRU286项目中的短读测序分析，生物信息学分析将能够改进SF3B1突变样本剪接异构体的检测（Aim 1）。此外，我们计划对SF3B1突变和野生型MDS样本与健康造血干细胞进行Iso-seq，比较MDS和CLL中受SF3B1突变影响的同种异构体。这将使我们能够发现SF3B1突变的CLL和MDS病例之间的差异，这可能是导致预后差异的原因（目的2）。此外，除了Iso-seq，我们计划进行基因组DNA的长读测序，以确定结构变异。我们对CRU286短读RNA-seq数据的分析表明，在CLL中，重复RNA在一般情况下显着去调控，但在SF3B1突变病例中也特别如此。这一点，再加上SF3B1和细胞应激反应之间的联系，使得基因组水平上的改变非常有可能通过长读测序来识别。总的来说，拟议的项目将理想地补充我们的功能分析和（epi）基因组数据集从CRU286项目。这将有助于破译SF3B1突变在CLL和MDS中的功能后果，从而可能有助于了解肿瘤进展的机制。

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the Western world. One of the most frequently mutated genes in CLL is the splicing factor SF3B1, which is associated with an adverse outcome in CLL. Mutations in SF3B1 have been shown to induce splicing alterations and are associated with alterations of the DNA damage response, which in turn influences chromosomal stability. Interestingly, SF3B1 is also recurrently mutated in myelodysplastic syndrome (MDS), a disease of the hematopoietic stem cell. However, in contrast to CLL, SF3B1 mutations in MDS are associated with a favorable prognosis. The overall goal of this study is to analyze the effect of SF3B1 mutations on RNA splicing, repeat expansion and genomic stability in CLL. To answer these questions, we will employ long-read sequencing using single molecule real time sequencing (SMRT) by PacBio. We will perform Isoform sequencing of RNA (Iso-seq) of CLL-samples mutated and wildtype for SF3B1 and of healthy B-cells. Bioinformatics analyses will enable an improved detection of splicing isoforms in SF3B1 mutated samples in conjunction with our analyses with short-read sequencing in the CRU286 project (Aim 1). In addition, we plan to perform Iso-seq of MDS samples mutated and wildtype for SF3B1 and healthy hematopoietic stem cells and compare the isoforms affected by mutated SF3B1 mutations in MDS and CLL. This will enable us to uncover differences between SF3B1 mutated CLL and MDS cases which might be responsible for the differences in prognosis (Aim 2).Furthermore, besides Iso-seq we plan to perform long-read sequencing of genomic DNA to identify structural variations. Our analyses of the CRU286 short-read RNA-seq data have shown that repetitive RNA is significantly de-regulated in CLL in general, but also specifically in SF3B1 mutated cases. This, together with the connection between SF3B1 and the cellular stress response, renders alterations on a genomic level very likely which will be possible to identify with long-read sequencing. Taken together, the proposed project will ideally complement our functional analyses and (epi)genomic data sets from the CRU286 project. It will help to decipher the functional consequences of SF3B1 mutations in CLL and MDS and thereby may give insight in mechanisms of tumor progression.