Charting the differentiation topology of SF3B1 mutated clonal hematopoiesis (CH) and myelodysplastic syndromes (MDS) via a multi-omics single-cell toolkit

摘要 对数千名MDS患者的基因组分析证实，RNA剪接因子的突变是 MDS患者中最常见的一类基因改变。同时，功能研究也有 研究表明，其中几个遗传损伤似乎推动了异常的造血自我更新和 差异化是MDS的特征。具体地说，剪接因子3b亚单位1(SF3B1)、a 核心剪接体成分，是MDS患者中最常见的，并导致错误的内含子 分支点识别。尽管取得了这些进展，但我们对这种基因改变对 在实际的疾病起始细胞内的下游基因表达程序受到了 正常野生型造血与携带体细胞驱动因子的异常克隆共存 突变。虽然这些限制中的一些现在开始用单细胞基因组学来解决， 执行分层基因组分析以同时捕获体细胞突变、基因表达、RNA 剪接，在单个细胞中染色质状态从未被执行过。RNA结合蛋白的表达是 反过来，依赖于细胞类型，需要同时分析基因表达，全长cdna和 单细胞水平的SF3B1突变状态，允许在适当的细胞环境中检查剪接。 为了应对这一挑战，我们开发了一系列多组体单细胞技术，能够 获取多层信息(例如，基因类型、转录本、甲基组、蛋白质表达) 同样的单细胞。此外，我们还解决了单细胞scrna-seq基因分型的特殊挑战。 通过发展转录本基因分型(GOT)获得高通量的细胞。重要的是，得到了转弯 突变型和野生型混合造血从限制到优势，使直接 同一个体内突变细胞和野生型细胞的比较。 利用来自MDS和CH患者的独特的骨髓样本队列，我们现在的目标是 应用和扩展多组学单细胞工具包以测试定义SF3B1体细胞突变如何导致克隆 增长优势。首先，我们将使用规范的SF3B1驱动程序执行跨MDS和CH样本的GET 突变。我们将通过测序将GOT与转录和表位的细胞索引(CITE- SEQ)(GOT-CITE)，将细胞表面标记的关键层添加到单细胞整个转录本中。第二, 剪接因子的突变与CH转化的风险更大有关。因此，我们 我将开发和实施GET-Splice，其中将使用长读测序来定义剪接变异 作为细胞身份的函数。第三，鉴于表观遗传模式对造血干细胞的高度重要性 细胞鉴定，我们将在染色质可及性的背景下开发和应用有针对性的单细胞基因分型 (GOT-CHA)。这将使我们能够解开SF3B1驱动的CH和MDS的监管基础。

SUMMARY Genomic analyses of thousands of MDS patients has established that mutations in RNA splicing factors are the most common class of genetic alterations in patients with MDS. In parallel, functional studies have revealed that several of these genetic lesions appear to drive aberrant hematopoietic self-renewal and differentiation that is characteristic of MDS. Specifically, mutations in splicing factor 3b subunit 1(SF3B1), a core spliceosome component, are among the most common in patients with MDS and lead to incorrect intronic branch point recognition. Despite these advances, our knowledge of the effects of this genetic alteration on downstream gene expression programs within actual disease initiating cells has been hampered by the coexistence of normal wildtype hematopoiesis together with the aberrant clone harboring somatic driver mutations. While some of these limitations are now beginning to be addressed with single cell genomics, performing a layered genomic analysis to simultaneously capture somatic mutations, gene expression, RNA splicing, and chromatin state in single cells has never been performed. Expression of RNA-binding proteins is in turn cell type dependent, necessitating the simultaneously profiling of gene expression, full-length cDNA and SF3B1 mutational status at the single cell level, allowing splicing to be examined in the proper cellular context. To address this challenge, we developed an array of multi-omic single-cell technologies that are capable of capturing multiple layers of information (e.g., genotypes, transcriptomes, methylomes, protein expression) from the same single cells. Moreover, we addressed the specific challenge of genotyping in scRNA-seq in single cells at high throughput by developing Genotyping of Transcriptomes (GoT). Importantly, GoT turns the admixture of mutant and wildtype hematopoiesis from a limitation to an advantage, enabling the direct comparison of mutant and wildtype cells within the same individual. Capitalizing on a unique cohort of bone marrow samples from individuals with MDS and CH, we now aim to apply and extend the multi-omics single-cell toolkit to test define how SF3B1 somatic mutations lead to clonal growth advantage. First, we will perform GoT across MDS and CH samples with canonical SF3B1 driver mutations. We will integrate GoT with Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE- seq) (GoT-CITE), to add the critical layer of cell surface markers to single-cell whole transcriptomes. Second, mutations in splicing factors are specifically associated with greater risk of transformation in CH. Therefore, we will develop and implement GoT-Splice, where long-read sequencing will be used to define splicing variation as a function of cell identity. Third, given the high importance of epigenetic patterning to hematopoietic stem cell identity, we will develop and apply targeted single-cell genotyping in the context of chromatin accessibility (GoT-ChA). This will allow us to unravel the regulatory underpinnings of SF3B1-driven CH and MDS.