Murthy Tushar, Paul Kiran V, Minella Alexander C, Pillai Manoj M
Driskill Graduate Program, Northwestern University, Chicago, IL, USA.
Yale Cancer Center, Yale University, New Haven, CT, USA.
Methods Mol Biol. 2019;1881:83-99. doi: 10.1007/978-1-4939-8876-1_7.
Mutational landscape of CLL is now known to include recurrent non-synonymous mutations in SF3B1, a core splicing factor. About 5-10% of newly diagnosed CLL harbor these mutations which are typically limited to HEAT domains in the carboxyl-terminus of the protein. Importantly, the mutations are not specific to CLL but also present in several unrelated clonal disorders. Analysis of patient samples and cell lines has shown the primary splicing aberration in SF3B1-mutant cells to the use of novel or "cryptic" 3' splice sites (3SS). Advances in genome-editing and next-generation sequencing (NGS) have allowed development of isogenic models and detailed analysis of changes to the transcriptome with relative ease. In this manuscript, we focus on two relevant methods to study splicing factor mutations in CLL: development of isogenic scalable cell lines and informatics analysis of RNA-Seq datasets.
目前已知慢性淋巴细胞白血病（CLL）的突变图谱包括核心剪接因子SF3B1中的复发性非同义突变。约5%-10%新诊断的CLL患者携带这些突变，这些突变通常局限于该蛋白羧基末端的HEAT结构域。重要的是，这些突变并非CLL所特有，在几种不相关的克隆性疾病中也存在。对患者样本和细胞系的分析表明，SF3B1突变细胞中的主要剪接异常是使用新的或“隐蔽的”3'剪接位点（3SS）。基因组编辑和下一代测序（NGS）技术的进步使得相对容易地构建同基因模型并详细分析转录组变化成为可能。在本手稿中，我们重点介绍两种研究CLL中剪接因子突变的相关方法：构建可扩展的同基因细胞系以及对RNA-Seq数据集进行信息学分析。
