ALS-Associated KIF5A Mutations Abolish Autoinhibition Resulting in a Toxic Gain of Function [RNA-seq]

Purpose: The goal of this study was to identify gene expression changes in iMNs containing a patient KIF5a mutation compared to an isogenic control. Further, splicing changes were evaluated in the mutant vs WT sample. Methods: RNA samples were extracted and sequenced on an Illumina NovaSeq6000 instrument at the Yale Center for Genome Analysis. The sample sequence quality control check was performed using FastQc. The STAR aligner program was used for read mapping with reference file and HTSeq was used to count the reads for each transcript in strand specific mode. The Differential expression analysis was performed using the DESeq2 package to compare the gene expression profiles between wild type and mutant was performed using the Wald test to generate p-values and Log2 fold changes. The STAR aligned RNA Sequence sample bam files were used to detect differential alternative splicing events with the Multivariate Analysis of Transcript Splicing (MATS) method. Pathway analysis was performed with Metascape Results: With the RNA Sequence pipeline, we analyzed the RNA Sequence from wild-type parental line (Iso Control) and the heterozygous mutant line (KIF5AR1007K). The RNA Sequence analysis of the iMNs revealed 57 genes displaying altered expression. Using the method, Multivariate Analysis of Transcript Splicing (MATS), we identified 1,919 transcripts with altered splicing compared to the isogenic control iMNs. Of these, 1,000 exons showed decreased skipping, while 919 showed increased skipping in KIF5AR1007K lines. Pathway analysis of each of these groups suggests that this group of altered genes, as a whole, represents cytoskeletal and transport defects in the cell, both of which are hallmarks of neurodegenerative disease. Overall design: mRNA profiles of DIV15 iMNs harboring a KIF5AR1007K mutation and its isogenic control

研究目的：本研究旨在鉴定相较于同基因对照（isogenic control），携带患者来源KIF5a突变的诱导运动神经元（iMNs）中的基因表达变化，并进一步评估突变样本与野生型（WT）样本间的可变剪接差异。研究方法：本研究于耶鲁大学基因组分析中心使用Illumina NovaSeq6000测序平台对提取的RNA样本进行测序。采用FastQC软件完成测序序列的质量控制；使用STAR比对工具结合参考基因组文件完成读段比对，随后采用HTSeq软件以链特异性模式统计每个转录本的读段计数。使用DESeq2软件包进行差异表达分析，以比较野生型与突变样本的基因表达谱，并通过Wald检验生成P值及Log₂倍变化值。此外，基于STAR比对得到的RNA测序BAM文件，采用转录剪接多元分析（Multivariate Analysis of Transcript Splicing，MATS）方法检测差异可变剪接事件。路径富集分析通过Metascape工具完成。研究结果：本研究通过RNA测序分析流程，对野生型亲本细胞系（同基因对照，Iso Control）及杂合突变细胞系（KIF5AR1007K）的RNA测序数据进行分析。对诱导运动神经元（iMNs）的RNA测序分析显示，共有57个基因存在表达量异常。采用转录剪接多元分析（MATS）方法，本研究共鉴定出1919个与同基因对照iMNs相比存在剪接异常的转录本，其中1000个外显子的外显子跳跃事件减少，919个外显子的外显子跳跃事件在KIF5AR1007K细胞系中增加。对上述两组异常基因的路径富集分析结果显示，该类异常基因整体上与细胞的细胞骨架及物质运输缺陷相关，而这两类缺陷均为神经退行性疾病的典型特征。实验设计：携带KIF5AR1007K突变的DIV15诱导运动神经元（iMNs）及其同基因对照的mRNA表达谱。