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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE196537 | GEO | 2022/02/17

REPOSITORIES: GEO

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Project description:Background: To determine differentially expressed and spliced RNA transcripts in chronic lymphocytic leukemia specimens a high throughput RNA-sequencing (HTS RNA-seq) analysis was performed. Methods: Ten CLL specimens and five normal peripheral blood CD19+ B cells were analyzed by HTS RNA-seq. The library preparation was performed with Illumina TrueSeq RNA kit and analyzed by Illumina HiSeq 2000 sequencing system. Results: An average of 48.5 million reads for B cells, and 50.6 million reads for CLL specimens were obtained with 10396 and 10448 assembled transcripts for normal B cells and primary CLL specimens respectively. With the Cuffdiff analysis, 2091 differentially expressed genes (DEG) between B cells and CLL specimens based on FPKM (fragments per kilobase of transcript per million reads and false discovery rate, FDR q<0.05, fold change >2) were identified. Expression of selected DEGs (n=32) with up regulated and down regulated expression in CLL from RNA-seq data were also analyzed by qRT-PCR in a test cohort of CLL specimens. Even though there was a variation in fold expression of DEG genes between RNA-seq and qRT-PCR; more than 90% of analyzed genes were validated by qRT-PCR analysis. Analysis of RNA-seq data for splicing alterations in CLL and B cells was performed by Multivariate Analysis of Transcript Splicing (MATS analysis). Skipped exon was the most frequent splicing alteration in CLL specimens with 128 significant events (P-value <0.05, minimum inclusion level difference >0.1). Conclusion: The RNA-seq analysis of CLL specimens identifies novel DEG and alternatively spliced genes that are potential prognostic markers and therapeutic targets. High level of validation by qRT-PCR for a number of DEG genes supports the accuracy of this analysis. Global comparison of transcriptomes of B cells, IGVH non-mutated CLL (U-CLL) and mutated CLL specimens (M-CLL) with multidimensional scaling analysis was able to segregate CLL and B cell transcriptomes but the M-CLL and U-CLL transcriptomes were indistinguishable. The analysis of HTS RNA-seq data to identify alternative splicing events and other genetic abnormalities specific to CLL is an added advantage of RNA-seq that is not feasible with other genome wide analysis. Ten CLL specimens and five normal peripheral blood CD19+ B cells were analyzed by HTS RNA-seq. The library preparation was performed with Illumina TrueSeq RNA kit and analyzed by Illumina HiSeq 2000 sequencing system.

Project description:MSI (Microsatellite Instability) colorectal cancer (CRC) show improved survival, are less prone to metastasis and show poor response to chemotherapy (compared to MSS tumors). The underlying reasons for these characteristics are still not understood and no specific therapeutic approach for MSI colon tumours (15% of CRC overall) has yet been developed. The MSI process is oncogenic when it affects DNA repeat sequences that have a functional role, e.g. Small Coding Repeats (SCR). MSI also frequently affects Long Non-Coding Repeats (LNCR) in tumour DNA. In contrast to SCR, only a few LNCR are endowed with biological activity. Consequently, this area has received very little attention. Our group recently identified HSP110 mutant chaperone protein in MSI CRC that was generated by somatic deletion of a LNCR. Of interest, HSP110 mutant (due to exon skipping) have anti-oncogenic properties and the survival of MSI CRC patients receiving chemotherapy is positively associated with HSP110 mutations in tumour DNA. The aim of the current project is to identify additional clinically relevant MSI-associated splicing aberrations due to mutations in LNCR located in splice acceptor sites. The four main steps are as follows: 1. To identify exon/intron sites affected by aberrant splicing events due to MSI in CRC . All RNASeq data will be exploited to identify recurrent splicing aberrations (mostly exon skipping) that occur specifically in MSI colon tumours; 2. To investigate for possible functional links between MSI and any detected aberrant splicing events . All specific aberrant splicing events detected by RNAseq in MSI CRC samples will be first confirmed (quantitative RT-PCR) in order to eliminate false positive cases. For validated exon candidates, the allelic profiles of adjacent intronic LNCR will be analysed (PCR and fluorescence genotyping) in CRC cell lines and primary tumours (MSI and MSS), as well as in matching normal mucosa samples in order to assess their polymorphic status; 3. To identify splicing events and LNCR mutations with clinical relevance in MSI CRC patients . All LNCR with a confirmed role in gene splicing in MSI CRC will be analysed. The clinical relevance of candidate genes will be assessed using multivariate survival regression models for Relapse- Free Survival, with interaction terms (response to chemotherapy); 4. To initiate functional studies on a limited number of clinically relevant, cancer-related genes whose splicing is perturbed in MSI cancer cells, and to develop biological tools to simplify screening in future clinical assays Similar to HSP110, we will focus on 4 or 5 mutant proteins that are promising drug therapeutic targets. Functional assays will be developed to further elucidate their role in the pathophysiology of MSI tumours. We also aim to develop biological tools for these candidate genes, such as the detection of wild-type or mutant proteins by immunohistochemistry.

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

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