Gene expression and splicing alterations analyzed by high throughput RNA sequencing of chronic lymphocytic leukemia specimens

BMC Cancer

Research Article

Matteo Pelegrini¹  Ryan T. Phan²  Phillipp Nham²  Gwen Jordaan³  Wei Liao³  Sanjai Sharma⁴ 

[1] Department of Molecular, Cell and Developmental Biology, UCLA, Los Angeles, CA, USA;Department of Pathology, VA Greater Los Angeles Healthcare System, Los Angeles, CA, USA;Division of Hematology-Oncology, UCLA-VA Greater Los Angeles Healthcare System, Los Angeles, CA, USA;Division of Hematology-Oncology, UCLA-VA Greater Los Angeles Healthcare System, Los Angeles, CA, USA;UCLA West Los Angeles VA Medical Center, 11301 Wilshire Blvd, Bldg 304, Rm E1-115, 90073, Los Angeles, CA, USA;

关键词: CLL;    RNA-sequencing;    Differential gene expression;    Alternative splicing;   

DOI  :  10.1186/s12885-015-1708-9

 received in 2014-11-09, accepted in 2015-10-07,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundTo determine differentially expressed and spliced RNA transcripts in chronic lymphocytic leukemia specimens a high throughput RNA-sequencing (HTS RNA-seq) analysis was performed.MethodsTen 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.ResultsAn 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).ConclusionThe 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.

【 授权许可】

CC BY   
© Liao et al. 2015