Determination of genetic aberrations and novel transcripts involved in the pathogenesis of oligodendroglioma using array comparative genomic hybridization and next generation sequencing.

Abstract

The aim of the present study was to determine the genetic aberrations and novel transcripts, particularly the fusion transcripts, involved in the pathogenesis of low-grade and anaplastic oligodendroglioma. In the present study, tissue samples were obtained from patients with oligodendroglioma and additionally from archived tissue samples from the Brain Tumor Tissue Bank of the Brain Tumor Foundation of Canada. Six samples were obtained, three of which were low-grade oligodendroglioma and the other three anaplastic oligodendroglioma. DNA and RNA were extracted from each tissue sample. The resulting genomic DNA was then hybridized using the Agilent CytoSure 4×180K oligonucleotide array. Human reference DNA and samples were labeled using Cy3 cytidine 5′-triphosphate (CTP) and Cy5 CTP, respectively, while human Cot-1 DNA was used to reduce non-specific binding. Microarray-based comparative genomic hybridization data was then analyzed for genetic aberrations using the Agilent Cytosure Interpret software v3.4.2. The total RNA isolated from each sample was mixed with oligo dT magnetic beads to enrich for poly(A) mRNA. cDNAs were then synthesized and subjected to end-repair, poly(A) addition and connected using sequencing adapters using the Illumina TruSeq RNA Sample Preparation kit. The fragments were then purified and selected as templates for polymerase chain reaction amplification. The final library was constructed with fragments between 350–450 base pairs and sequenced using deep transcriptome sequencing on an Illumina HiSeq 2500 sequencer. The array comparative genomic hybridization revealed numerous amplifications and deletions on several chromosomes in all samples. However, the most interesting result was from the next generation sequencing, where one anaplastic oligodendroglioma sample was demonstrated to have five novel fusion genes that may potentially serve a critical role in tumor pathogenesis and progression.