Lab-Pediatrics Laboratory Research

Abstract

Chromosomal rearrangements resulting in novel fusion genes are among the most prevalent form of genetic alterations known in cancer, and numerous examples exist in both adult and childhood malignancies. To date, however, none have been reported in pediatric high-grade glioma (pHGG), so we have undertaken to search for novel structural rearrangements using three distinct techniques. Firstly, we took a candidate approach and screened a series of 83 pHGG for the fusion previously described in adult glioblastoma between PDGFRA and KDR (VEGFR2) at 4q12. Using RT-PCR and sequencing, we identified the second reported instance of KDR:PDGFRA in a single case of glioblastoma (age 1.2 years). Next, we applied the iCNA algorithm to identify copy number aberrations with intragenic breakpoints using Affymetrix 500K SNP data from a cohort of 100 pHGG, identifying two candidates arising from genomic amplification and intrachromosomal rearrangement in an analogous mechanism to KDR:PDGFRA. We finemapped the breakpoints using custom Agilent oligonucleotide arrays and characterized the fusions DHX57:MAP4K3 (2p22) and CSGALNACT2:RET (10q11) in cases of anaplastic astrocytoma (2 years) and recurrent glioblastoma (12.8 years), respectively. Finally, we sequenced the entire genomes of five pediatric glioma cell lines at .30× coverage using the Illumina HiSeq2000 platform, and screened for rearrangements using the BreakDancer (BD) package. We identified a median of 165 intragenic structural variants per genome that were filtered based on BD confidence score, number and orientation of reads and by visual inspection using IGV software. Candidate fusions being systematically validated and screened in our pHGG cohorts include interchromosomal rearrangements resulting in TULP4:RPTOR (t6;17 -SF188), GORASP2:CDADC1 (t2;13 -KNS42) and C15ORF57:CBX3 (t15;7 -UW479). These data highlight the presence of hitherto unrecognized fusion genes in pHGG which may play important roles in the unique biology of the tumors as well as provide excellent candidates for novel therapeutic strategies. Recently, we and others identified the first recurrent somatic mutations in a histone gene (H3F3A) in one-third of pediatric glioblastoma (pedGBM), the most devastating brain tumor in childhood. The observed mutations in H3F3A, coding for the non-canonical histone variant H3.3, led to amino acid substitutions at two critical residues of the histone tail (K27M and G34R), at or near sites of important post-translational modifications. Furthermore, we have shown that pedGBMs carrying K27M or G34R mutations are characterized by distinct gene expression and DNA methylation profiles. This clearly shows that different signalling pathways predominate in K27M or G34R mutated tumors. To address the functional impact of these different H3.3 mutations in more detail, we generated GBM cell lines stably expressing either K27M or G34R mutated H3.3. K27M mutated cells show a reduction of trimethylated H3K27 (H3K27me3) as indicated by western blot. Using immunohistochemistry on a tiss...

Integrative omics data analysis uncovers biomarker genes and potential candidate drugs for G3 medulloblastoma

“…Moreover, MB patients with MYC gene amplification tend to have a worse survival rate than other patients 40 . COL3A1, COL1A1, and COL1A2 are also important biomarkers and play a pre-growth role 41 . Elevated levels of PPARG have been found in the clinical samples of MB 42 .…”