Dataset Information

Treatment-associated TP53 DNA-binding domain missense mutations in the pathogenesis of secondary gliosarcoma.

ABSTRACT: Gliosarcoma is a rare variant of glioblastoma (GBM) that exhibits frequent mutations in TP53 and can develop in a secondary fashion after chemoradiation of a primary GBM. Whether temozolomide (TMZ)-induced mutagenesis of the TP53 DNA-binding domain (DBD) can drive the pathogenesis of gliosarcoma is unclear.We identified a case of a primary GBM that rapidly progressed into secondary gliosarcoma shortly after chemoradiation was initiated. Bulk tumor was collected and gliomasphere cultures derived from both the pre- and post-treatment tumors. We performed targeted DNA sequencing and transcriptome analyses of the specimens to understand their phylogenetic relationship and identify differentially expressed gene pathways. Gliomaspheres from the primary GBM were treated with TMZ and then analyzed to compare patterns of mutagenesis in vivo and ex vivo.The pre- and post-treatment tumors shared EGFR, CDKN2A, and PTEN mutations, but only the secondary gliosarcoma exhibited TP53 DBD missense mutations. Two mutations, R110C, and R175H, were identified, each in distinct clones. Both were base transitions characteristic of TMZ mutagenesis. Gene expression analysis identified increased JAK-STAT signaling in the gliosarcoma, together with reduced expression of microRNAs known to regulate epithelial-mesenchymal transition. Ex vivo treatment of the GBM spheres with TMZ generated numerous variants in cancer driver genes, including TP53 and CDH1, which were mutated in the post-treatment tumor.TMZ-induced TP53 gain-of-function mutations can have a driving role in secondary gliosarcoma pathogenesis. Analysis of variants identified in ex vivo TMZ-treated gliomaspheres may have utility in predicting GBM evolutionary trajectories in vivo during standard chemoradiation.

SUBMITTER: Pain M

PROVIDER: S-EPMC5788663 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Treatment-associated <i>TP53</i> DNA-binding domain missense mutations in the pathogenesis of secondary gliosarcoma.

Pain Margaret M Wang Huaien H Lee Eunjee E Strahl Maya M Hamou Wissam W Sebra Robert R Zhu Jun J Yong Raymund L RL

Oncotarget 20171220 2

<h4>Background</h4>Gliosarcoma is a rare variant of glioblastoma (GBM) that exhibits frequent mutations in <i>TP53</i> and can develop in a secondary fashion after chemoradiation of a primary GBM. Whether temozolomide (TMZ)-induced mutagenesis of the <i>TP53</i> DNA-binding domain (DBD) can drive the pathogenesis of gliosarcoma is unclear.<h4>Methods</h4>We identified a case of a primary GBM that rapidly progressed into secondary gliosarcoma shortly after chemoradiation was initiated. Bulk tumor ...[more]

PMID: 29416795

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Project description:BACKGROUND: We have developed an oligonucleotide microarray (genosensor) utilizing a double tandem hybridization technique to search for 9 point mutations located in the most frequently altered codons of the TP53 gene. Isolated and multiplexed PCR products, 108 and 92 bp long, from exons 7 and 8, respectively, were obtained from 24 different samples. Single-stranded target DNA was then prepared from isolated or multiplexed PCR products, through cyclic DNA synthesis. Independent ssDNA's were annealed with the corresponding pairs of labeled stacking oligonucleotides to create partially duplex DNA having a 7-nt gap, which contains the sequence that will be interrogated by the capture probes forming double tandem hybridization. In the case of multiplexed ssPCR products, only two stacking oligonucleotides were added per target, therefore the gap for the PCR products having two consecutive codons to be interrogated in exon 7 was 12 nt long, so only single tandem hybridization was produced with these respective probes. RESULTS: 18 codon substitutions were found by DNA sequencing. In 13 of them a perfect correlation with the pattern of hybridization was seen (In 5 no signal was seen with the wt probe while a new signal was seen with the appropriate mutant probe, and in 8 more, as expected, no signal was seen with any probe due to the absence of the corresponding probe in the array). In 3 other cases a mutation was falsely suggested by the combination of the absence of the wild type signal along with a false signal in the other probe. In the other 2 cases the presence of the mutation was not detected due to the production of a false hybridization signal with the wild type probe. In both cases (false mutation or no mutation detected) relatively stable mismatched target/probe duplexes should be formed. These problems could be avoided by the addition of probes to improve the performance of the array. CONCLUSION: Our results demonstrate that a simple TP53 microarray employing short (7-mer) probes, used in combination with single or double tandem hybridization approach and a simple or multiplex target preparation method, can identify common TP53 missense mutations from a variety of DNA sources with good specificity.

Project description:Maturity-onset diabetes of the young type 3 (MODY3) is a non-ketotic form of diabetes associated with poor insulin secretion. Over the past years, several studies have reported the association of missense mutations in the Hepatocyte Nuclear Factor 1 Alpha (HNF1A) with MODY3. Missense mutations in the POU homeodomain (POUH) of HNF1A hinder binding to the DNA, thereby leading to a dysfunctional protein. Missense mutations of the HNF1A were retrieved from public databases and subjected to a three-step computational mutational analysis to identify the underlying mechanism. First, the pathogenicity and stability of the mutations were analyzed to determine whether they alter protein structure and function. Second, the sequence conservation and DNA-binding sites of the mutant positions were assessed; as HNF1A protein is a transcription factor. Finally, the biochemical properties of the biological system were validated using molecular dynamic simulations in Gromacs 4.6.3 package. Two arginine residues (131 and 203) in the HNF1A protein are highly conserved residues and contribute to the function of the protein. Furthermore, the R131W, R131Q, and R203C mutations were predicted to be highly deleterious by in silico tools and showed lower binding affinity with DNA when compared to the native protein using the molecular docking analysis. Triplicate runs of molecular dynamic (MD) simulations (50ns) revealed smaller changes in patterns of deviation, fluctuation, and compactness, in complexes containing the R131Q and R131W mutations, compared to complexes containing the R203C mutant complex. We observed reduction in the number of intermolecular hydrogen bonds, compactness, and electrostatic potential, as well as the loss of salt bridges, in the R203C mutant complex. Substitution of arginine with cysteine at position 203 decreases the affinity of the protein for DNA, thereby destabilizing the protein. Based on our current findings, the MD approach is an important tool for elucidating the impact and affinity of mutations in DNA-protein interactions and understanding their function.

Dataset Information

Treatment-associated TP53 DNA-binding domain missense mutations in the pathogenesis of secondary gliosarcoma.

Publications

Treatment-associated <i>TP53</i> DNA-binding domain missense mutations in the pathogenesis of secondary gliosarcoma.

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