The identification of mitochondrial DNA variants in glioblastoma multiforme

Abstract

BackgroundMitochondrial DNA (mtDNA) encodes key proteins of the electron transfer chain (ETC), which produces ATP through oxidative phosphorylation (OXPHOS) and is essential for cells to perform specialised functions. Tumor-initiating cells use aerobic glycolysis, a combination of glycolysis and low levels of OXPHOS, to promote rapid cell proliferation and tumor growth. Glioblastoma multiforme (GBM) is an aggressively malignant brain tumor and mitochondria have been proposed to play a vital role in GBM tumorigenesis.ResultsUsing next generation sequencing and high resolution melt analysis, we identified a large number of mtDNA variants within coding and non-coding regions of GBM cell lines and predicted their disease-causing potential through in silico modeling. The frequency of variants was greatest in the D-loop and origin of light strand replication in non-coding regions. ND6 was the most susceptible coding gene to mutation whilst ND4 had the highest frequency of mutation. Both genes encode subunits of complex I of the ETC. These variants were not detected in unaffected brain samples and many have not been previously reported. Depletion of HSR-GBM1 cells to varying degrees of their mtDNA followed by transplantation into immunedeficient mice resulted in the repopulation of the same variants during tumorigenesis. Likewise, de novo variants identified in other GBM cell lines were also incorporated. Nevertheless, ND4 and ND6 were still the most affected genes. We confirmed the presence of these variants in high grade gliomas.ConclusionsThese novel variants contribute to GBM by rendering the ETC. partially dysfunctional. This restricts metabolism to anaerobic glycolysis and promotes cell proliferation.

Segregation of Naturally Occurring Mitochondrial DNA Variants in a Mini-Pig Model

“…Indeed, parallels can be drawn with undifferentiated tumors, where reduced OXPHOS and a greater reliance on glycolysis promotes aerobic glycolysis, i.e., the Warburg effect to mediate cell proliferation. As with developing tumors, which also harbor mtDNA variants (Yeung et al 2014), this would be advantageous for the developing embryo when its prime role is to divide to increase cellular number in an exponential manner (Cagnone et al 2011;Krisher and Prather 2013). Indeed, the use of OXPHOS inhibitors promotes cell proliferation in dividing preimplantation pig embryos (Machaty et al 2001).…”

“…Next-generation sequencing reactions were performed, as described (Sobinoff et al 2014;Yeung et al 2014). Briefly, purified long-PCR fragment pairs were combined at equal concentrations, prior to generation of the amplicon libraries.…”

“…Susceptibility was determined by normalizing the number of variants identified in each mtDNA region to the size of the region (in base pairs) on which the variant was located, as described (Yeung et al 2014). …”

“…Variant selection was also performed using CLC Genomics Workbench (Version 7.5), as described (Sobinoff et al 2014;Yeung et al 2014). For quality control, reads were filtered to exclude those of a nucleotide length of ,15 bp, with one nucleotide being trimmed from each end of all reads.…”

“…Reaction conditions were 94°for 2 min, 35 cycles of 94°for 15 sec, 63°f or 30 sec, and 68°for 8 min 45 sec. Products were purified using the Isolate II PCR kit (Bioline), as described in the manufacturer's instructions.Next-generation sequencing reactions were performed, as described (Sobinoff et al 2014;Yeung et al 2014). Briefly, purified long-PCR fragment pairs were combined at equal concentrations, prior to generation of the amplicon libraries.…”