Qualitative and Quantitative Analysis of IDH1 Mutation in Progressive Gliomas by Allele-Specific qPCR and Western Blot Analysis

Abstract

To date, diagnosis of IDH1 mutation is based on DNA sequencing and immunohistochemistry, methods limited in terms of sensitivity and ease of use. Recently, the diagnosis of IDH1 mutation by real-time polymerase chain reaction was introduced as an alternative method. In this study, real-time polymerase chain reaction was validated as a tool for detection of IDH1 mutation, and expression levels were analyzed for correlation with course of the disease. A total of 113 tumor samples were obtained intraoperatively from 84 patients with glioma having a diagnosis of diffuse glioma (World Health Organization II), anaplastic glioma (World Health Organization III), secondary glioblastoma ± chemotherapy, primary glioblastoma ± chemotherapy (World Health Organization IV). Tumor samples were snap frozen and processed for sectioning and RNA and protein isolation. Presence of IDH1 mutation was determined by DNA sequencing. Hereafter, quantitative expression of IDH1 messenger RNA was assessed using real-time polymerase chain reaction with specific primers for IDH1 mutation and –wt; protein expression was verified by Western Blot analysis and immunohistochemistry. Additionally, 19 samples of low-grade glioma and their consecutive high-grade glioma were analyzed at different time points of the disease. IDH1 mutation was identified in 63% of samples by DNA sequencing. In correlation with the real-time polymerase chain reaction results, a cutoff value was determined. Above this threshold, sensitivity and specificity of real-time polymerase chain reaction in detecting IDH1 mutation were 98% and 94%, respectively. Quantitative analysis revealed that IDH1 mutation expression is upregulated in secondary glioblastoma (mean ± standard error of mean: 3.52 ± 0.55) compared to lower grade glioma (II = 1.54 ± 0.22; III = 1.67 ± 0.23). In contrast, IDH1 wt expression is upregulated in all glioma grades (concentration >0.1) compared to control brain tissue (0.007 ± 0.0016). Western Blot analysis showed a high concordance to both sequencing and real-time polymerase chain reaction results in qualitative analysis of IDH1 mutation status (specificity 100% and sensitivity 100%). Moreover, semiquantitative protein expression analysis also showed higher expression levels of mutated IDH1 in secondary glioblastoma. In our study, real-time polymerase chain reaction and Western Blot analysis were found to be highly efficient methods in detecting IDH1 mutation in glioma samples. As cost-effective and time-saving methods, real-time polymerase chain reaction and Western Blot analysis may therefore play an important role in IDH1 mutation analysis in the future. IDH1 mutation expression level was found to correlate with the course of disease to a certain extent. Yet, clinical factors as recurrent disease or prior radiochemotherapy did not alter IDH1 mutation expression level.

Energy Metabolism in Cancer: The Roles of STAT3 and STAT5 in the Regulation of Metabolism-Related Genes

“…Aberrant TCA cycle function is implicated in a wide variety of pathological processes such as obesity. Moreover, several TCA cycle enzymes are deregulated in cancer (Figure 3) [63][64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80][81][82]. tumour cells, which require a continuous supply of small metabolites for the synthesis of proteins, nucleic acids, and lipids.…”

“…Aberrant TCA cycle function is implicated in a wide variety of pathological processes such as obesity. Moreover, several TCA cycle enzymes are deregulated in cancer (Figure 3) [63][64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80][81][82]. The electron transport chain (ETC) controls combustion, which generates energy to be used to drive the oxidation of NADH, which produces three ATP molecules and FADH2 (which produces two ATP molecules) at the expense of reducing oxygen.…”

Antidepressant effects of total iridoids of Valeriana jatamansi via the intestinal flora-blood–brain barrier pathway

“…Western blotting was used as described (Perrech et al. 2019 ) to measure ZO-1 and occludin protein levels. The brain tissue was homogenised on ice until fully lysed, and protein concentration was quantified using a BCA kit.…”

MiR-30c-5p/ATG5 Axis Regulates the Progression of Parkinson’s Disease

“…Brain tissues or cells were collected for determining protein level (Perrech et al, 2019 ). Tissue homogenate or cell lysate in the RIPA lysis buffer was centrifuged at 4°C (15,000× g , 15 min).…”