Development of a sensitive, specific and cost-effective T-ARMS PCR assay for the genotyping of R132H of IDH1 gene in glioma patients

Abstract

The discovery of isocitrate dehydrogenase isoform 1 (IDH1) mutation as a key molecular marker has resulted in a change in glial tumour classification [1]. IDH1 mutations are commonly in gliomas, particularly in low-grade gliomas and secondary glioblastoma [2]. IDH1 p.R132H (c.395G>A) accounted for more than 90% of the mutation in IDH1/2 mutation and had a significant association with clinical outcomes. IDH1/2 mutations cause gain-of-function resulting in the formation of an oncometabolite, R-2-hydroxyglutarate instead of α-ketoglutarate implying a disruption of oxidative decarboxylation of Kreb’s cycle and other cellular mechanisms [3,4,5]. Immunohistochemistry (IHC) and Sanger sequencing is the most common approach used in molecular diagnostics. Nevertheless, both IHC and Sanger sequencing methods have shortcomings. IHC is prone to miss out on the mutation if the tumour samples are of poor quality, and it has also been reported to have low sensitivity when compared to sequencing-based techniques [6,7].
 
 This study aimed to develop a sensitive, specific and cost-effective assay for genotyping IDH1 p.R132H mutations in glioma patients in order to shorten the time required for confirmatory diagnosis to be made. The tetra primer amplification refractory mutation system polymerase chain reaction (T-ARMS PCR) was used in this study to develop and validate the clinical applicability of the assay. A total of 61 glial specimens were collected and genomic DNA was isolated from all of them. All the samples were subjected to endpoint PCR and Sanger sequencing for mutation detection. T-ARMS PCR was developed and optimized prior to the screening of all the samples, and comparative mutation analysis was carried out.
 Overall, IDH1 p.R132H mutation was found to be 45.90% (n=28/61) prevalent and was found to be significantly associated with gender, tumour subtypes and grading, and location of the lesions (p=<0.05). With an F1 score of 0.966, we reported T-ARMS PCR with sensitivity, specificity and accuracy of 100% (95% CI: 87.94-100.00%), 93.94% (95% CI: 80.39-98.92%) and 96.72%, respectively. To compare with published studies, a meta-analysis is T-ARMS PCR detected one case with IDH1 p.R132C (c.394C>T), howbeit the case could have a double mutation of p.R132C (c.394C>T) and p.R132H (c.395G>A) or single mutation of p.R132Y (c.394_395CG>TA). However, this study was able to detect IDH1 p.R132G (c.394C>G) mutation via PCR by Sanger sequencing whereas T-ARMS PCR excluded the mutation, suggesting the assay is very specific to IDH1 p.R132HH (c.395G>A) only.
 Hereby, the performance of the T-ARMS PCR assay sheds a light that can be adapted for preoperative or intraoperative diagnosis.