Yit, Pua Jing
(2022)
Evaluation of genetic alterations of
IDH1, TP53 and CASP9 genes as
biomarkers in glioma patients.
Masters thesis, Universiti Sains Malaysia.
Abstract
Isocitrate dehydrogenase isoform 1 (IDH1) hotspot mutations are commonly
found in gliomas, especially in low-grade gliomas and secondary glioblastoma. The
IDH1 p.R132H (c.395G>A) gathered a significant association with clinical outcomes
in previous literature. The p53 pathway is usually dysregulated in most of the gliomas.
Recent clinical reports showed that glioma patients have the germline mutation of the
CASP9 gene and has been observed that the loss of CASP9 occurs in conjunction with
several tumor-suppressor genes. The development of gliomas are primarily influenced
by a number of significant genetic alterations. However, only a small number of genes
were examined in depth. The TP53 and IDH1 genes were noted as having mutations
most frequently observed in gliomas. CASP9 gene mutation described in recent
clinical reports has illustrated their potential contribution to the gliomagenesis. One of
these genes may be a predictive biomarker that may be applied in laboratory settings.
To improve early mutation detection for IDH1 hotspot mutation, this study developed
tetra primer amplification refractory mutation system polymerase chain reaction (TARMS
PCR) assay as a sensitive and specific laboratory testing method to accelerate
the process of early screening for glioma. This study identified a novel biomarker in
glioma patients through the screening of genetic alteration analysis in all the coding
exons in IDH1, TP53 and CASP9 genes. A total of thirty-six (n=36) specimens of hair
follicles, blood, and glioma tissue were recruited and analysed using Sanger
sequencing. Genetic alterations analysis of the genes of interest were confirmed using
Chi-square test and Fisher’s exact test, and bioinformatics analysis from open source databases such as The Cancer Genome Altas (TCGA) and the Chinese Glioma
Genome Atlas (CGGA). For the development of the T-ARMS PCR assay, a total of
sixty-one (n=61) glioma specimens were collected from the patients to determine their
clinical applicability. This study suggested CASP9 p.Q221R (Ex 5 +32G>A) as a novel
prognostic biomarker for glioma patients other than the current biomarker of IDH1
p.R132H. A follow-up study was conducted through a semi-quantitative determination
of CASP9 protein expression from the glioma tissue and showed a significant
difference in the CASP9 protein expression (p=0.024) in the patient group with mutant
CASP9 p.Q221R as compared to the wildtype. This study also reported T-ARMS PCR
assay is an early screening method with a sensitivity, specificity, and accuracy of 100%
(95% CI: 87.94-100.00%), 93.94% (95% CI: 80.39-98.92%) and 96.72%, respectively,
and the F1 score of 0.966. A forest plot analysis was used to compare the performance
of the T-ARMS PCR assay developed in this study with other published assays. In
short, this study demonstrated CASP9 p.Q221R as a novel biomarker for glioma
patients besides the current biomarker IDH1 p.R132H and hypothesized to be
associated with the survival of glioma patients. The performance of the T-ARMS PCR
assay was comparable to the gold standard and could be adapted for preoperative or
intraoperative diagnosis as a sensitive and specific screening tool.
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