Establishment and Validation of the Detection of TERT Promoter Mutations by Human Gliomas U251 Cell Lines

Gliomas are the most common type of primary brain tumor, yet the prognosis for glioma patients remains poor. Mutations in the promoter region of the telomerase reverse transcriptase gene (TERTp) are associated with diagnosis and poor prognosis in gliomas. Here, we developed a precise and rapid Sange...

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Main Authors: Huili Bai, Shunjie Bai, Xiaosong Li, Yangli Zhang, Ying Li, Fang He, Wei Cheng
Format: Article
Language:English
Published: Hindawi Limited 2021-01-01
Series:BioMed Research International
Online Access:http://dx.doi.org/10.1155/2021/3271395
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spelling doaj-9e3c86adffb54df9902443eb699060ba2021-06-14T00:16:46ZengHindawi LimitedBioMed Research International2314-61412021-01-01202110.1155/2021/3271395Establishment and Validation of the Detection of TERT Promoter Mutations by Human Gliomas U251 Cell LinesHuili Bai0Shunjie Bai1Xiaosong Li2Yangli Zhang3Ying Li4Fang He5Wei Cheng6The Center for Clinical Molecular Medical DetectionDepartment of Clinical LaboratoryThe Center for Clinical Molecular Medical DetectionThe Center for Clinical Molecular Medical DetectionThe Center for Clinical Molecular Medical DetectionDepartment of pharmacyThe Center for Clinical Molecular Medical DetectionGliomas are the most common type of primary brain tumor, yet the prognosis for glioma patients remains poor. Mutations in the promoter region of the telomerase reverse transcriptase gene (TERTp) are associated with diagnosis and poor prognosis in gliomas. Here, we developed a precise and rapid Sanger sequencing assay to screen or TERTp mutations. We established the Sanger sequencing approach for the detection of TERTp mutations based on human glioma cell lines U251 and assessed the analytical validation by determining the accuracy, sensitivity, precision, and specificity. In our study, we verified the accuracy of Sanger sequencing by the real-time polymerase chain reaction method. Our data showed that TERTp mutations were detected at an analytical sensitivity of 10% per mutant. The precision and specificity validation also showed the desired results. In total, 147 glioma patients were investigated for TERTp mutations, and of each patient, clinical data and molecular characteristics were analyzed. We found that anaplastic oligodendroglioma had the highest frequency of TERTp mutations (66.7%). No differences in TERTp mutation frequency were observed between frozen tissue specimens and formalin-fixed and paraffin-embedded tissue. TERTp mutations were associated with older patients (≥45 years), whereas isocitrate dehydrogenase (IDH) mutations were inclined to a younger age (<45 years), frontal location, and pathologic stage II-III patients. IDH mutations were significantly associated with O6-methylguanine-DNA methyltransferase (MGMT) methylation (P=0.003) and lower Ki-67 protein expression (P=0.011). Moreover, MGMT methylation was enriched in IDH-mutant/TERTp-mutant gliomas, and Ki-67 protein expression was the highest in the IDH-wild type/TERTp-mutant group. Taken together, the findings of this study indicate the establishment of a rapid, precise, and practical Sanger sequencing technique for TERTp mutations in gliomas that may show promising results in clinical applications.http://dx.doi.org/10.1155/2021/3271395
collection DOAJ
language English
format Article
sources DOAJ
author Huili Bai
Shunjie Bai
Xiaosong Li
Yangli Zhang
Ying Li
Fang He
Wei Cheng
spellingShingle Huili Bai
Shunjie Bai
Xiaosong Li
Yangli Zhang
Ying Li
Fang He
Wei Cheng
Establishment and Validation of the Detection of TERT Promoter Mutations by Human Gliomas U251 Cell Lines
BioMed Research International
author_facet Huili Bai
Shunjie Bai
Xiaosong Li
Yangli Zhang
Ying Li
Fang He
Wei Cheng
author_sort Huili Bai
title Establishment and Validation of the Detection of TERT Promoter Mutations by Human Gliomas U251 Cell Lines
title_short Establishment and Validation of the Detection of TERT Promoter Mutations by Human Gliomas U251 Cell Lines
title_full Establishment and Validation of the Detection of TERT Promoter Mutations by Human Gliomas U251 Cell Lines
title_fullStr Establishment and Validation of the Detection of TERT Promoter Mutations by Human Gliomas U251 Cell Lines
title_full_unstemmed Establishment and Validation of the Detection of TERT Promoter Mutations by Human Gliomas U251 Cell Lines
title_sort establishment and validation of the detection of tert promoter mutations by human gliomas u251 cell lines
publisher Hindawi Limited
series BioMed Research International
issn 2314-6141
publishDate 2021-01-01
description Gliomas are the most common type of primary brain tumor, yet the prognosis for glioma patients remains poor. Mutations in the promoter region of the telomerase reverse transcriptase gene (TERTp) are associated with diagnosis and poor prognosis in gliomas. Here, we developed a precise and rapid Sanger sequencing assay to screen or TERTp mutations. We established the Sanger sequencing approach for the detection of TERTp mutations based on human glioma cell lines U251 and assessed the analytical validation by determining the accuracy, sensitivity, precision, and specificity. In our study, we verified the accuracy of Sanger sequencing by the real-time polymerase chain reaction method. Our data showed that TERTp mutations were detected at an analytical sensitivity of 10% per mutant. The precision and specificity validation also showed the desired results. In total, 147 glioma patients were investigated for TERTp mutations, and of each patient, clinical data and molecular characteristics were analyzed. We found that anaplastic oligodendroglioma had the highest frequency of TERTp mutations (66.7%). No differences in TERTp mutation frequency were observed between frozen tissue specimens and formalin-fixed and paraffin-embedded tissue. TERTp mutations were associated with older patients (≥45 years), whereas isocitrate dehydrogenase (IDH) mutations were inclined to a younger age (<45 years), frontal location, and pathologic stage II-III patients. IDH mutations were significantly associated with O6-methylguanine-DNA methyltransferase (MGMT) methylation (P=0.003) and lower Ki-67 protein expression (P=0.011). Moreover, MGMT methylation was enriched in IDH-mutant/TERTp-mutant gliomas, and Ki-67 protein expression was the highest in the IDH-wild type/TERTp-mutant group. Taken together, the findings of this study indicate the establishment of a rapid, precise, and practical Sanger sequencing technique for TERTp mutations in gliomas that may show promising results in clinical applications.
url http://dx.doi.org/10.1155/2021/3271395
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