TERT promoter mutation analysis in glioma samples by allele-specific biochip hybridization

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Abstract

Somatic mutations in the promoter of the telomerase reverse transcriptase gene TERT can cause reactivation of the telomerase enzyme, which stimulates neoplastic processes in the body. C228T and C250T mutations of the TERT gene promoter (TERTp) are most often found in brain gliomas, for which they are important diagnostic and prognostic markers. To detect TERTp mutations, an approach involving amplification of the promoter region and subsequent hybridization with immobilized probes on a biological microarray (biochip) has been developed. Using this approach, the mutational status of TERTp in 94 glioma samples (astrocytoma, oligodendroglioma, glioblastoma) was investigated. To verify the genotyping results, we used data from Illumina platform targeting sequencing and Sanger direct sequencing. In total, TERTp mutations were detected in 62 of 94 samples (66%), most commonly in patients with glioblastoma (71%). The C228T mutation (69%) was significantly more frequent compared to the C250T mutation (31%). The results of biochip validation on a collection of clinical samples show that it can be used as a convenient and reliable diagnostic tool in genetic analysis of CNS tumors.

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About the authors

V. O. Varachev

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tanased06@rambler.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

I. O. Barinova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tanased06@rambler.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

О. Yu. Susova

N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of the Russian Federation

Email: tanased06@rambler.ru
Russian Federation, Kashirskoe shosse 23, Moscow, 115478

A. A. Mitrofanov

N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of the Russian Federation

Email: tanased06@rambler.ru
Russian Federation, Kashirskoe shosse 23, Moscow, 115478

S. A. Surzhikov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tanased06@rambler.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

I. V. Grechishnikova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tanased06@rambler.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

А. S. Zasedatelev

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tanased06@rambler.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

А. V. Chudinov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tanased06@rambler.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

Т. V. Nasedkina

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Author for correspondence.
Email: tanased06@rambler.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

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2. Fig. 1. Structure of the TERT gene promoter. The C>T nucleotide substitution occurs at position –146 (C250T) or at position –124 (C228T) before the translation initiation site (ATG), which leads to the formation of new binding sites for transcription factors of the E-26 family (ETS, E-26 transformation-specific); 1 – transcription initiation site, 2 – coding sequence of the TERT gene. The formation of new binding sites for transcription factors leads to an increase in the level of mRNA transcription by 2–4 times.

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3. Fig. 2. Detection of mutations in the TERT gene promoter in glial tumor samples: the upper part of the figure shows the hybridization patterns on the biochip, the lower part – the normalized values ​​of the fluorescence signals. (a) – sample with the wild-type genotype; (b – sample with the C250T mutation in the heterozygous state; (c – sample with the C228T mutation in the heterozygous state. Fluorescent signals of the biochip cells were recorded using a biochip analyzer with excitation of 760 ± 25 nm and recording of 810–830 nm (Cy7). M – cell containing the fluorescent dye Cy5, wt – wild type, mut – mutation.

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4. Fig. 3. Determination of mutations in the TERT gene promoter by direct Sanger sequencing. The wild-type sequence is at the top, the sequence with the mutation in the heterozygous state is at the bottom. (a) – Mutation C250T, (b) – mutation C228T.

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