Detection of the IDH1/2 gene mutations in tumor samples with low abundance of the mutant allele

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Abstract

Identification of driver mutations in tumors is an extremely important task in oncology for the choice of treatment strategy and assessment of therapy efficacy. In many cases, especially in disease monitoring, there is a need to detect a small number of copies of the mutant allele against the background of excessive content of wild-type DNA. In this work we investigated the possibilities of highly sensitive detection of mutations in IDH1 and IDH2 genes at suppression of wild-type DNA amplification using oligomers of “locked” nucleic acid (LNA) with subsequent hybridization of fluorescently labeled polymerase chain reaction (PCR) product on a biological microchip (biochip). The limit of detection of mutant DNA is 0.1% in the wild-type DNA background. The effectiveness of this approach is demonstrated by analyzing 26 samples of chondroid tumors and glial brain tumors with low representation of the mutant allele, previously undetected mutations R132C, R132L and R132H were identified in three cases.

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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

D. A. Guskov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

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

O. Y. 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. P. Shekhtman

Russian Children’s Clinical Hospital

Email: tanased06@rambler.ru
Russian Federation, Leninsky prosp. 117, Moscow, 119117

D. V. Rogozhin

Russian Children’s Clinical Hospital

Email: tanased06@rambler.ru
Russian Federation, Leninsky prosp. 117, Moscow, 119117

S. A. Surzhikov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

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

A. V. Chudinov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

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

A. S. Zasedatelev

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

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

T. 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. Suppression of amplification of the wild-type R132 IDH1 allele in a sample containing ~50% of the mutant R132H allele at different concentrations of LNA oligomer. (a) – Amplification curves of the wild-type R132 allele and the mutant R132H allele: wild-type (wt) allele – curves 1 (0 nM), 2 (50 nM) and 3 (100 nM); mutant allele (R132H) – curves 4 (0 nM), 5 (50 nM) and 6 (100 nM); (b) – difference in the threshold cycle values ​​of the amplification curves of the wild-type (wt) and mutant allele (R132H) allele at different concentrations of LNA compared to zero concentration.

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3. Fig. 2. Analysis of melting curves with a TaqMan probe for suppression of the amplification of the wild-type allele R132 IDH1 (wt) in a sample containing 15–20% of the mutant allele (R132H) at different concentrations of LNA oligomer: 1 – wild-type sample (0 nM); samples with the R132H mutation – curves 2 (0 nM), 3 (50 nM), and 4 (100 nM). An increase in the R132H peak is noticeable with increasing LNA oligomer concentration.

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4. Fig. 3. Detection of the R132H IDH1 mutation in a sample containing 0.5% of the mutant allele at different concentrations of the LNA oligomer. Hybridization patterns: (a) 0 nM LNA, (b) 50 nM LNA, (c) 100 nM LNA; (d) normalized values ​​of fluorescent signals.

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5. Fig. 4. Detection of mutations in the IDH1 gene in tumor samples (chondrosarcoma) with a low content of the mutant allele. The upper part of the figure shows the hybridization patterns on the biochip, the lower part – the normalized values ​​of the fluorescence signals. Sample with the R132C mutation: (a) – 0 nM LNA oligomer in the PCR reaction, (b) – with the addition of 100 nM LNA oligomer. Sample with the R132L mutation: (c) – 0 nM LNA oligomer in the PCR reaction, (d) – with the addition of 100 nM LNA oligomer. Wells with the fluorescent dye Cy5, which act as a marker, are located in the corners of the biochip.

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