Detection of BRAF, NRAS, KIT, GNAQ, GNA11 and MAP2K1/2 mutations in Russian melanoma patients using LNA PCR clamp and biochip analysis
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Marina Emelyanova1, Lilit Ghukasyan1, Ivan Abramov1,2, Oxana Ryabaya2, Evgenia Stepanova2, Anna Kudryavtseva1,3, Asiya Sadritdinova1,3, Cholpon Dzhumakova2, Tatiana Belysheva2, Sergey Surzhikov1, Lyudmila Lyubchenko2, Alexander Zasedatelev1,2 and Tatiana Nasedkina1,2
1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russian Federation
2Blokhin Cancer Research Center, Ministry of Health of the Russian Federation, Moscow, Russian Federation
3P. Hertsen Moscow Oncology Research Institute, Moscow, Russian Federation
Marina Emelyanova, email: firstname.lastname@example.org
Keywords: biochip, somatic mutations, melanoma, diagnostic tool, targeted therapy
Received: December 30, 2016 Accepted: March 30, 2017 Published: April 10, 2017
Target inhibitors are used for melanoma treatment, and their effectiveness depends on the tumor genotype. We developed a diagnostic biochip for the detection of 39 clinically relevant somatic mutations in the BRAF, NRAS, KIT, GNAQ, GNA11, MAP2K1 and MAP2K2 genes.
We used multiplex locked nucleic acid (LNA) PCR clamp for the preferable amplification of mutated over wild type DNA. The amplified fragments were labeled via the incorporation of fluorescently labeled dUTP during PCR and were hybridized with specific oligonucleotides immobilized on a biochip. This approach could detect 0.5% of mutated DNA in the sample analyzed. The method was validated on 253 clinical samples and six melanoma cell lines.
Among 253 melanomas, 129 (51.0%) BRAF, 45 (17.8%) NRAS, 6 (2.4%) KIT, 4 (1.6%) GNAQ, 2 (0.8%) GNA11, 2 (0.8%) MAP2K1 and no MAP2K2 gene mutations were detected by the biochip assay. The results were compared with Sanger sequencing, next generation sequencing and ARMS/Scorpion real-time PCR. The specimens with discordant results were subjected to LNA PCR clamp followed by sequencing. The results of this analysis were predominantly identical to the results obtained by the biochip assay. Infrequently, we identified rare somatic mutations.
In the present study we demonstrate that the biochip-based assay can effectively detect somatic mutations in approximately 70% of melanoma patients, who may require specific targeted therapy.
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