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. 2022;14(2):41-49.
doi: 10.17691/stm2022.14.2.04. Epub 2022 Mar 28.

A Complex of Pyrosequencing-Based Methods for Detection of Somatic Mutations in Codons 600 and 601 of the BRAF gene

O P Dribnokhodova  1 , A S Esman  2 , V I Korchagin  2 , A Yu Bukharina  3 , E A Dunaeva  2 , G V Leshkina  4 , E V Borisova  5 , Ya A Voiciehovskaya  6 , A I Daoud  7 , V N Khlyavich  8 , K O Mironov  9
Affiliations

A Complex of Pyrosequencing-Based Methods for Detection of Somatic Mutations in Codons 600 and 601 of the BRAF gene

O P Dribnokhodova et al. Sovrem Tekhnologii Med. 2022.

Abstract

The aim of the study is to develop methods for the differentiation of mutations in the BRAF codon 600 and to increase the sensitivity of the K601E mutation detection.

Materials and methods: The nucleotide sequence of the BRAF codons 592-602 was identified using the PyroMark Q24 genetic analysis system. The mutations search in codon 600 was conducted using the 600-S primer in line with the following order of adding nucleotides: GCTGTCАTCTGCTAGCTAGAC (corresponding to nucleotides 1799-1786). The K601E mutation was detected using the 601-S primer in line with the following order of nucleotide addition: GCTACTCACTGTAG (corresponding to nucleotides 1801-1793). The analytical characteristics of the proposed methods for somatic mutations' detection were determined using dilutions of plasmid DNA samples containing the BRAF gene region without mutations or with one of the following mutations: V600E, V600R, V600K, V600M, and K601E. Validation was performed on 132 samples of biological material obtained from the thyroid nodules.

Results: The developed methods allow to determine 2% of the V600E or V600M mutations, 1% of the V600K and V600R mutations, and 3% of the K601E mutations in samples with high DNA concentration; it is also possible to confidently detect at least 5% of the mutant allele for all mutations in low concentration samples (less than 500 copies/PCR). During biological material testing, 53 samples with the V600E mutation were detected; the proportion of the mutant allele was 4.9-50.0%.

Conclusion: A complex of methods for determination of the nucleotide sequence of the BRAF codons 592-601 and the algorithm for testing samples and analyzing mutations in the BRAF codons 600-601 was developed. The method provides sufficient sensitivity to detect frequent mutations in codons 600 and 601 and allows them to be precisely differentiated.

Keywords: BRAF; fine-needle aspiration biopsy; oncogenetics; pyrosequencing; thyroid cancer.

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Conflict of interest statement

Conflicts of interest. The authors declare that they have no conflicts of interest.

Figures

Figure 1.
Figure 1.. Sequenced region and examples of sample pyrograms:
(а) a wild sample, which was sequenced using the 600-S primer; (b) a sample with the c.1799 T>A p.V600E mutation, 30% of the mutant allele, 600-S; (c) a sample with the c.1798_1799delinsAA p.V600K mutation, 30% of the mutant allele, 600-S; (d) a sample with the c.1798_1799delinsAG p.V600R mutation, 30% of the mutant allele, 600-S; (e) a sample with the c.1798 G>A p.V600M mutation, 30% of the mutant allele, 600-S; (f) a wild sample, sequenced using the 601-S primer; (g) a sample with the c.1801 A>G p.K601E mutation, 30% of the mutant allele, 601-S. The X-axis is the sequence of nucleotides supply into the reaction mixture; the Y-axis is the signal level detected by the device. The nucleotide sequences used for mutation analysis are shown above the pyrograms. The arrows indicate signals for nucleotides with the values changing in case of a mutation. (h) shows the arrangement of methods for the BRAF pyrosequencing: the reference sequence is NC_000007.14, the arrows indicate sequencing primers, the dotted line shows sequenced regions
Figure 2.
Figure 2.. Correlation between the expected and the measured fractions of the mutant allele for the V600E mutation at sequencing with the 600-S primer
The X-axis is the expected proportion of the mutant allele in the sample (%); the Y-axis is the measured proportion of the mutant allele (%); crosses are wild samples; circles are dilutions with the V600E mutation, 10,000 copies/PCR; triangles are dilutions with the V600E mutation, 100 copies/PCR. The dotted line shows the trend for 100 copies/PCR, the solid line demonstrates the trend for 10,000 copies/PCR
Figure 3.
Figure 3.. Scattering graph of signal ratios in T3/C2 and T8/C2 positions on pyrograms for samples with mutations in codon 600
The X-axis is the ratio of signals in the T3/C2 positions; the Y-axis is the ratio of signals in the T8/C2 positions of the pyrogram; triangles are dilutions with the V600R mutation, rhombuses are dilutions with the V600K mutation, squares are dilutions with the V600M mutation, dark circles are dilutions with the V600E mutation, light circles are samples of thyroid nodules with the V600E mutation
See this image and copyright information in PMC

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