RU2777663C1 - QUANTITATIVE METHOD FOR DETERMINING THE EXPRESSION OF GNAO1 ALLELES OF A HEALTHY FORM AND WITH c.607 G>A MUTATION - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology; in particular, developed is a quantitative PCR-based method using specific primers and probes to determine the levels of expression of wild-type GNAO1 transcripts and the clinical c.607 G>A variant.

EFFECT: invention can be used to evaluate the effectiveness of medicinal products for RNA therapy aimed at specific inhibition of the expression of GNAO1 transcripts with the c.607 G>A mutation.

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Description

Technical field

The invention relates to the field of genetics and molecular biology. A quantitative method based on PCR using specific primers and fluorescently labeled probes was developed to determine the expression levels of wild-type and clinical variant c.607 G>A GNAO1 transcripts. The present invention can be used to evaluate the effectiveness of RNA therapy preparations aimed at specifically suppressing the expression of GNAO1 transcripts with the c.607 G>A mutation.

The invention was created with the financial support of the Ministry of Science and Higher Education of the Russian Federation under Agreement No. 075-15-2019-1661 dated 10/31/2019.

State of the art

In 2013, a link was found between mutations in the GNAO1 gene and an orphan neurological disorder called GNAO1-associated encephalopathy. This disease manifests itself in infancy and is characterized by epileptic encephalopathy (OMIM 615473) and/or brain development disorder with involuntary movements (OMIM 617493). The GNAO1 gene is predominantly expressed in the brain and encodes the Gαo1 protein. As of 2021, 25 variants of pathogenic mutations of this gene have been described in the literature. Mutations in patients occur de novo and have a dominant manifestation. One of the most common mutations is variant p.607 G>A (polymorphism rs587777057). This mutation occurs only in the heterozygous state and results in the G203R amino acid substitution near the GTP-binding center of the protein.

To study GNAO1-associated encephalopathy with the c.607 G>A variant, as well as to test RNA-targeted therapy drugs (antisense oligonucleotides, small interfering RNAs, artificial miRNAs), a quantitative method for assessing the expression levels of healthy and mutant GNAO1 transcript in biological samples is required ( patient skin biopsy, patient-specific neurons, cellular test systems). The development of such a method is possible on the basis of real-time polymerase chain reaction (PCR).

To assess the level of expression of the GNAO1 gene in the studied samples, Bio-Rad developed two sets of reagents. a) “PrimePCR SYBR® Green Assay: GNAO1, Human” (qHsaCID0006500), the supplied PCR mix contains primers specific for the GNAO1 gene transcript. Signal detection when using the kit is carried out due to the accumulation of a fluorescent signal from the DNA-intercalating dye SYBR Green. b) “PrimePCR Probe Assay: GNAO1, Human” (qHsaCIP0026191), the supplied PCR mixture contains primers specific to the GNAO1 gene transcript, as well as a DNA probe labeled with one of five fluorophores to choose from. Both sets are applicable only for assessing the overall level of GNAO1 gene expression in samples and do not allow discrimination between wild-type and c.607 G>A transcripts.

Closest to the present invention is the Thermo Fisher Scientific reagent kit for the allele-specific detection of the rs587777057 polymorphism by multiplex PCR with the TaqMan detection system. The kit (“TaqMan™ SNP Genotyping Assay, human”, Assay ID: C_338418529_10) is a pair of primers specific to the GNAO1 genomic region and two probes with different fluorescent labels for the G and A isoforms of the rs587777057 polymorphism. The presence of the wild-type GNAO1 allele (isoform G) is detected in the VIC channel, the presence of the clinical variant c.607 G>A (isoform A) is detected in the FAM channel. This method for detecting the rs587777057 polymorphism is intended for genotyping DNA samples and cannot be used to study GNAO1 transcripts, which is its significant drawback. Moreover, this method implies only qualitative, but not quantitative, detection of the G/A isoforms of the rs587777057 polymorphism. Finally, the kit lacks a universal probe that detects both polymorphism isoforms and acts as an internal positive control of the reaction.

Technical task and technical result

The objective of this invention is to develop a quantitative method based on PCR to assess the level of expression of G and A isoforms of GNAO1 transcripts in heterozygous polymorphism rs587777057.

The present invention consists in creating a set of primers and fluorescently labeled probes (PCR kit No. 1) with the TaqMan detection system, specific for wild-type GNAO1 transcripts (isoform G), clinical variant c.607 G>A (isoform A) and both isoforms transcript as an internal positive control.

The invention also consists in developing a method for quantifying the level of expression of GNAO1 transcript isoforms with the rs587777057 (G/A) polymorphism. In order to quantify the method, in addition to PCR kit No. 1, it is necessary to use DNA standards with a known concentration. For the quantitative comparison of several similar biological samples, primers and fluorescently labeled probes (PCR kit No. 2) specific for housekeeping gene transcripts were developed. Optimized multiplex-PCR reaction conditions provide high specificity and efficiency of the primers and probes of the PCR kit No. 1 and No. 2. This, in turn, determines the accuracy of the qualitative and quantitative assessment of the expression of GNAO1 transcript isoforms with the rs587777057 (G/A) polymorphism.

This invention can be used for scientific purposes to study the mechanism of GNAO1 c.607 G>A encephalopathy and its modeling, for diagnostic purposes to determine the level of expression of healthy and mutant alleles in the tissues of a patient with heterozygous rs587777057 polymorphism, as well as to develop and evaluate the effectiveness of drugs RNA therapy (antisense oligonucleotides, small interfering RNAs, artificial miRNAs) aimed at allele-selective suppression of the expression of the GNAO1 mutant transcript p.607 G>A.

Disclosure of the essence of the invention

The present invention is a quantitative method based on multiplex PCR to assess the level of expression of wild-type and c.607 G>A GNAO1 transcripts (rs587777057 polymorphism) in biological samples. The essence of the developed method is the use of primers and probes that are highly specific for the G and A isoforms of GNAO1 transcripts (PCR kit No. ” (PCR kit No. 2).

PCR kit No. 1 for the detection of GNAO1 transcripts is schematically shown in Figure 1. A universal pair of forward (GNAO1_F) and reverse (GNAO1_R) primers provide amplification of GNAO1 transcripts regardless of the rs587777057 polymorphism isoform. To differentiate wild-type and mutated GNAO1 transcripts, the GNAO1-G-FAM and GNAO1-A-VIC probes were developed, which are strictly specific to the G and A isoforms of the rs587777057 polymorphism, respectively. The kit uses the GNAO1-ROX probe as an internal control for the passage of PCR, as well as for detecting the total level of GNAO1 transcripts. Optimized multiplex PCR conditions guarantee high specificity (Fig. 2) and efficiency (Fig. 3) of the primers and three probes in one reaction and allow the joint qualitative detection of healthy and mutant GNAO1 transcripts in biological samples (Fig. 4).

Sequences of PCR kit No. 1:

GNAO1_F 5'-atcagctcaacgactctgcc-3' (SEQ ID NO: 1)

GNAO1_R 5'-acgtcctcgaagcaatggat-3' (SEQ ID NO: 2)

GNAO1-ROX 5'-ROX-aaccactggcatcgtagaaaccca-BHQ2-3' (ROX - SEQ ID NO: 3 - BHQ2)

GNAO1-G-FAM 5'-FAM-cgtcggaggccagc-BHQ1-3' (FAM - SEQ ID NO: 4 - BHQ1)

GNAO1-A-VIC 5'-VIC-cgtcagaggccagc-BHQ1-3' (VIC - SEQ ID NO: 5 - BHQ1);

where ROX, VIC, FAM are fluorescent labels of different colors (fluorophores), and BHQ1 and BHQ2 are fluorescence quenchers.

Evaluation of the effective concentration of GNAO1 transcript isoforms in the studied samples is carried out according to the calibration graph, which is constructed by analyzing serial dilutions of DNA standards with a known concentration using PCR kit No. 1. Any two plasmid constructs of known length containing the wild-type GNAO1 coding frame and the c.607 G>A mutation can serve as DNA standards. The known nucleotide size and concentration of the DNA standards make it possible to convert the recorded signal in the samples into the number of copies of G and A isoform transcripts per reaction or 100 ng of total RNA.

In order to compare the expression of GNAO1 in several samples derived from the same cell or tissue type, the effective concentration of the G and A isoforms is additionally normalized to the relative amount of material introduced into the PCR reaction. To normalize the samples, a PCR kit No. 2 was developed, consisting of primers and probes specific for transcripts of the GAPDH and EF1A “housekeeping” genes.

Sequences of PCR kit No. 2:

GAPDH_F 5'-tcggagtcaacggatttggt-3' (SEQ ID NO: 6)

GAPDH_R 5'-ttcccgttctcagccttgac-3' (SEQ ID NO: 7)

GAPDH-VIC 5'-VIC-tgattccacccatggcaaattcca-BHQ1-3' (VIC - SEQ ID NO: 8 - BHQ1)

EF1A_F 5'-aaggatgttcgtcgtggcaa-3' (SEQ ID NO: 9)

EF1F_R 5'-gccgtgtggcaatccaatac-3' (SEQ ID NO: 10)

EF1A-ROX 5'-ROX-aaatgacccaccaatggaagcagc-BHQ2-3' (ROX - SEQ ID NO: 11 - BHQ2);

where ROX, VIC are fluorescent labels of different colors (fluorophores), and BHQ1 and BHQ2 are fluorescence quenchers.

The described method based on multiplex PCR allows both qualitative (Fig. 4) and quantitative (Fig. 5) analysis of the expression of wild-type GNAO1 transcripts and those with the c.607 G>A mutation (rs587777057 polymorphism). The material for research can be a skin biopsy of a patient with the rs587777057 polymorphism, patient-specific cell cultures obtained using the technology of induced pluripotent stem cells (Fig. 4 and 5), continuous cell lines with transient or stable expression of GNAO1. Also, the developed quantitative detection method can be used to compare the expression level of healthy and mutant GNAO1 alleles under different exposures. For example, to evaluate the effectiveness of RNA therapy preparations aimed at allele-selective suppression of the expression of GNAO1 transcripts with the c.607 G>A mutation.

Brief description of the drawings

Figure 1. Illustration of a technical solution for the detection of transcripts of the GNAO1 gene of the wild type and with the c.607 G>A mutation, as well as the total level of GNAO1 transcripts (PCR set No. 1). Schematically shows the region of the GNAO1 transcript (NM_020988.3) with single nucleotide polymorphism rs587777057. White arrows symbolically indicate the location of the forward and reverse PCR primers specific to the GNAO1 transcript sequence. Gray arrows show the position of PCR probes labeled with ROX, VIC, FAM fluorophores for universal and selective detection of GNAO1 transcripts.

Figure 2. Selectivity of the detection method for wild-type and c.607 G>A GNAO1 using a set of specific primers and probes for multiplex PCR (PCR kit No. 1). As DNA standards, two known concentration plasmid DNAs containing the wild-type and mutated GNAO1 coding region (pGNAO1 wild-type and pGNAO1 p607 G>A, respectively) were used. Serial, 10-fold dilutions of DNA standards in the range of 0.05 ng - 0.5 fg per reaction were added to the reaction with PCR master mix #1. Amplification was performed on a CFX96 Real-Time PCR Detection System; the fluorescent signal was recorded in the ROX, FAM, VIC channels. Representative curves of PCR product accumulation are shown. In the ROX channel, wild-type and mutated GNAO1 is detected. In the FAM and VIC channels, wild-type GNAO1 and c.607 G>A GNAO1 are detected strictly selectively in the selected concentration range, respectively.

Figure 3. Efficiency of multiplex PCR for detection of wild type and c.607 G>A GNAO1 GNAO1 using a set of specific primers and probes. The reaction was carried out in PCR master mix #1 and a standard curve was built for the amplification of the fluorescence signal in the ROX, FAM, VIC channels with 10-fold dilutions of DNA standards (pGNAO1 wild type and pGNAO1 c.607 G>A) in the range of 0.05 ng - 0.5 fg per reaction. The efficiency for each of the probes (GNAO1-ROX, GNAO1-G-FAM, GNAO1-A-VIC) was calculated from the standard curve. Shown are representative standard curves and mean PCR efficiencies for n=5 independent experiments.

Figure 4. Qualitative detection of wild-type and c.607 G>A GNAO1 transcripts by multiplex PCR. Neurons obtained by the technology of induced pluripotent stem cells from a skin biopsy of a healthy donor and a patient with the GNAO1 c.607 G>A mutation were used as material for the study. Sample preparation was carried out according to the standard method, qualitative analysis of expression was performed using PCR master mix #1.

Figure 5. Quantification of the expression level of transcripts in cells expressing wild-type GNAO1 and c.607 G>A mutation.

Neuronal cells obtained from a skin biopsy of a healthy donor and a patient with a heterozygous mutation GNAO1 c.607 G>A were used as material for the study. Sample preparation was carried out according to the standard method, analysis of the expression of GNAO1 and housekeeping genes c was performed using PCR master mix #1 and #2, respectively. The number of copies of each of the GNAO1 variants in the reaction was determined from a standard curve constructed using serial dilutions of DNA standards. Each sample was normalized to the amount of material introduced into the PCR reaction, determined using housekeeping genes and a standard curve with a control sample. The level of expression of wild-type and c.607 G>A GNAO1 transcripts in samples is expressed as copies per 100 ng of total RNA.

Implementation of the invention

Example 1. Preparation of primers, probes and DNA standards. To quantify the level of expression of healthy and mutant GNAO1 transcripts in biological samples, we used specific sets of primers and probes for multiplex PCR with the TaqMan detection system, as well as plasmid DNA standards.

- For the selective detection of GNAO1 transcripts, PCR kit No. 1 was developed (Fig. 1): forward primer GNAO1_F (5'-atcagctcaacgactctgcc-3'; SEQ ID NO: 1), reverse primer GNAO1_R (5'-acgtcctcgaagcaatggat-3'; SEQ ID NO: 2), GNAO1-ROX universal probe (5'-ROX-aaccactggcatcgtagaaaccca-BHQ2-3'; SEQ ID NO: 3), and GNAO1-G-FAM selective probes (5'-FAM-cgtcggaggccagc-BHQ1-3' ; SEQ ID NO: 4) and GNAO1-A-VIC (5'-VIC-cgtcagaggccagc-BHQ1-3'; SEQ ID NO: 5) for healthy and mutant transcripts, respectively. Oligonucleotides were synthesized and purified by DNA Synthesis (Russia). Lyophilized DNA was resuspended in sterile PCR water to a final concentration of 100 μM and stored at -20°C.

- DNA standards were used to determine the copy number of GNAO1 transcripts in the sample. For this, plasmid constructs were created with the coding region of the GNAO1 gene (CCDS10756.1) of the wild type and the c.607 G>A mutation (pGNAO1 and pGNAO1 c.607 G>A, respectively). The size of each of the constructs is 5079 base pairs, and one ng of the plasmid is equivalent to 1.8×10^8 copies of the gene.

- To normalize the material introduced into the PCR reaction, PCR kit No. 2 was used, consisting of primers and probes specific to the GAPDH and EF1A “housekeeping” gene transcripts. For GAPDH detection: GAPDH_F forward primer (5'-tcggagtcaacggatttggt-3'; SEQ ID NO: 6), GAPDH_R reverse primer (5'-ttcccgttctcagccttgac-3'; SEQ ID NO: 7), GAPDH-VIC probe (5'- VIC-tgattccacccatggcaaattcca-BHQ1-3'; SEQ ID NO: 8); for EF1A detection: EF1A_F forward primer (5'-aaggatgttcgtcgtggcaa-3'; SEQ ID NO: 9), EF1F_R reverse primer (5'-gccgtgtggcaatccaatac-3'; SEQ ID NO: 10), EF1A-ROX probe (5'- ROX-aaatgacccaccaatggaagcagc-BHQ2-3'; SEQ ID NO: 11).

Example 2 Preparing Samples for Analysis

Samples of the patient's skin biopsy with a heterozygous GNAO1 c.607 G>A substitution and cell cultures obtained from the biopsy (fibroblasts, induced pluripotent stem cells, neuronal progenitors, neurons of different degrees of differentiation), as well as transplanted cell cultures can be used as material for research. with transient or stable expression of a mutant and healthy GNAO1 variant.

- Isolation of total RNA is carried out using commercial RNA isolation kits according to the method recommended by the manufacturer, taking into account the characteristics of the sample and its quantity.

- The concentration of isolated RNA is measured spectrophotometrically.

- RNA is processed by DNase I using a commercial kit and protocol compatible with downstream reverse transcription and amplification steps. DNase I inactivation is carried out according to the manufacturer's recommendations.

- Synthesis of cDNA from RNA template is carried out using a commercial kit for reverse transcription according to the manufacturer's recommendations. The reaction is carried out in a volume of 20 µl containing 100 ng/µl RNA, 1 µM Oligo(dT) ₁₅ primer (SB001; Eurogen), random decanucleotide primer (SB002; Eurogen), dNTP mix 1 mM each (PB006S; Eurogen), 2 mM DTT, MMLV reverse transcriptase, 1x manufacturer's buffer (SK022L; Eurogen), incubation at 37°C for 1 hour. Inactivation 10 min at 70°C. cDNA samples are diluted 5-fold with sterile TE buffer (PB026S; Eurogen) and stored at -20° C. until analysis.

Example 3 Real-time PCR

To quantitatively measure the expression levels of GNAO1 alleles in experimental samples, multiplex real-time PCR is performed using sets of primers and probes specific for GNAO1 transcripts and housekeeping gene transcripts, as well as DNA standards.

- PCR master mix #1 consists of 400 nM primers and 200 nM probes for selective detection of GNAO1 transcripts (PCR kit #1 described in example 1); 3 mM MgCl2 and 1x PCR Reaction Mix (Synthol, part number M-428).

- To 20 µl of PCR master mix #1, add 5 µl of experimental or standard sample. As an experimental sample using cDNA obtained in example 2; the introduced amount of the sample corresponds to approximately 100 ng of the original RNA per reaction. As a standard sample, serial 10-fold dilutions of the DNA standards described in example 1 are used; the amount of sample applied is in the range of 0.05 ng - 0.5 fg of plasmid per reaction, which corresponds to 9x10^6 - 90 copies of GNAO1 per reaction.

- PCR master mix #2 consists of 400 nM primers and 200 nM probes for detecting GAPDH and EF1A housekeeping gene transcripts (PCR set #1 described in Example 1); 5 mM MgCl2 and a single PCR reaction mix (Synthol, part number M-428).

- To 20 µl of PCR master mix #2 add 5 µl of experimental or standard sample. As an experimental sample using cDNA obtained in example 2; the introduced amount of the sample corresponds to approximately 100 ng of the original RNA per reaction. Serial 5-fold dilutions of control cDNA are used as a standard; the introduced amount of the sample corresponds to about 0.8, 2, 20 and 100 ng of the original RNA per reaction.

- Standard and experimental samples are analyzed in three technical replicates each.

- PCR reactions are mixed in 96-well thin wall plates (SSI-3401-00, SSI) and sealed with UltraFlux® Standard optically clear film (SSI-3622-00, SSI).

- Real-time PCR was performed on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories) using the following amplification program: denaturation 95°C - 3 min; further 40 cycles: 95°C - 20 sec, 60°C - 40 sec, reading of the fluorescent signal. For PCR master mix #1, signal detection is performed in channels FAM (target - GNAO1 wild type), VIC (GNAO1 c.607 G>A), ROX (total GNAO1). For PCR master mix #2, signal detection is carried out in channels VIC (target - GAPDH), ROX (EF1A).

Example 4 Method Specificity and Efficiency

The specificity and efficiency of the set of primers and probes for the allelic detection of GNAO1 under the conditions of PCR master mix #1 and the amplification mode from example 3 were evaluated using DNA standards (pGNAO1 and pGNAO1 p.607 G>A) from example 1.

- Specificity of detection of GNAO1 transcripts with c.607 G>A mutation and wild type by probes GNAO1-A-VIC (5'-VIC-cgtcagaggccagc-BHQ1-3') and GNAO1-G-FAM (5'-FAM-cgtcggaggccagc-BHQ1 -3') respectively retained in the range of 90 copies to 9x10^6 copies of the GNAO1 coding region per reaction (FIG. 2).

- Cycle threshold (Cq) data for serial dilutions of DNA standards were used to construct calibration curves and determine the performance of multiplex-PCR for allelic detection of GNAO1. For probes GNAO1-A-VIC (5'-VIC-cgtcagaggccagc-BHQ1-3'), GNAO1-G-FAM (5'-FAM-cgtcggaggccagc-BHQ1-3') and GNAO1-ROX (5'-ROX-aaccactggcatcgtagaaaccca -BHQ2-3') is 105.4%, 107.6% and 101.2%, respectively (mean of n=5 independent experiments) (Fig. 3).

The efficiency of the set of primers and probes for detecting housekeeping gene transcripts under the conditions of PCR master mix #2 and the amplification mode from example 3 was evaluated using a standard cDNA obtained from the control sample in example 2.

- Cycle threshold (Cq) data for serial dilutions of control DNA were used to construct calibration curves and determine reaction efficiency. The efficiency of multiplex-PCR for the joint detection of GAPDH and EF1A transcripts is 91±4% and 89±5% (average over n=5 independent experiments), respectively.

Example 5. Quantification of results.

Quantitative determination of the expression level of GNAO1 alleles in experimental samples was determined as follows:

- The effective concentration of wild-type GNAO1 transcripts with a mutation and their total level in experimental samples was determined from the standard curves from example 4, built for serial dilutions of DNA standards and probes GNAO1-G-FAM, GNAO1-A-VIC and GNAO1-ROX, respectively .

- Copies of GNAO1 transcripts in different samples were normalized to the relative amount of material introduced into the PCR reaction, determined using primers/probes for housekeeping genes and a standard curve for the control cDNA sample from example 4.

- The level of expression of GNAO1 transcripts in experimental samples was expressed as copies of GNAO1 per 100 ng of total RNA (Fig. 5).

Claims (26)

1. A set of primers and fluorescently labeled probes specific for GNAO1 transcripts, designed for selective detection in a biological sample of wild-type GNAO1 transcripts (or isoform G of the rs587777057 polymorphism), a clinical variant of GNAO1 with the c.607 G>A mutation (or isoform A of the polymorphism rs587777057), as well as GNAO1 transcripts of both isoforms (G polymorphism rs587777057 and A polymorphism rs587777057), and consisting of: – direct GNAO1_F and reverse GNAO1_R primers providing amplification of GNAO1 transcripts regardless of the rs587777057 polymorphism isoform; – selective fluorescently labeled probes GNAO1-G-FAM and GNAO1-A-VIC, strictly specific to the G and A isoforms of the rs587777057 polymorphism, respectively; – universal fluorescently labeled probe GNAO1-ROX used for internal control of PCR and for detection of the total level of GNAO1 transcripts. 2. A set of primers and probes according to claim 1, containing: – as a forward primer, the sequence 5'-atcagctcaacgactctgcc-3'; – as a reverse primer, the sequence 5'-acgtcctcgaagcaatggat-3'; – as a universal fluorescently labeled probe for the detection of the total level of GNAO1 transcripts, the sequence 5'-ROX-aaccactggcatcgtagaaaccca-BHQ2-3'; – as a selective fluorescently labeled probe specific to the G isoform of the rs587777057 polymorphism, the sequence 5'-FAM-cgtcggaggccagc-BHQ1-3'; – as a selective fluorescently labeled probe specific to isoform A of polymorphism rs587777057, sequence 5'-VIC-cgtcagaggccagc-BHQ1-3'. 3. A method for quantitative determination of the level of expression of GNAO1 transcript isoforms with rs587777057 polymorphism in a biological sample based on multiplex real-time PCR, which includes the following steps: 1) qualitative detection with determination of the number of copies of GNAO1 transcripts in the sample using the kit according to claims 1, 2 and using DNA standards in the FAM channels (for wild-type GNAO1 or isoform G of the rs587777057 polymorphism), VIC (for GNAO1 with c. 607 G>A or isoforms A of polymorphism rs587777057) and ROX (for isoforms GNAO1 G of polymorphism rs587777057 and A of polymorphism rs587777057); 2) normalization of copies of GNAO1 transcripts to the relative amount of material introduced into the PCR reaction using a set of primers and fluorescently labeled probes specific to transcripts of the GAPDH and EF1A “housekeeping” genes, while the set contains: – forward GAPDH_F and reverse GAPDH_R primers specific for GAPDH housekeeping gene transcripts; – forward EF1A_F and reverse EF1F_R primers specific for transcripts of the EF1F housekeeping gene; – fluorescently labeled probe GAPDH-VIC, specific for transcripts of the GAPDH housekeeping gene; – fluorescently labeled EF1A-ROX probe specific for EF1A housekeeping gene transcripts. 3) Determination of the level of expression of wild-type and c.607 G>A GNAO1 transcripts in samples expressed in copies per 100 ng of total RNA. 4. The method according to claim 3, in which the set of primers and fluorescently labeled probes specific for GAPDH and EF1A housekeeping gene transcripts contains: – as a forward primer specific to GAPDH housekeeping gene transcripts - GAPDH_F, sequence 5'-tcggagtcaacggatttggt-3'; – as a reverse primer specific to GAPDH housekeeping gene transcripts - GAPDH_R, sequence 5'-ttcccgttctcagccttgac-3'; – as a direct primer specific to transcripts of the “housekeeping” gene EF1F - EF1A_F, sequence 5'-aaggatgttcgtcgtggcaa-3'; – as a reverse primer specific to transcripts of the “household” gene EF1F - EF1F_R, sequence 5'-gccgtgtggcaatccaatac-3'; – as a fluorescently labeled probe specific for transcripts of the GAPDH housekeeping gene - GAPDH-VIC, sequence 5'-VIC-tgattccacccatggcaaattcca-BHQ1-3'; – as a fluorescently labeled probe specific for transcripts of the EF1A housekeeping gene - EF1A-ROX, sequence 5'-ROX-aaatgacccaccaatggaagcagc-BHQ2-3'.

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