RU2739889C1 - Diagnostic technique of 35delg (rs80338939) mutation of gjb2 gene - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention refers to biotechnology, namely to molecular biology, medical genetics and otorhinolaryngology. Disclosed are synthetic oligonucleotides for diagnosing (genotyping) of 35delG (rs80338939) mutation of GJB2 gene in human biomaterial. Primers are combined into kits for detecting DNA in blood and other biological materials by polymerase chain reaction.

EFFECT: invention enables highly sensitive diagnosis of congenital hereditary deafness in biological material; using this method provides also highly sensitive and objective description of human genotype with determination of homozygous and heterozygous carriage of this mutation.

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Description

The invention relates to the field of biotechnology, namely to molecular biology, medical genetics and otorhinolaryngology.

Hearing loss is one of the most common sensory disorders in humans and has the highest rate of disability in the world [1]. Approximately half of cases of hearing loss have a genetic etiology with autosomal dominant, autosomal recessive, X-linked or mitochondrial inheritance patterns [2]. Congenital deafness is one of the most common human diseases, registered with a frequency of 1: 1000 newborns. Hearing loss is both a medical and a social problem. According to the WHO, there are 466 million people in the world with disabling hearing loss, which is about 5% of the entire population of the globe. There are more than 13 million people in Russia with hearing impairments, of which more than 3 million need hearing aids.

Among hereditary hearing impairments, the most common form (about 80%) of hereditary hearing loss - non-syndromic autosomal recessive deafness (DFNB1) [3, 4], refers to sensorineural hearing impairments in which the process of sound perception suffers. Studies in European and Asian populations have revealed mutations in the genes of the connexin protein family: GJB2 (gap junction p2) and GJB6 (gap junction β6), which are the most common cause of the disease. These genes are localized on chromosome 13 in the region 13q11-q13, encoding connexin 26 (Cx26) and connexin 30 (Cx30), proteins of intercellular gap junctions [5].

GJB2, encoding the connexin-26 protein, is responsible for more than half of the cases, followed by SLC26A4, MYO15A, OTOF, Cdh23, and TMC1 [6]. These mutations occur with different frequencies in different populations [7], with c.35delG, c.l67delT, and c.235delC prevailing in the populations of Caucasian, Ashkenazi Jews, and East Asia, respectively [8-12]. In addition, mutations in Pagent's syndrome in SLC26A4 account for 10% of inherited hearing loss in most of the world's population. In China, almost 50% of patients with non-syndromic hearing loss have GJB2 or SLC26A4 mutations [12]. Identifying these mutations is of primary interest for genetic counseling. Although a large number of cases are caused by hotspot mutations in these genes, molecular epidemiological studies have shown that rare mutations can also contribute to hearing loss.

More than 300 pathogenic variations of GJB2 have been registered in the human gene mutation database [13]. Many of them have a high ethnogeographic specificity of prevalence [14]. The most common GJB2 mutation is the deletion of one guanine (G) from the six (GGGGGG) at the 35 position of the gene - 35delG (rs80338939), leading to the formation of a stop codon and the termination of the synthesis of the connexin-26 protein, which determines the passive transport of electrolytes between cells. As a result, apoptotic death of the sensitive hairy neuroepithelium of the organ of Corti occurs, manifested by the blockade of mechano-electrical transduction in hair cells.

It is known that the recessive pathogenic variant GJB2 c.35delG (NM_004004.5) is common in deaf patients of Caucasian origin [14, 15]. Previous studies have shown that the c.35delG carrier frequency in Europe is about 1 in 50 [16], and in Southern Europe it is 1 in 31 [17]. In a meta-analysis of data published before 2008, the average carrier frequencies c.35delG were found to be 1.89, 1.52, 0.64, 1 and 0.64% for the populations of Europe, America, Asia, Oceania and Africa. respectively [15]. The founder effect plays an important role in the prevalence and accumulation of c.35delG in populations of Caucasian origin, since other populations show lower or absent c.35de1G values. According to the generally accepted hypothesis, c.35delG descended from a common ancestor in the Middle East or Mediterranean approximately 10,000-14,000 years ago and spread throughout Europe with Neolithic migrations. The specific prevalence of c.35delG and the detection of widespread STR and SNP haplotypes carrying the c.35delG mutation in Mediterranean, Middle Eastern, North European populations, and in individuals of European descent in the United States confirm this hypothesis [17-19].

The prevalence of c.35delG in deaf patients has been reported in several studies conducted in the European part of Russia [20-22]. High frequencies of c.35delG are observed in the populations of the northwestern and central parts of Russia with a tendency to decrease from west to east [23].

Anamnestic and clinical data are insufficient to make a correct diagnosis in the case of hereditary deafness. In addition, timely and accurate diagnosis of this hereditary defect will make it possible to begin treatment and rehabilitation measures with the highest quality and at the earliest possible time to ensure full social adaptation of persons with hereditary hearing impairments.

A large number of methods are known for determining genetic polymorphism, using various methods to analyze the unique nucleotide sequence of human DNA. This includes methods based on the use of enzymes (amplified fragment length polymorphism (AFLP); restriction fragment length polymorphism (RFLP), etc.), chemical methods (chemical cleavage of heteroduplexes, chemical ligation), methods based on different electrophoretic mobility of polymorphic DNA regions , detection on a solid phase, chromatographic methods, physical methods.

In particular, polymorphisms of the GJB2 gene have been investigated in many works [9-11, 23]. Among the methods for detecting mutations in this gene, a method for detecting 35delG, 167delT, and 235delC mutations of the GJB2 gene is known using heteroduplex analysis [24]. Significant disadvantages of the method are low sensitivity, the use of expensive gels, and the impossibility of determining the exact localization of SNPs within the studied fragment. Also known is the SSCP method (analysis of conformational polymorphism of single-stranded DNA), proposed in 1989 and widely used for the detection of single-nucleotide deletions [25]. However, this analysis is rather laborious and requires expensive equipment and reagents, and its duration is about 5 days. Later, a method for detecting mutations by the method of a multiplex polymerase chain reaction (PCR) system with one tube appeared. This method was also used to identify common mutations in the Connexin-26 gene and was described in the work of Baris I. et al. [26]. This paper describes an experiment that includes DNA isolation from peripheral blood lymphocytes, multiplex polymerase chain reaction, and amplification of DNA fragments containing 35delG, 167delT, and 235delC mutations of the GJB2 gene. Using this method, it is possible to detect the heterozygous carriage of mutations in the GJB2 gene.

The following analogs of the present invention are known from the prior art.

Patent No. RU 2317547 C1 "A METHOD FOR DETECTING MUTATIONS IN THE GJB2 GENE ACCOMPANYING THE DEVELOPMENT OF NON-SYNDROMAL AUTOSOMIC-RECESSIVE DEAFNESS" [Dzhemileva L.U. et al., 2008].

The subject of this invention is a multiplex polymerase chain reaction in which three regions of the GJB2 gene are amplified simultaneously. In the presence of certain alleles, heterozygous or homozygous carriage of 35delG, 167delT, and 235delC mutations is diagnosed.

Patent No. RU 2555755 C1 "A METHOD FOR DETECTING MUTATIONS IN THE MYO7A GENE ACCOMPANYING THE DEVELOPMENT OF NON-SYNDROMAL AUTOSOMIC-RECESSIVE DEAF AND USHER'S SYNDROME" [Dzhemileva L.U. et al., 2015].

The method described in this invention includes isolation of DNA from peripheral blood lymphocytes by phenol-chloroform extraction, PCR, amplification of 18 regions of the MY07A gene, detection in denaturing acrylamide gel and sequencing. PCR is carried out using specially selected sequences of oligonucleotides flanking regions of 18 exons of the MYO7A gene with the possible content of various mutations.

Patent No. RU 2448163 C2 "METHOD FOR DETECTING 17 GJB2 AND GJB6 GENES MUTATIONS IN HEREDITARY NONSYNDROMAL DEAFNESS" [Barashkov N.А. et al., 2012]

The present invention is directed to the detection of 17 disease-associated mutations in the GJB2 and GJB6 genes using PCR amplification of the corresponding regions of these genes, which is carried out in 8 reaction mixtures using specific primer pairs, and subsequent analysis of the resulting amplicons, carried out or without prior cleavage with endonucleases (when determining mutations c.312del14, p. 333-334delAA, ΔGJB6-D13S1830, ΔGJB6-D13S1854), or after hydrolysis with the corresponding specific restrictases (when determining mutations IVS1 + 1G> A, c.35delG, c.71G> A, c .79G> A, c.l67delT, c.235delC, c.224G> A, c.299-300delAT, c.360delGAG, c.341A> G, c.269T> C, c. 101T> C, c.109G > A).

Patent No. RU 2505608 C1 “METHOD FOR DETECTING MUTATION p. -53-2A> G IN THE PRESTINA GENE (SEC26A5), CAUSING THE DEVELOPMENT OF NON-SYNDROMAL AUTOSOMO-RECESSIVE DEAF ”[Dzhemileva L.U. et al., 2014]

The method described in this invention includes the isolation of DNA from peripheral blood lymphocytes by phenol-chloroform extraction. A polymerase chain reaction is carried out with the ability to conduct an endpoint fluorescence analysis. Simultaneously amplify two regions of the SLC26A5 gene in a mixture of two pairs of fluorescently labeled oligonucleotide sequences:

flanking the region with possible mutation c.-53-2A> G in the SLC26A5 gene.

Patent No. RU 2688180 C1 "Method for detecting mutations in the GJB2 gene that cause autosomal recessive deafness type 1A" [Pshennikova V. G. et al., 2019]

This invention proposes a method comprising the detection of the three most common mutations in Yakutia c.-23 + lG> A, c.35delG and c.109G> A using primers and subsequent analysis of restriction fragment length polymorphism using endonucleases AsuHPI, Bsc4I , HindII.

Patent No. RU 2627115 C2 "Method for the simultaneous diagnosis of hereditary diseases" [M.T. Savvina. et al., 2017]

This invention proposes a method for the simultaneous diagnosis of hereditary diseases based on the use of a biochip with oligonucleotide targets immobilized on its surface, including the detection of point in genes CUL7, NBAS, DIA1, FAH and causing 3M syndrome, SOPH syndrome, hereditary enzymopenic methemoglobinemia type 1, 1 type and hereditary non-syndromic type 1A deafness, respectively.

Patent No. CN 103352080 B "Kit for the determination of genes in hereditary hearing loss" [Zheng Weiguo Meng Xiang et al., 2014]

The invention discloses a kit capable of detecting 17 non-syndromic hearing loss predisposition genes. The kit consists of 17 pairs of specific primers for genotyping of genes for susceptibility to hearing loss and allows simultaneous detection of 17 mutations in the four most common Chinese genes in one test tube for 3 hours. The kit includes primer combinations of 17 polymorphic regions of hereditary hearing loss on the GJB2 (CX26) gene, the SLC26A4 gene (PDS), the GJB3 gene and the 12SrRNA gene (MTRNR1), they can be used to accurately assess the wild type, pure mutant type or hybrid type from 17 sites and for the diagnosis and screening of genes for hearing loss. The essence of the proposed set consists in the detection of the site of polymorphism of the third generation genetic marker (single nucleotide polymorphism) and combination with the detection technique, applying fluorescence to the non-syndromic gene of hereditary hearing impairment.

The prototype of the claimed invention is the method described in the application RU 2016136727 A, LLC "TVOI GEN", Zhmyrko EV, 2018 "A set of oligonucleotide primers, probes and a method for detecting gene mutations by allele-specific PCR amplification in hereditary non-syndromic deafness." Identification of gene mutations, using a set of primers and probes, by allele-specific PCR amplification in hereditary non-syndromic deafness is carried out according to the results of real-time PCR; in this case, the presence / absence of heterozygous carriage of the mutation is determined by the emitted fluorescence signal, in the presence of a signal through the FAM channel, the genotype is interpreted as the presence of a gene mutation in the test sample, in the presence of a signal through the HEX channel, as a norm, and in the presence of two signals, as a heterozygous state, carriage of a gene mutation.

The analysis shows that in the described analogs, as in the prototype, the disadvantage is the relatively low reliability of the research results due to the small difference between the obtained fluorescence curves for different alleles.

The present invention is aimed at increasing the reliability of diagnosis for the presence or absence of heterozygous / homozygous 35delG mutation (rs80338939) of the GJB2 gene, determining the human genotype in biological material (whole blood, saliva), as well as increasing the availability of such studies, since the method can be carried out on a standard known equipment.

The advantage lies in the uniqueness of the composition of the reaction mixture, in particular, in the nucleotide sequence of primers and probes for the diagnosis of heterozygous / homozygous 35delG (rs80338939) mutation of the GJB2 gene in human biomaterial. Determination of variable positions in DNA is carried out in a more reliable way using standard equipment.

A method for the diagnosis of heterozygous and homozygous 35delG mutations of the GJB2 gene in blood and other biological materials of a person by RT-PCR is proposed.

The use of the polymerase chain reaction (PCR) method for this purpose is the most promising. The principle of the PCR method is based on the use of the process of DNA amplification, which consists in repeated cycles of temperature denaturation of DNA, annealing of primers with complementary sequences, and subsequent extension of polynucleotide chains from these primers with Taq polymerase. The main advantage of the PCR method as a molecular biological study is its extremely high sensitivity.

Signaling probes containing fluorescent labels Fam and Hex were introduced into the mixture for amplification for each variant of the determined genetic polymorphism. After the end of the PCR, a round of thermal melting of the duplexes formed by the amplicons and signal probes is carried out, as a result of which the fluorescence level changes, which is recorded and represented by the instrument software in the form of a graph. If the signaling probe is partially complementary to the target DNA, the melting temperature of such a duplex will be lower than the melting temperature of the duplex if the probe is completely complementary. The analysis results are interpreted based on the melting point of the signal probes.

The use of three fluorescent dyes makes it possible to simultaneously determine two alleles and estimate the amount of genomic DNA in one test tube. A study using reagent kits for the determination of genetic polymorphisms consists of the following stages: DNA extraction (sample preparation) and real-time PCR amplification.

Work on the creation of oligonucleotides that are used in such kits is usually organized as follows.

1) With the help of open and commercial databases of nucleotide sequences of human genes or as a result of independent determination of the nucleotide sequence of the studied gene, a gene region is selected in which genetic polymorphism is possible (35delG mutation).

2) Based on the selected region of the gene, using the software, the sequence of oligonucleotides used for the PCR reaction is selected.

3) Manufacturing of primers is ordered from the service center.

4) Practical experiments prove the suitability of the selected sequences for specific purposes (for example, for the diagnosis of heterozygous / homozygous 35delG mutation of the GJB2 gene in human biomaterial).

The proposed approach to the detection of genetic polymorphism is the use of two allele-specific oligonucleotides labeled with different fluorescent labels, as well as an oligonucleotide carrying a fluorescence quencher and hybridizing to a template next to the allele-specific oligonucleotide. Hybridization of the allele-specific oligonucleotide to the template leads to the transfer of energy from the donor fluorophore located on it to the fluorescence quencher of the nearby “quenching” oligonucleotide. The registration of the amplification results is carried out at the end of the PCR by taking the fluorescence spectrum when the temperature of the reaction mixture changes in the range from 25 to 80 degrees Celsius (the so-called "melting curves"). When obtaining graphs of fluorescence, both heating and cooling of the reaction mixture in the specified temperature range is possible. The proposed invention makes the determination of variable positions in DNA more reliable and reduces the cost of such studies due to the use of standard equipment.

The technical result, which the present invention is aimed at, is a reliable diagnosis of the presence or absence of a heterozygous / homozygous 35delG mutation of the GJB2 gene, determination of the human genotype in biological material (whole blood, blood plasma, saliva, tissue samples), as well as increasing the availability of such studies because the method can be carried out on standard known equipment. In addition, this method provides the ability to conduct research in one test tube, which reduces research costs.

This result is achieved by using a set of synthetic oligonucleotides for diagnostics of the heterozygous / homozygous 35delG mutation of the GJT32 gene causing hearing loss in PCR, which includes one pair of forward and reverse primers (35delGsl and 35delGal, respectively) and two allele-specific wild-type probes corresponding to the genotype type and mutant (35delGptl and 35delGpt2, respectively), as well as a quencher probe (35delGpt3), hybridizing to a template next to allele-specific probes:

where FAM is a FAM fluorescent dye attached to nucleotide A at the 3'-end of the probe, HEX is a fluorescent HEX dye attached to nucleotide C at the 3'-end of the probe, BHQ1 means a dark fluorescence quencher attached to the 5'-terminal nucleotide.

Each set of reagents for the determination of genetic polymorphisms by the method of polymerase chain reaction in real time includes:

1) Test tubes with a reaction mixture sealed with paraffin. This mixture includes the specified primers and probes specific to a certain region of the GJB2 gene for each variant of the determined genetic polymorphism, a mixture of four types of deoxyribonucleotide triphosphates, a reaction buffer (80 mM Tris-HCl (pH 8.6 at 25 ° C), 20 mM (NH ₄ ) ₂ SO ₄ , 3 mM MgCl ₂ ).

Both primers and probes are synthetic oligonucleotides. Primers specific to the marker region of the gene are introduced into the reaction directly for amplification (production) of the product. The probes (also specific to a given DNA region) contain a fluorescent label, the fluorescence intensity of which indicates the amount of the product being formed, which, in turn, depends on the efficiency of the primers, the amount of starting material, and other reaction parameters. The primers in the reaction act as components that ensure the passage of the reaction, and the probes as the component for monitoring the progress of the reaction.

2) A solution of the enzyme Taq-polymerase.

3) Mineral oil.

4) Negative control sample - a sample that is introduced into the experiment to control possible contamination of reagents with products of previous reactions. A positive result in this sample indicates the need to replace reagents and rearrange the experiment.

In addition, a system for amplifying a fragment of human genomic DNA is included in the composition of amplification mixtures specific for each genetic polymorphism, which makes it possible to control the amount of DNA in the amplification tube and eliminate genotyping errors. The system for amplification of human genomic DNA includes a DNA probe that contains a fluorescent label (Cy5) and a fluorescence quencher.

Moreover, the mixture for amplification is specific for each genetic polymorphism. PCR buffer, Taq-AT polymerase, mineral oil - universal reagents for all reagent kits for the determination of genetic polymorphisms.

Similar kits, as well as reaction mixtures, primers for the diagnosis of heterozygous / homozygous 35delG mutation of the GJB2 gene in blood and other biological materials of a person are unknown.

Implementation (implementation) of the invention.

1) Biological material (whole blood, saliva) before carrying out PCR using the proposed set of reagents is carried out through the procedure of sample preparation using another set (the set of reagents for sample preparation is not the subject of this patent); during this procedure, DNA is isolated from the biological material, which, in turn, is used for PCR;

2) The required number of tubes with the prepared reaction mixture containing specific synthetic oligonucleotide primers and probes is marked according to the number of analyzed samples;

3) Taq-polymerase solution is added to all labeled tubes without damaging the paraffin layer;

4) In all labeled tubes (except for K- tubes (negative control sample), the DNA extracted according to clause 1) is added;

5) A negative control sample is introduced into a test tube marked with K-;

6) All tubes are installed in the thermal cycler unit, amplification is carried out according to the modes prescribed in the instructions for the kit. The detection of results is carried out by a detecting thermal cycler automatically during amplification (devices: detecting thermal cyclers DT-322, DT-96 (NPO DNA-Technology LLC).

The analysis of the results is carried out in accordance with the instructions for the device.

When a specific product is formed, the DNA probe is destroyed, the effect of the quencher on the fluorescent label stops, which leads to an increase in the level of fluorescence. The number of destroyed probes (and, consequently, the level of fluorescence) increases in proportion to the number of specific amplicons formed and is measured at each cycle of amplification.

Tubes (prepared as described above) were installed in a DT-prime detecting amplifier (NPO DNA-Technology LLC, Moscow) and a reaction was performed with the following cycling parameters:

The method of genotyping used by us is a variant of the classical method of "adjoining samples". When determining substitutions of single nucleotides, first, PCR was carried out with primers common to both variants of the sequence, then the temperature of the reaction mixture was reduced to hybridize the resulting template with oligonucleotide probes. To determine the sequence variant, two types of oligonucleotides hybridizing to a matrix side by side were used. One of the oligonucleotides was labeled with a fluorophore, the other with a fluorescence quencher. In the reaction, one common oligonucleotide with a fluorescence quencher and a pair of allele-specific (sequence-specific) oligonucleotides carrying a fluorophore were used. Oligonucleotide probes corresponding to one or another variant of the sequence were labeled with different fluorophores, which made it possible to determine both variants in one tube. The genotype was determined after PCR and hybridization by measuring the level of fluorescence during temperature denaturation of oligonucleotide duplexes and the resulting templates (or vice versa - hybridization). This measurement was performed in real time and resulted in melting curves. The reaction conditions were selected so as to maximize the difference in the melting temperatures of perfect and imperfect duplexes. Thus, if the analyzed sample contained only one variant of the sequence, i.e. was homozygous for this polymorphism, the melting point for the sample forming a perfect duplex was significantly higher than for the sample forming an imperfect duplex. If a heterozygous sample was analyzed, the melting points were practically the same.

On the basis of experimental data when comparing blood samples by RT-PCR "in real time", the results were obtained and analyzed. Detection and accounting of results is carried out automatically by the detecting amplifier. After the end of the amplification program, a corresponding information message will appear on the screen and it will be offered to proceed to the analysis of the results.

For negative control samples, the program records an unreliable result. Upon receipt of a positive value (determination of the genotype), the results of the entire production series are considered unreliable. In this case, it is necessary to take measures to eliminate possible contamination.

The melting points of the amplification products are plotted as temperature bars. Samples homozygous for the Fam channel polymorphism will be automatically colored blue; homozygous for the Hex channel - in green; heterozygous - red; doubtful and unreliable - in yellow (see Fig. 1-2). In this case, the assignment of a sample to a homozygote or heterozygote for a given allele is carried out according to the form of DNA melting curves (according to the maximum of the first derivative of the fluorescence graphs); the presence of one peak on the graph indicates that the sample is homozygous for the allele corresponding to the more refractory fluorescently labeled allele-specific oligonucleotide probe, and the presence of two peaks on the graph indicates the heterozygosity of the sample.

FIG. 1 shows an example of interpretation of results (Fam channel).

FIG. 2 shows an example of interpretation of results (Hex channel).

List of sources used

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List of nucleotide sequences

SEQ ID NO 1

CTC AGG TGA ACA AGC TAC TTT

SEQ ID NO 2

TTS STS TTS TTS TSA TGT STS

SEQ ID NO 3

CTG GGG GGT GTG AA -FAM

SEQ ID NO 4

CTG GGG GTG TGA AC -HEX

SEQ ID NO 5

BHQ1- GGG GCA CGC TGC AGA C - (P)

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Claims (3)

A method for diagnosing the 35delG (rs80338939) mutation of the GJB2 gene in a human biomaterial selected from whole blood, saliva, based on real-time polymerase chain reaction (RT-PCR), characterized in that primers and probes are used in RT-PCR. polymorphic region rs80338939 of the GJB2 gene:

where FAM is a fluorescent FAM dye attached to nucleotide A at the 3'-end of the sample; HEX is a HEX fluorescent dye attached to nucleotide C at the 3'-end of the probe, and BHQ1 means a dark fluorescence quencher attached to the 5'-terminal nucleotide; Based on the obtained graphs of the DNA melting curves, a mutation is determined, while in the presence of one peak on the graph, homozygous carriage is diagnosed, and in the presence of two peaks, heterozygous carriage is diagnosed.

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