RU2688180C1 - Method of detecting mutations of gjb2 gene causing autosomal-recessive deafness 1a type - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, particularly to medical genetics and otorhinolaryngology, and aims at detecting mutations of the GJB2 gene causing autosomal-recessive deafness of type 1A. Disclosed is a method involving detection of three most common mutations in Yakutia c.-23 + 1G> A, p.35delG and c.109G> A using primers and followed by analysis of polymorphism of restriction fragment lengths using endonucleases AsuHPI, Bsc4I, HindII.EFFECT: invention provides fast and accurate detection of GJB2-mutations responsible for 98 % of all pathogenic variants causing autosomal recessive deafness of type 1A in Yakutia.1 cl, 6 dwg, 1 tbl

Description

The invention relates to medicine, namely medical genetics and otolaryngology, in particular, the identification of the most common GJB2 mutations in Yakutia, causing autosomal recessive deafness 1A type (ARG 1A).

Known solutions for identifying mutations in the GJB2 gene, causing hereditary hearing impairments, for example, a method for detecting mutations in the GJB2 gene, accompanied by the development of nonsyndromic autosomal recessive deafness (see RU No. 2317547, cl. G01N 33/50, С12Q 1/68, publ. 20.02.2008), which allows to simultaneously detect three GJB2 mutations c.35delG (p.Gly12ValfsX2), p.167delT (p.Leu56ArgfsX26) and p.235delС (p.Leu79Cysfs), by carrying out a multiplex polymerase reaction. In this case, mutational variability can be easily detected by changing the length of fragments in one nucleotide after electrophoretic separation of the amplifying DNA segments with possible content of these deletions without processing the PCR products of restriction endonucleases, which greatly simplifies the detection method. However, of the three mutations presented, the variants p. 177delT and p. 2335delC are not characteristic of the Yakut population, as the earlier analysis of the spectrum and frequency of mutations of the GJB2 gene in patients with congenital hearing impairment from Yakutia 363 unrelated) showed that in the spectrum of 8 identified pathogenic variants (mutations) of this gene, three mutations are most common (allelic frequency> 1%): c.-23 + 1G> A (42.28%), p.35delG (5.92%) and c.109G> A (1.92%), which account for 98% of all pathogenic alleles (51.10%) of the number of chromosomes studied, are not related three patients. In the studied sample of patients, the p.167delT mutation, which is a major among deaf Ashkenazi Jews, was detected only in one Russian patient in a heterozygous state (0.13%, 1/726), and a characteristic mutation in the countries of Central and Eastern Asia , neither among patients nor among the control group was detected (see Analysis of the spectrum and frequency of GJB2 mutations in patients with congenital hearing impairment in the Republic of Sakha (Yakutia) / VG Pshennikova, NA Barashkov, F.M. Teryutin and others // Medical genetics. - 2015. - V. 14. - №6 (156). - P. 10-2327; Spectrum and Frequency of the GJB2 Barashkov, VG Pshennikova, OL Posukh et al // PLoS One. - 2016. - Vol. 11 (5) : e0156300. doi: 10.1371 / journal.pone.0156300).

There is a method for detecting 17 mutations of the GJB2 and GJB6 genes in hereditary nonsyndromic deafness (see RU No. 2448163, class G01N 33/50, C12Q 1/68, publ. 20.04.2012), in which the diagnostic panel of hereditary nonsyndromic deafness includes the spectrum from 17 allelic variants of the GJB2 gene (of which c.71G> A, c.79G> A and c.341A> G have no clinical significance), where PCR amplification of GJB2 and GJB6 gene regions is carried out in 8 reaction mixtures of 10 pairs of oligonucleotide sequences that is technically time consuming and costly to use in routine DNA diagnostics of ARG 1 in Yakutia.

The method of simultaneous diagnostics of hereditary diseases (see RU No. 2627115, Cl. G01N 33/50, С12Q 1/68, publ. 03.08.2017) allows you to simultaneously diagnose five hereditary diseases based on the use of biochip with oligonucleotide targets on its surface for the detection of point mutations localized in five genes, among which the detection of c.-23 + 1G> A mutation in the GJB2 gene responsible for ARG 1A is included. The method is carried out taking into account the ethnicity of patients, since the composition of mutations for this biochip is optimized and strictly specific only for the Yakut population. Note that in the distribution of patients with congenital hearing impairments from Yakutia by ethnicity, the most frequent GJB2 mutation in Yakut patients (n = 296) was s.-23 + 1G> A (51.8%), the second is c.109G > A (2.3%), and the third - c.35delG (1.6%). In Russian patients (n = 51), three frequent GJB2 mutations were also detected: p.35delG (22.3%), pp-23 + 1G> A (5.3%), and p.313_326 del14 (2.1 %). However, the widespread introduction of biochip technology into practice is hampered by the high cost of the entire complex of automated equipment (and special robots) necessary for their production and application of biological macromolecules to the platform.

The problem to which the claimed invention is directed is to create a method for identifying the most common mutations of the GJB2 gene in Yakutia, causing autosomal recessive deafness of type 1A.

The technical result obtained by solving the problem, expressed in the rapid and accurate identification of GJB2 mutations responsible for 98% of all pathogenic variants causing autosomal recessive deafness 1A type in Yakutia.

To solve this problem, a method for identifying the most common GJB2 mutations in Yakutia is c.-23 + 1G> A, p.35delG and c.109G> A, causing autosomal recessive deafness of type 1A, in order to carry out the amplification of significant GJB2 - regions containing these mutations, the following primers are used: c.-23 + 1G> A (F) - 5'-CCGGGAAGCTCTGAGGAC-3 ', (R) - 5'-GCAACCGCTCTGGGTCTC-3'; c.35delG (F) - 5'-ACTCAGGTGAACAAGCTACT-3 ', (R) - 5'-TCTTTCCAATGCTGGTGGAGTGTTTGTTCCCA-3' and c.109G> A (F) - 5'-TCTTTTTAGAGAGCAAACCGAGAGAGAGAG-3, 3-star, 3-star -TGCTCATCTCCCCACACCTCCTTTGCAGCGTCAA-3 ', followed by analysis of restriction fragment length polymorphism using endonucleases AsuHPI, Bsc4I, HindII.

Comparative analysis of the signs of the claimed solution with the signs of analogues indicates the compliance of the stated decision to the criterion of "novelty."

The combination of features of the invention provides a solution to the claimed technical problem, namely, obtaining a molecular-genetic method for detecting mutations of the GJB2 gene, causing autosomal recessive deafness 1A type, the most common in Yakutia.

The advantage of the proposed method over existing analogues is that it allows the routine method to quickly and accurately identify GJB2 mutations responsible for 98% of all pathogenic variants causing autosomal recessive type 1A deafness in Yakutia, which is based on the results of years of molecular genetic studies congenital hearing impairment in Yakutia.

The claimed technical solution is illustrated by drawings, where figure 1 shows the region of overlapping sequence of oligonucleotide primers for detecting the c.-23 + 1G> A mutation (IVS1 + 1G> A) located at the splicing site of the intron region adjacent to exon 1 of the GJB2 gene. The frames indicate the direct and reverse oligodeoxynucleotide primers; Figure 2 shows the overlap region of the original sequence of oligonucleotide primers for the detection of c.35delG mutation (p.Gly12ValfsX2) in exon 2 of the GJB2 gene. The frames indicate the forward and reverse oligodeoxynucleotide primers. The bottom shows a modification of the reverse primer, where the thymine nucleotide in the 38 position is selected with a dotted frame, replaced by guanine to create an artificial polindromny site of the restriction enzyme Bsc4I site; Figure 3 shows the overlap region of the original sequence of oligonucleotide primers for detection of c.109G> A (p.Val37Ile) in exon 2 of the GJB2 gene. The frames indicate the forward and reverse oligodeoxynucleotide primers. The bottom shows a modification of the reverse primer, where two nucleotides of thymine in the 113 position and the subsequent guanine, replaced by adenine and cytosine, respectively, are highlighted in the dotted frame, respectively, to create an artificial polindromny restriction site of the HindII endonuclease; 4 shows the detection of c.-23 + 1G> A mutation (IVS1 + 1G> A) in the intron region adjacent to exon 1 of the GJB2 gene. At the same time, A is an electrophoregram of a PCR-RFLP analysis for detecting a c.-23 + 1G> A mutation (4% agarose gel). Tracks: 1 and 8 - molecular weight marker pUC19 DNA / MspI; 2 and 3 - control of biallelic (homozygous) c.-23 + 1G> A; 4 - proband; 5 - mother; 6 - father; 7 - control without c.-23 + 1G> A. B - genealogy of the proband's family with identified genotypes for c.-23 + 1G> A mutation. In the presence of biallelic c.-23 + 1G> A, the restriction site is absent - 363 mon; in the presence of a monoallel c.-23 + 1G> A, there are two bands - 363 mon and 293 mon; in the absence of c.-23 + 1G> A, one band is visualized - 293 mo; 5 shows the detection of c.35delG mutation (p.Gly12ValfsX2) of the GJB2 gene. At the same time, A is an electrophoregram PCR-RFLP analysis for the detection of c.35delG mutation (10% polyacrylamide gel). Tracks: 1 and 8 - control without mutation c.35delG; 2 - mother; 3 - proband; 4 - control on the biallelic (homozygous) mutation of c.35delG; 5 - sibs; 6 - father; 7 - control on monoallelic (heterozygous) c.35delG mutation. B - genealogy of the proband's family with identified genotypes for c.35delG mutation. In the presence of biallelic c.35delG, the restriction site is absent - 337 mon; in the presence of a monoallel c.35delG, there are two bands - 337 mon and 305 mon; in the absence of a c.35delG deletion, one band is visualized - 305 mon; 6 shows the detection of the c.109G> A mutation (p.Val37Ile) of the GJB2 gene (exon 2). At the same time, A is an electrophoregram of a PCR-RFLP analysis for detecting the mutation c.109G> A (4% agarose gel). Tracks: 1 - molecular weight marker pUC19 DNA / MspI, 2 - control of biallelic (homozygous) mutation c.109G> A, 3 - proband, 4 - mother, 5 - father, 6 - sibs, 7 - control without c.109G> A. B - genealogy of the proband's family with identified genotypes for the c.109G> A mutation. In the presence of biallelic c.109G> A, the restriction site is absent - 175 mon; in the presence of a monoallel c.109G> A, there are two bands — 175 mon and 142 mon; in the absence of c.109G> A, one band is visualized - 142 pn.

The algorithm of the claimed method consists of three stages of sequential search for mutations in the GJB2 gene - c.-23 + 1G> A (mutation of mRNA splicing), c.35delG (p.Gly12ValfsX2) and p.109G> A (p.Val37Ile):

Stage 1 - search for the c.-23 + 1G> A mutation in the intron region adjacent to exon 1. As a result of the search, when this mutation is detected in the biallelic state (c. [- 23 + 1G> A]; [- 23 + 1G> A]), the DNA diagnosis of ARG 1A will be positive. If a mutation is detected in the monoallic state (c. [- 23 + 1G> A]; [Wt]) or in its absence (c. [Wt]; [Wt]), the DNA diagnostics of ARG 1A will be negative. In negative cases, you should proceed to the second stage. Detection of c.-23 + 1G> A mutation in the GJB2 gene is shown in Figure 4;

Stage 2 - search for c.35delG mutation in the protein-coding region of the gene in exon 2. As a result of this stage, if c.35delG is detected in the biallelic state (c. [35delG]; [35delG]) or in a compound-heterozygous state with a mutation c.-23 + 1G> A (c. [- 23 + 1G> A]; [35delG]), then the DNA diagnosis of ARG 1A will be positive. If c.35delG is detected in the monoallelic state (c. [35delG]; [Wt]) or in its absence (c. [Wt]; [Wt]), the DNA diagnostics of ARG 1A will be negative. In negative cases, proceed to the next step. The detection of the c.35delG mutation is presented in FIG. 5;

Stage 3 - search for a mutation p.109G> A, also in exon 2. As a result, when detecting p.109G> A in the biallelic state (c. [109G> A]; [109G> A]) or in compound heterozygous condition with c.-23 + 1G> A or c.35delG mutation (c. [- 23 + 1G> A]; [109G> A], c. [35delG]; [109G> A]) DNA diagnostics of ARG 1A will be positive. If a p.109G> A mutation is detected in the monoallelic state (c. [109G> A]; [Wt]) or in its absence (c. [Wt]; [Wt]), the DNA diagnosis of ARG 1A will be negative. The detection of the c.109G> A mutation is presented in Figure 6.

In conclusion, all the stages of the search for GJB2 mutations (c.-23 + 1G> A, c.35delG, c.109G> A), when the DNA diagnosis remains uninformative, under monoallelic states (Mut / Wt) there is a probability of being random heterozygous the carrier of the mutation, and the presence of another mutation in the second allele, which is in the compound-heterozygous state, or in the trans position outside the analyzed region of the GJB2 gene. In cases where none of the three mutations (Wt / Wt) will be identified, the hearing loss may be associated with other, more rare GJB2 mutations in Yakutia, or the hearing loss is not associated with mutational changes in the GJB2 gene.

The method is as follows.

1) With the informed consent of the survey (in children - after the informed consent of the parents or guardians), venous blood is taken from the cubital vein to isolate genomic DNA samples.

2) Genomic DNA is extracted using phenol-chloroform extraction, described in S. Matthew (see Matthew CC / Mathew // Methods in Molecular Biology / Ed. Walker JMYL: Human Press. - 1984. - Vol. 2. - p. 31-34).

3) Further, the obtained DNA without impurities (with ΔA ₂₆₀ / ΔA values ₂₈₀ = 1.8-2.0) is used as a template for the polymerase chain reaction to amplify significant regions of the GJB2 gene. For the amplification of the intron-exon 1 region, possibly containing variant c.-23 + 1G> A, the oligonucleotide sequences described in A. Sirmaci et al. (See Sirmaci A. The c.IVS1 + 1G> A mutation in the GJB2 gene A. Sirmaci, D. Akcayoz-Duman, M. Tekin // J. Genet., 2006. — Vol. 85 (3). P. 213 -216): (F) - 5'-CCGGGAAGCTCTGAGGAC-3 ', (R) 5'-GCAACCGCTCTGGGTCTC-3'. (see figure 1). For amplification of exon 2 sites, possibly containing variants c.35delG and c.109G> A, the original oligonucleotide sequences are used, the design of which is presented in Figures 2 and 3, respectively: c.35delG (F) - 5'-ACTCAGGTGAACAAGCTACT-3 ', ( R) - 5'-TCTTTCCAATGCTGGTGGAGTGTTTGTTCCCA-3 ';(c.109G> A) (F) - 5'-TCTTTTCCAGAGCAAACCGCCCAGAG-3 ', (R) - 5'-TGCTCATCTCCCCACACCTCTTTTGAGCGTCAA-3'.

The method is carried out using the standard composition of the reaction mixture: 1 μg of genomic DNA, the appropriate amount of each oligonucleotide, 125 μM deoxynucleoside triphosphate (Promega, USA) are placed in a buffer for PCR of the following composition (concentrations indicated for 10x buffer): 67 mM Tris-HCl, pH 8 , 6-8.8 at 20 ° С, 6.7 mM MgCl2, 16.6 mM (NH ₄ ) ₂ SO4, 0.01% Tween 20. To the resulting mixture add 5 units of thermophilic DNA polymerase. The composition of the reaction mixture is presented in the table. The amplification mode described in the method of detection of 17 mutations of the genes GJB2 and GJB6 in hereditary nonsyndromic deafness (see RU No. 2448163, cl. G01N 33/50, С12Q 1/68, publ. 04/20/2012).

4) After carrying out the amplification of the required fragments, RFLP analysis is performed for the detection of mutations using endonucleases: for c.-23 + 1G> A - AsuHPI (see Fig. 1); for detection of c. 35delG - Bsc4I (see Fig. 2); for the detection of c.109G> A - HindII (see Fig. 3). The reaction is carried out according to the protocol of the manufacturer of these endonucleases. The results of RFLP analysis are evaluated by electrophoresis: c.-23 + 1G> A and c.109G> A on a horizontal (15 x 15) 4% agarose gel; 35delG on a vertical (20 x 20) 10% polyacrylamide gel. Before applying the electrophoresis sample in the ratio of 1: 5 with paint containing 0.25% bromophenol blue, 0.25% xylene cyanol and 15% ficoll.

5) The results are detected by staining the gels with ethidium bromide followed by visualization in UV light on the transilluminator, as follows: figure 4 shows the PCR-RFLP electropherogram analysis for detecting the c.-23 + 1G> A mutation, where the presence of 363 mon alleles and 293 mon will indicate a heterozygous carriage, detection of the 363 mon allele will indicate a homozygous state; 5 shows an electrophoregram of a PCR-RFLP analysis for detecting a p.35delG mutation, where the presence of 337 mon and 305 mon alleles would indicate a heterozygous carriage, 337 mon alleles - a homozygous state; 6 shows an electrophoregram of PCR-RFLP analysis for detecting c.109G> A mutation, where the presence of 175 mon and 145 mon alleles would indicate heterozygous carriage, 175 mon alleles - on the homozygous state.

The time of the study (from the beginning of the DNA extraction of the test to the detection of the GJB2 variant) is 5 (stage 1) - 7 (all 3 stages) days.

The effectiveness of our proposed method for detecting three GJB2 mutations, c.-23 + 1G> A, c.35delG, c.109G> A, was tested on DNA samples of 393 patients with congenital hearing impairments from Yakutia, with previously identified genotypes of the GJB2 gene obtained as a result of direct sequencing by Sanger of significant regions of the GJB2 gene (intron region adjacent to exon 1 and exon 2). Of these, genotypes for these mutations were identified: c. [- 23 + 1G> A]; [- 23 + 1G> A] - 149 (37.9%); c. [35delG]; [35delG] - 14 (3.6%); c. [109G> A]; [109G> A] - 4 (1.0%); c. [- 23 + 1G> A]; [35delG] - 18 (4.6%); c. [- 23 + 1G> A]; [109G> A] - 2 (0.5%); c. [35delG]; [109G> A] -1 (0.3%); c. [- 23 + 1G> A]; [Wt] - 18 (4.6%); c. [- 23 + 1G> A]; [option without clinical significance] - 10 (2.5%); c. [35delG]; [Wt] - 3 (0.8%); c. [109G> A]; [Wt] - 3 (0.8%); without mutations c. [Wt]; [Wt] - 125 (31.8%) (see Analysis of the spectrum and frequency of GJB2 mutations in patients with congenital hearing impairment in the Republic of Sakha (Yakutia) / VG Pshennikova, N. A. Barashkov, FM Teryutin and others // Medical genetics. - 2015. - V. 14. - №6 (156). - P. 10-2327; Spectrum and Frequency of the GJB2 Gene Pathogenic Variants in Barashkov, VG Pshennikova, OL Posukh et al // PLoS One., 2016. - Vol. 11 (5): e0156300. doi : 10.1371 / journal.pone.0156300). As a result of the test of the proposed method on the DNA samples of patients identified with genotypes for mutations c.-23 + 1G> A, c.35delG, c.109G> A of the GJB2 gene, all genotypes fully corresponded to the results previously obtained using direct sequencing by Sanger gene Gjb2 Thus, the proposed method in the application turned out to be accurate and informative and can be used in the routine DNA diagnosis of hereditary deafness / hearing loss in a regional context.

Table

Reaction mixture for PCR

Option
gene
Gjb2 PCR
buffer (x10)
(µl) DNTP mix
(µl) Primer Taq polymerase
(µl) H ₂ O
(µl) Betaine
(µl) DNA
(100 ng / ml) F
(µl) R
(µl) c.-23 + 1G> A 0.8 0.5 0.39 0.54 0.2 2 2.57 one p.35delG 0.8 0.5 0.25 0.32 0.2 11.3 - one c.109G> A 0.8 0.5 0.2 0.2 0.2 4.1 eight one

Claims (1)

A method for detecting GJB2 gene mutations causing autosomal recessive type 1A deafness, including detection of the three c.-23 + 1G> A mutations, p.35delG and c.109G> A, most common in Yakutia, for carrying out significant GJB2 for amplification - the following primers are used: c.-23 + 1G> A (F) - 5'-CCGGGAAGCTCTGAGGAC-3 ', (R) - 5'-GCAACCGCTCTGGGTCTC-3'; c.35delG (F) - 5'-ACTCAGGTGAACAAGCTACT-3 ', (R) - 5'-TCTTTCCAATGCTGGTGGAGTGTTTGTTCCCA-3'; (C.109G> A) (F) - 5'-TCTTTTCCAGAGCAAACCGCCCAGAG-3 ', (R) - 5'-TGCTCATCTCCCCACACCTCCTTTGCAGCGTCAA-3', followed by analysis of restriction fragment length polymorphism using endonuclease AsuHPI, Bsc4I, HindII.

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