RU2791878C1 - Method for preimplantation genetic testing of non-syndromic sensorineural hearing loss - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for diagnosing a monogenic disease, non-syndromic sensorineural hearing loss, under conditions of preimplantation genetic testing (PGT) is described. Diagnostics of pathogenic variants NC_000013.10:g.20763691del (NM_004004.6:c.35delG, p.Gly12fs) and NC_000013.10:g.20763554del (NM_004004.6:c.167del, p.Leu56fs) in the GJB2 gene is carried out for use within the framework of PGT of a monogenic disease, non-syndromic neurosensory hearing loss using direct and indirect diagnostics. The method has high specificity and efficiency, as well as versatility in relation to bio-samples of various types: single cells, the product of whole genome amplification, total DNA isolated from different tissues.

EFFECT: in this case, the method can be used not only for the detection of a specific pathogenic variant, but also for any other pathogenic variant or several in the GJB2 gene using indirect detection.

1 cl, 1 tbl, 1 ex

Description

The invention relates to preimplantation genetic testing of monogenic diseases. Currently, there are more than 350 million people in the world suffering from a rare disease (according to the RARE Project). The total number of such diseases, according to the estimates of the European Organization for Rare Diseases (EURORDIS), varies from 5 to 7 thousand. At the same time, about 80% of rare diseases have a genetic cause. The known genetic basis of the disease makes it possible to predict with high accuracy not only the health of an already born child, but also to assess the risk of having such a child by analyzing the genotypes of the parents, as well as to conduct genetic diagnostics at the earliest stages. Preimplantation genetic testing (PGT) of a monogenic disease is becoming a powerful tool for the prevention of such diseases.

The present invention relates to a method for preimplantation genetic testing of non-syndromic sensorineural hearing loss. Non-syndromic sensorineural hearing loss is an autosomal recessive hereditary hearing disorder. It is known that only about 3% of people in the world are carriers of mutations in the GJB2 gene, and that mutations in this gene are responsible for about a third of cases of severe hearing loss among children and young people. The disease non-syndromic neurosensory hearing loss leads to partial or complete hearing loss in the absence of disorders from other organs and systems. With an autosomal recessive type of inheritance of the disease, the probability of having a child with this disease in the family is 25%.

Non-syndromic sensorineural hearing loss can be caused by pathogenic genetic variants in the GJB2 gene located on chromosome 13. [Vona, B. et al. Non-syndromic hearing loss gene identification: A brief history and glimpse into the future. Molecular and Cellular Probes, 2015; 29(5), 260-270] This gene encodes the protein Connexin 26, which is involved in the creation of intercellular contacts, providing the transport of ions and small molecules between the cells of the inner ear.

PGT for non-syndromic neurosensory hearing loss is performed for families with a confirmed molecular genetic nature of the disease. It is important to note that the substantiation of the pathogenicity and causation of genetic variants occurs before the PGT of a monogenic disease and is not included either in the goals and objectives of the PGT of a monogenic disease, or in the complex of measures for conducting PGT of a monogenic disease. Pathogenicity is assessed according to the international standard - according to the criteria described in 2015 by the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) in the search for the molecular genetic cause of the disease. PGT is recommended for families at high risk of having a child with a severe (incurable) hereditary disease with established pathogenic variants that cause this risk. PGT allows you to select from all IVF (in vitro fertilization) embryos, embryos without pathogenic variants in the compound heterozygote and, therefore, without the risk of developing the disease.

The main problem in the genetic diagnosis of embryos is the small initial amount of biomaterial, since the biopsy contains from one to three cells. In this case, to improve the efficiency and accuracy of the analysis, it is important both to completely eliminate the possibility of contamination and to neutralize the possible effect of uneven and/or incomplete amplification, as well as degradation of the biomaterial. This requires the development of a test system with special characteristics. At the same time, the test system is being developed taking into account the possibility of using different types of biomaterial - total deoxyribonucleic acid (DNA) isolated from different tissues, the product of Whole Genome Amplification (WGA), as well as single cells. The combination of versatility with respect to the biomaterial with stepwise amplification of target fragments makes it possible to analyze several pathogenic variants for a single sample, including on single cells, and also to identify the situation of incomplete amplification, contamination, or degradation of the sample. Another feature of PGT is the lack of information about the biological characteristics of the embryo: unlike an adult, an embryo can have any chromosomal abnormalities that complicate the task of assessing the status of an embryo for a specific genetic variant. Therefore, a test system for PGT of a monogenic disease should make it possible to identify such cases and evaluate their impact on the reliability of the diagnostic result.

The closest technical solution is to conduct PGT non-syndromic neurosensory hearing loss, proposed by G. Altarescu and colleagues [G. Altarescu et al, 2009] for the detection of mutations in the GJB2 gene using multiplex nested PCR in 2 rounds on single cells, using direct mutation diagnostics and indirect diagnostics using the analysis of the inheritance of alleles of polymorphic markers linked to a pathogenic variant. An important difference of our approach is the whole genome amplification before diagnostics, which allows, if necessary, to carry out reactions on the biomaterial of the same embryo under different conditions in order to achieve amplification of the maximum number of polymorphic markers. Whole genome amplification also allows additional testing for recommended embryos (eg screening for chromosomal abnormalities) without re-biopsy of the embryo. Also, the advantage of our approach is the higher specificity of primers, which minimizes the likelihood of amplification of nonspecific PCR products.

The PGT method presented by us for non-syndromic neurosensory hearing loss solves the problem of developing a more accurate method for preimplantation genetic testing of this monogenic disease without the use of expensive devices and reagents, which could be used on various types of biomaterial: DNA isolated from different tissues, a product of whole genome amplification (WGA), single cells.

The technical result was the creation of a test system for the diagnosis of pathogenic variants (the nucleotide number in the reference sequence of genomic DNA is indicated by the prefix NC, the nucleotide number in the reference sequence of the coding transcript is indicated by the prefix NM): NC_000013.10:g.20763691del(NM_004004.6:c.35delG , p.Gly12fs) and NC_000013.10:g.20763554del(NM_004004.6:c.167del, p.Leu56fs) in the GJB2 gene [Snoeckx, R.L. et al. GJB2 Mutations and Degree of Hearing Loss: A Multicenter Study. The American Journal of Human Genetics, 2005; 77(6), 945-957] with a dual detection system - direct and indirect. Such a system is necessary when working with a small amount of biomaterial, since unstable amplification can lead to loss of information or reduced accuracy of the analysis. Direct diagnosis involves the analysis of the direct presence-absence of a pathogenic variant. In this case, for genetic variants NC_000013.10:g.20763691 del(NM_004004.6:c.35delG, p.Gly12fs) and NC_000013.10:g.20763554del(NM_004004.6:c.167del, p.Leu56fs) of the deletion type (absence of several nucleotides, English. Deletion del) primers were selected that allow detection of the mutation by the difference in the lengths of the fragments using capillary electrophoresis. Indirect diagnosis consists in the analysis of the inheritance of molecular genetic markers linked to a mutation, i.e. inherited with it. To do this, at a distance of no more than 3 mB (which corresponds to 3% crossover on average) from the GJB2 gene, polymorphic loci called STR (short tandem repeat - short tandem repeat) were selected in each direction with a heterozygosity of at least 0.70 to ensure maximum informativity of indirect diagnostics. STRs are repeats of 2 or more nucleotides located one after another (for example, an adenine-cytosine (AC) pair repeated several times in a row: ACACACACA) and are present in large numbers in the human genome. The number of repeats in each of them may vary from individual to individual, and may also be different in the same person on 2 homologous chromosomes. Heterozygosity above 0.70 means that there is a high probability that in the same person the number of nucleotide repeats in this STR on one chromosome will differ from the number of repeats in the same STR on the homologous chromosome, in other words, the alleles of this marker in this person will differ in length. When amplifying a fragment containing such a marker, amplicons of two different lengths will be obtained. By analyzing the number of repeats in several markers surrounding the pathogenic variant and examining how they are inherited in the tested family, linkage between the alleles of the markers and the pathogenic variant can be established. The diagnostic value of studying the number of repeats in these markers in embryos is that by which allele of each of the markers was inherited by the embryo, it can be judged whether the embryo inherited the GJB2 gene carrying the pathogenic variant, or whether it inherited the GJB2 gene from another , a homologous chromosome that does not contain a pathogenic variant. For each of these loci, primers were selected for amplification by the type of nested or semi-nested PCR in 2 rounds, which makes it possible to increase the accuracy and efficiency of amplification. The test system included 12 STR loci for the GJB2 gene: D13AC19.7, D13S1316, D13AC20.1, D13S141, D13S175, D13S633, D13S250, D13S1236, D13S1275, D13S292, D13S1243, D13S283. Primers for amplification are located on the 13th chromosome in the region of coordinates 19907040-25600789 (according to hg19). The sequence of primers for amplification of DNA fragments containing the listed STR loci are indicated in the claims in the list of SEQ ID NO 1-45. It is important to note that during the selection of primers, a number of special requirements were observed: the length of the product with external primers for the first round of PCR should not exceed 500 bp. (for production from fragments obtained by whole genome amplification), product length from internal primers for the second round of PCR from 120 to 350 bp, high specificity of external primers, annealing temperature does not differ by more than 1°C.

Preparatory stage of the PGT

At the preparatory stage, the test system is developed: selection of amplification conditions that are optimal for primers to work, analysis of the efficiency and specificity of PCR amplification in both rounds, assessment of the universality of the test system for various types of biosamples (DNA, NGA product, single cells). When testing the test system, stock dilutions of primers were prepared with a concentration of 100 mM, and working dilutions of primer combinations (a combination of pairs of primers for rounds 1 and 2 of PCR) with a concentration of JtM of each primer in solution. Since different types of matrices can be used as part of the diagnostics of clinical material, two biopsies of single cells in a special lysis buffer (1 * PCR Buffer, 0.1% Tween-20, 0.1% Triton X-100, 1 µg Proteinase K), two samples of the products of whole genome amplification of embryonic biopsies (WGA), as well as total DNA of family members isolated from blood, for compiling a pedigree and identifying the linkage of a pathogenic variant with alleles of polymorphic markers.

In nested and semi-nested PCR, amplification is carried out in two steps. At the first stage, multiplex PCR is performed with all external primers for all loci included in the test system to enrich the sample with all target fragments. At the second stage, individual amplification of each fragment with internal primers is carried out.

Semi-nested PCR

For the first stage, external highly specific primers were selected to amplify fragments from 300 to 500 bp. For the second stage, primers were selected to amplify fragments no longer than 350 base pairs, and labels were introduced for detection by fragment analysis. The sequence of primers for amplification of DNA fragments containing STR loci are indicated in the claims in the list of SEQ ID NO 1-45. The PCR mixture for the first round of amplification contained 1×PCR buffer with Mg2+ (Evrogen, Russia), 0.1 mM of each deoxynucleotide, 0.15 μM of each primer, 2.5 U/μl of HsTaq DNA polymerase (Evrogen, Russia), 6% dimethyl sulfoxide (DMSO ) and 1 µl total DNA or 2.5 µl WGA or 5 µl lysis buffer with sample as template. The first stage of amplification was carried out according to the following protocol: the stage of denaturation at 94°C for 2 minutes, 30 cycles with a decrease in the annealing temperature of the primers from 62 to 45°C in each, the stage of completion of all templates at 72°C for 10 minutes. Next, the products of the 1st stage were separated into individual tubes with one pair of primers per specific locus.

The PCR mixture for the second stage included 1×PCR buffer with Mg2+ (Evrogen, Russia), 0.5×RediLoad™ loading buffer (Thermo Fisher Scientific, USA), 0.2 mM of each deoxynucleotide, 0.2 μM of each primer, 1U/μl of HsTaq DNA polymerase (Evrogen, Russia), 6% dimethyl sulfoxide (DMSO) and 1 μl of the PCR product of the first stage of amplification as a template. The second stage of amplification was carried out according to the following protocol: denaturation stage at 95°C for 2 minutes, 35 cycles: denaturation at 95°C for 30 seconds, primer annealing at 57°C for 30 seconds, template synthesis at 72°C for 1 minute, completion of all templates 72°C 5 minutes. Evaluation of the efficiency and specificity of amplification was performed using electrophoresis in 2% agarose gel. The result of electrophoresis in agarose gel allows you to determine the required degree of dilution of the amplification products for application to fragment analysis (amplification products of DNA of family members).

Fragment analysis of amplification products was performed using capillary electrophoresis on a 3130x1 Genetic Analyzer (Applied Biosystems, USA). Based on the results of the fragment analysis, a pedigree is compiled and informative polymorphic STR loci for each family are noted, which will later be used in clinical diagnostics. Loci are divided into non-informative (the carrier of the pathogenic variant is homozygous for this locus), semi-informative (alleles for this marker are the same on some and parental chromosomes), informative (alleles of this marker are different on all chromosomes of the parents, which makes it possible to distinguish each of them when analyzing the genotype of the embryo ). Detection of the pathogenic variant NC_000013.10:g.20763691 del (NM_004004.6:c.35delG, p.Gly12fs) was carried out using amplification primers:

Outer: 5'-GTAGCACACGTTCTTGCAGC-3' and 5'-TTCCAGAGCAAACCGCC-3'

Internal: 5'-(FAM)ACGGTGAGCCAGATCTTTCC-3' and 5'-TTCCAGAGCAAACCGCC-3'

Detection of the pathogenic variant NC_000013.10:g.20763554del (NM_004004.6:c.167del, p.Leu56fs) was carried out using amplification primers:

Outer: 5'-GTAGCACACGTTCTTGCAGC-3' and 5'-TTCCAGAGCAAACCGCC-3'

Internal: 5'-GTAGCACACGTTCTTGCAGC-3' and 5'-(FAM)GATCTGGCTCACCGTCCT-3'

Example 1

Patients A

Family A turned to the Center for Genetics and Clinical Research, in which a child was born with a disease of non-syndromic neurosensory hearing loss with compound heterozygous carriage of pathogenic variants NC_000013.10:g.20763691del(NM_004004.6:c.35delG,p.Gly12fs) and NC_000013.10:g. 20763554del(NM_004004.6:c.167del, p.Leu56fs) in the GJB2 gene. The couple was recommended to undergo PGT for the disease non-syndromic neurosensory hearing loss as part of IVF to select embryos that did not inherit the disease.

Family haplotyping

At the first stage, biomaterial (peripheral blood) of family members was obtained to detect the pathogenic variant and identify linkage groups of alleles of polymorphic markers. 12 STR loci were analyzed. Of these, 7 were informative for the mother and/or father. Thus, embryo samples were tested only for informative markers.

The definition of linkage groups was carried out as follows. Alleles of polymorphic markers that match in the parent-carrier of the pathogenic variant in the GJB2 gene and in a child with non-syndromic sensorineural hearing loss are recognized as linked to the corresponding pathogenic variant and to each other. Alleles of polymorphic markers that do not match in such parents and a child with a disease are considered linked to each other and to the normal allele of the GJB2 gene. Indirect diagnostics makes it possible to draw a conclusion about the inheritance of a normal or mutant allele of a gene even if the allele is lost or the reaction fails during amplification of DNA fragments for direct diagnostics.

As a result of haplotyping and determination of linkage groups for family A from the example, the results presented in Table 1 were obtained. The alleles indicated on the same line are located on the same chromosome, that is, they represent the linkage group. Thus, for each family member, 2 linkage groups are presented, corresponding to each of the two thirteenth chromosomes. N in the table indicates the absence of a pathogenic variant, mut - its presence. Numerals indicate the lengths of amplicons in base pairs; their lengths depend on the number of repeats in the STR marker. The pathogenic variant NC_000013.10:g.20763691del(NM_004004.6:c.35delG, p.Gly12fs) in the GJB2 gene is designated in the table as GJB2 c.35delG. The pathogenic variant NC_000013.10:g.20763554del (NM_004004.6:c.167del, p.Leu56fs) in the GJB2 gene is designated in the table as GJB2 c.167del.

As a result of haplotyping, it was concluded that the following alleles of STR markers were linked in the patient's partner with the pathogenic variant: D13AC19.7 - 197, D13AC20.1 - 218, D13S141 - 124, D13S175 - 101, D13S1236-117, D13S1275-207, D13S1243-251.

The following alleles of STR markers were linked in the patient with the pathogenic variant: D13AC19.7 - 191, D13AC20.1 - 216, D13S141 - 124, D13S175 -101, D13S1236-115, D13S1275-201, D13S1243 - 251.

Preimplantation genetic testing

In the IVF cycle, 3 embryos were obtained, a biopsy was performed on the 5th day of development (in the IVF clinic), the biopsy in WGA buffer (1xPBS (Invitrogen, USA), 1% polyvinylpyrrolidone (PVP) (Fertipro, Belgium)) was sent to the laboratory "Genetiko ". To control contamination at different stages of working with a sample, the laboratory developed a system of controls: control of contamination of the biopsy buffer, control of contamination during transportation (one test tube with buffer is not opened by the embryologist), control of contamination of each sample (sample of the medium from the last wash drop of biopsy material). All these controls, together with the samples, go through the stage of whole genome amplification, after which the smallest amount of DNA that contaminated the controls will be noticeable. Genome-wide amplification was performed using the commercial SurePlex kit (lllumina, USA).

The product of genome-wide amplification, as well as the DNA of all family members, was amplified at stage 1 in multiplex PCR with primers for the detection of the pathogenic variant and primers for polymorphic markers informative for the A family in accordance with the protocol for the test system developed as part of the preparatory stage. At stage 2, amplification was performed for each marker separately in accordance with the developed protocol for the test system. In this way, the linkage groups inherited by each embryo were established. The results obtained are presented in table 1.

According to the results of direct and indirect diagnostics, 2 embryos (embryo 1 and 3) did not inherit the disease, and 1 embryo (embryo 2) had a haplotype corresponding to the inherited disease. Embryo 1 turned out to be a carrier of the pathogenic variant NC_000013.10:g.20763691 del(NM_004004.6:c.35delG,p.Gly12fs). With the consent of the parents, preimplantation genetic screening for chromosomal abnormalities was performed for embryos that did not inherit the disease. Based on the results of all tests carried out, embryo 1 was recommended for transfer. Embryo 3 was not recommended due to the identified chromosomal abnormalities.

Primer sequence list:

D13AC19.7

SEQ ID NO 1: 5'-CACTGTGCCTGGTCCG-3' (Fout);

SEQ ID NO 2: 5'-CAAGTGACAGCCCAATGCTGG-3' (Rout);

SEQ ID NO 3: 5'-*ACACCTGGCTCTGTAACGTC-3' (Rin),

D13S1316

SEQ ID NO 4: 5'-TGTGAGACCTCTCGCCATT-3' (Fout);

SEQ ID NO 5: 5'-CACATAGCTGAAGAAAGAATAGGTG-3' (Rout)

SEQ ID NO 6: 5'-CTACTGGGGAGGCTGG-3' (Fin);

SEQ ID NO 7: 5'-CATGTCTCTGAATCGCTTTT-3' (Rin),

D13AC20.1

SEQ ID NO 8: 5'-GCGTTGCTCTTGCATTCCAT-3' (Fout);

SEQ ID NO 9: 5'-AGGCTGTGTTTGATACTGTAATTTG-3' (Rout)

SEQ ID NO 10: 5'-*TCTCCAGTGGGATGAGCAGA-3'(Fin),

D13S141

SEQ ID NO 11: 5'-CAGCTTCCCCTTTCAGGCAG-3' (Fout);

SEQ ID NO 12: 5'-CACTGCCTGTATTCCCGAGG-3' (Rout)

SEQ ID NO 13: 5'-GTCCTCCCGGCCTAGTCTTA-3' (Fin);

SEQ ID NO 14: 5'-ACCACGGAGCAAAGAACAGA-3' (Rin),

D13S175

SEQ ID NO 15: 5'-CGCGTGTTTGCTCTGTAAGG-3' (Fout);

SEQ ID NO 16: 5'-ACATCTTGAAATGATTACCCGCAA-3' (Rout)

SEQ ID NO 17: 5'-TATTGGATACTTGAATCTGCTG-3' (Fin);

SEQ ID NO 18: 5'-TGCATCACCTCACATAGGTTA-3' (Rin),

D13S633

SEQ ID NO 19: 5'-TAAGACTCGAACTGCTTTTGCTTCT-3' (Fout);

SEQ ID NO 20: 5'-CCCTCCAATCTCTTTCATATCCCA-3' (Rout)

SEQ ID NO 21: 5'-ACCTAAAATCTCCCAAATTGT-3' (Fin);

SEQ ID NO 22: 5'-ACGCTTTACCCCTGAAATC-3' (Rin),

D13S250

SEQ ID NO 23: 5'-ACTGTCCCCCTATTTTTATTTCCT-3' (Fout);

SEQ ID NO 24: 5'-TGCTAGAGTATTGAAAACCAGACT-3' (Rout)

SEQ ID NO 25: 5'-ACAGGATATCCCCCAGCAG-3' (Fin);

SEQ ID NO 26: 5'-GAAGACAGGGTTATGCCCA-3' (Rin),

D13S1236

SEQ ID NO 27: 5'-AAGTGACCTGAACAGCCCAA-3' (Fout);

SEQ ID NO 28: 5'-CCCAACTCCCCTTCCAGTTC-3' (Rout)

SEQ ID NO 29: 5'-GCACTTGGCCTGGGTAA-3' (Fin);

SEQ ID NO 30: 5'-AAGGGGCTGGCTCTTCA-3' (Rin),

D13S1275

SEQ ID NO 31: 5'-AGCGTCTAAGTAAAGTGGGTGC-3' (Fout);

SEQ ID NO 32: 5'-TTGTCCATTTGTTGGAGTGTGAAT-3' (Rout)

SEQ ID NO 33: 5'-ATCACTTGAATAAGAAGCCATTTG-3' (Fin);

SEQ ID NO 34: 5'-CCAGCATGACCTTTACCAG-3' (Rin),

D13S292

SEQ ID NO 35: 5'-TGTCTTCTCAAGGATTCGGGGA-3' (Fout);

SEQ ID NO 36: 5'-GTTCCACTGTCCAGTTCTGGGTTT-3' (Rout)

SEQ ID NO 37: 5'-TAATGGCGGACCATGC-3' (Fin);

SEQ ID NO 38: 5'-TTTGACACTTTCCAAGTTGC-3' (Rin),

D13S1243

SEQ ID NO 39: 5'-GTTCTAGCCAGAGTGAGGACC-3' (Fout)

SEQ ID NO 40: 5'-GCAGTACATCATGACAAGTTGAT-3' (Rout)

SEQ ID NO 41: 5'-TGCTGACAGGCTACAGAACTTT-3' (Fin);

SEQ ID NO 42: 5'-CTCTTGTGCAGGTATAGGGG-3' (Rin),

D13S283

SEQ ID NO 43: 5'-TGTCATTGTCAGCTCACTTCTA-3' (Fout);

SEQ ID NO 44: 5'-GCCATTCCAAGCGTGT-3' (Rout)

SEQ ID NO 45: 5'-TCTCATATTCAATATTCTTACTGCA-3' (Fin)

Claims (10)

A method for preimplantation genetic testing of non-syndromic sensorineural hearing loss, involving the detection of the inheritance of pathogenic variants NC_000013.10:g. p.Leu56fs), in the GJB2 gene, including a double detection system - direct and indirect, where direct detection is carried out using amplification primers: for the pathogenic variant NC_000013.10:g.20763691del (NM_004004.6:c.35delG, p.Gly 12fs): external: 5'-GTAGCACACGTTCTTGCAGC-3' and 5'-TTCCAGAGCAAACCGCC-3', internal: 5'-(FAM)ACGGTGAGCCAGATCTTTCC-3' and 5'-TTCCAGAGCAAACCGCC-3'; for the pathogenic variant NC_000013.10:g.20763554del (NM_004004.6:c. 167del, p.Leu56fs): external: 5'-GTAGCACACGTTCTTGCAGC-3' and 5'-TTCCAGAGCAAACCGCC-3', internal: 5'-GTAGCACACGTTCTTGCAGC-3' and 5'-(FAM)GATCTGGCTCACCGTCCT-3', and indirect detection is carried out using primers for the analysis of the inheritance of molecular genetic markers of the STR type linked to the pathogenic variant, selected from SEQ ID NO 1-45, while using primers directed to those STRs whose alleles are different on the chromosomes of at least one parent -mutation carrier, where the outer primers are designated as Fout (forward primer) and Rout (reverse primer), and the internal primers are designated as Fin (forward primer) and Rin (reverse primer), while the diagnosis is carried out in two stages of semi-nested PCR: on the first at the stage, multiplex PCR is carried out with external primers, at the second stage, individual PCR of each fragment is carried out with internal primers for STR, and simple PCR and fragment analysis are carried out to determine the pathogenic variant in the GJB2 gene.