Frequency of c.35delG Mutation in GJB2 Gene (Connexin 26) in Syrian Patients with Nonsyndromic Hearing Impairment

Background Hearing impairments (HI) are the most common birth defect worldwide. Very large numbers of genes have been identified but the most profound is GJB2. The clinical interest regarding this gene is very pronounced due to its high carrier frequency (0.5–5.4%) across different ethnic groups. This study aimed to determine the prevalence of common GJB2 mutations in Syrian patients with profound sensorineural HI. Methods We carried out PCR, restriction enzyme based screening, and sequencing of 132 Syrian patients diagnosed clinically with hereditary deafness for different GJB2 mutations. Results The result revealed that, in GJB2 gene, c.35delG is the most prevalent among affected studied subjects (13.64%), followed by c.457G>A (2.4%). Conclusion The benefit of this study on the one hand is its first report of prelingual deafness causative gene mutations identified by sequencing technology in the Syrian families. It is obvious from the results that the deployment in biomedical research is highly effective and has a great impact on the ability to uncover the cause of genetic variation in different genetic diseases.


Introduction
Hearing impairments (HI) are one of the most common detected neonatal defects in the world [1]. Of every 1000 newborns, at least two have permanent bilateral sensorineural HI of more than 40 dB [2]. The causes of HI are equally shared between environment and genetics [3]. Genes causing HI are numerous and they cause isolated HI (nonsyndromic HI) in 70% of cases or in association with other medical abnormalities (syndromic HI) in the remaining 30% of cases [4].
HI occurs more in developing than developed countries [4]. Reported mutations occur more frequently in GJB2 gene, which encodes the gap junction protein connexin 26 (beta-2, GJB2) [5]. GJB2 related HI is sometimes severe but the spectrum of variation severity is wide [6]. This gene is of special interest because of its high carrier frequency in many different ethnic groups (0.5-5.4%) [7,8].
35delG is the most frequent mutation reported in the GJB2 gene [8]. It is found in more than half of all GJB2 mutations discovered so far [9]. This mutation creates a frameshift resulting in a short nonfunctional truncated form of the protein [10]. Additional mutations are continuously discovered worldwide but very few were done in Middle Eastern countries. This study aimed to determine the prevalence of common GJB2 mutations in Syrians patients with profound sensorineural HI.

Material and Methods
This study was approved by the Ethical committee of the medical faculty (Syria). All subjects were recruited from Aleppo, which lies in the northwestern region of Syria, with the

Results
Two different DNA sequence variants c.35delG and c.457G>A were detected in 54 Syrian families.

Discussion
The purpose of this study was to identify the genetic basis of hereditary hearing impairment in Syrian patients with nonsyndromic HI, by applying Sanger sequencing leading to the results described above. The 35delG variant which is common worldwide was detected in 16.7% of Syrian families with nonsyndromic moderate to profound HI. Our results differ from a previous study by Al-Achkar et al. who found 35delG mutation in 30% of Syrian families, almost double that in our study [12]. This conflict is probably due to the difference in study areas. Geographical difference in prevalence of congenital deafness was also found previously in Italy [13]. 35delG mutation takes place by deletion of one guanine (G) in a run of six guanines extending from position 30 to position 35 in the GJB2 gene causing a frameshift of the coding sequence leading to premature chain termination at the twelfth amino acid [9]. Other studies in small groups found that this variant is responsible for nonsyndromic recessive deafness in a Muslim-Israeli village in the lower Galilee [14]. The frequency of 35delG variant is very high in Spain, Italy, and Israel [15]. It accounts for approximately 50% of cases [16] which suggests an evidence for an ancient deletion mutation that had spread in Europe and Middle-East.
The c.457G>A variant was found in about one-sixth of Syrian patients with HI. Same similar DNA variant had already been described in many other Asian countries [16,17]. c.457G>A p.Val153Ile (rs11033186) in the ClinVar database is classified as a benign/likely benign variant. According to ExAc database world frequency of p.457G>A is 0.01057 (1279/121044 studied chromosomes, of which 922/16488 in South Asia and 299/66564 in Europe). However our results are not strong enough to change the annotation of this variant to likely pathogenic, and more studies are needed in this regard.
Based on these results we suggest a routine screening for GJB2 gene mutations in Syrian population which could enhance the chance of detecting affected newborns with sporadic nonsyndromic HI at an early stage and consequently improves the current diagnostic and therapeutic options. Since GJB2 gene has only one coding exon molecular diagnosis of mutations can be made simple and cost-effective for a daily medical practice. Important for future studies is an increase in selected sample sizes in order to obtain more information regarding possible novel genes and their mutations involved in the hearing process and their faults in hearing impairment.

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