Deafness is among the major sensory-neurological deficits; the World Health Organization (WHO) estimates that approximately 360 million individuals with mild to severe deafness live in the world. Deafness is a heterogeneous disorder, i.e., caused by environmental and genetic factors [1]. Despite the genetic diversity of deafness in different populations, the DFNB1 locus is the cause of approximately 50% of non-syndromic deafness [2]. The GJB2 gene is located at the DFNB1 locus and has two exons [1]; it produces a protein product called connexin 26 [2]. Connexin 26 acts as an essential regulator of potassium ion (K+) homeostasis in the inner ear. Without K+ exchanges, auditory cells fail to respond to sound responses [2]. This study aimed to evaluate GJB2 gene mutations by PCR-sequencing in 31 non-syndromic deaf individuals.

In this descriptive cross-sectional study, a total of 85 unrelated deaf individuals from 85 families who were referred to Guilan General Welfare Office and Rasht Welfare Office, Rasht Deafness Center, and White Silence Deafness Center (in Anzali port) in Gilan Province, Iran, were identified. Among them, 31 patients with non-syndromic deafness and bilateral deafness, deafness from birth, and having one or more deaf relatives, ranging in age from 17 to 50 years were selected. Accordingly, the necessary blood samples were collected from them. After DNA purification, the entire length of the GJB2 gene was amplified by PCR. Besides, sequencing was performed with the reverse primer. The results of sequencing using CLC main workbench v3.5 software and online blast software (BLAST) were examined for mutations in deaf subjects, compared to the reference gene available at the NCBI site.
 Ethical considerations: With the permission of the Ethics Committee of the Islamic Azad University, Tonekabon Branch (Code: IR.IAU.TON.REC.1398.015) and with the consent of the deaf subjects or their parents, the sampling of deaf individuals was performed.

In this study, 31 non-syndromic deaf individuals (out of 85 deaf subjects) from Gilan Province were included; of them, 22(71%) and 9(29%) subjects were females and males, in sequence. The mean age of the examined deaf subjects was 33.33 years. In this study, 35 delG mutations in the GJB2 gene were detected in 16 patients (Table 1).



Of them, 14 patients were homozygous (Figure 1) and two patients were heterozygous (Figure 2). In one patient, the V153I mutation was observed as heterozygous. In one of two patients with a 35delG heterozygous mutation, the pathogenic variant of G200R was also observed as heterozygous (35delG/G200R).

Approximately 52% of the explored deaf individuals from Rasht and Bandar Anzali deaf centers presented mutations in the GJB2 gene; the most common mutation among these individuals was 35delG mutation with a frequency of 48.38%. Moreover, 35delG mutation in some studies in Isfahan with a frequency of 6.12% [1], in the Kurdish population with an allelic frequency of 13.33% [14], in Qom with a prevalence of 61.9% [15], in Tabriz with a frequency of 39% [11], and Lorestan with a frequency of 9.4% [16] was reported. The present study was more similar to Tabriz and Qom data concerning the frequency of 35delG mutation. However, in some parts of Iran, including Kurdistan, Azerbaijan, Isfahan, and neighboring countries (Turkey & Pakistan), the prevalence of this mutation is lower.
The allelic frequency of V153I mutation in several studies was measured as follows: Fars Province: about 6% [4], Isfahan: about 2.04% [1], and Lorestan: about 2.8% [16]. The V153I mutation was reported as a common mutation in some studies; however, in the present study, it was reported in only one allele and may not be highly prevalent in deaf individuals in Gilan Province.
In two studies in Shanghai, China [19] and Iran, the G200R variant was introduced as a pathogenic variant [20]. The frequency of this pathogenic variant in Morocco was reported to be almost 0.66% [21]. In a study in Iran, this pathogenic variant was reported in two alleles with a frequency of 0.05%. This pathogenic variant is in the second quarter (TM4) [22]; its importance in pathogenicity remains unknown. Mahdieh et al. found a small frequency of this variant in more than 10 studies in Iran; however, only in Gilan Province, this pathogenic variant was observed in abundance in an allele.
Due to the high rate of mutation in the GJB2 gene in Rasht and Anzali Cities in Gilan Province, it can be expected that mutation in this gene is critical in the occurrence of deafness in these cities. Due to the high prevalence of 35delG mutation concerning the genetic screening of deafness in Guilan Province, it is necessary to study the GJB2 gene as the main gene affecting non-syndromic deafness.

This study was approved by the Ethics Committee of the Islamic Azad University, Tonekabon Branch (Code: IR.IAU.TON.REC.1398.015).

This study was extracted from MSc. thesis of the first author at the Department of Genetics, Faculty of Biological Sciences, Tonekabon Branch, Islamic Azad University, Tonekabon.

Conceptualization: Najmeh Ranji and Ali Nazemi; Research and sampling method: Pedram Pouryari Biyachal; Data Analysis, Text Writing, and Review: All Authors.

The authors declared no conflicts of interest.

We thank the General Department of Welfare of Guilan Province, Welfare of Rasht City, Deaf Centers of Guilan Province located in Rasht and Bandar Anzali, and Mr. Sobhan Darvishi.

 

Reference

1. Abtahi SHR, Malekzadeh A, Soheilipour S, Salehi M, Taleban R, Rabieian R, et al. Evaluation of GJB2 and GJB6 mutations in patients afflicted with non-syndromic hearing loss. Int J Pediatr 2019; 7(2):9053-60. [DOI:10.22038/IJP.2018.34154.3017]
2. Amritkumar P, Jeffrey JM, Chandru J, Kalaimathi M, Ramakrishnan R, Karthikeyen NP, et al. Role of DFNB1 mutations in hereditary hearing loss among assortative mating hearing impaired families from South India. BMC Med Genet. 2018; 19:105. [DOI:10.1186/s12881-018-0609-6] [PMID] [PMCID]
3. Abe S, Nishio SY, Yokota Y, Moteki H, Kumakawa K, Usami SI. Diagnostic pitfalls for GJB2-related hearing loss: A novel deletion detected by Array-CGH analysis in a Japanese patient with congenital profound hearing loss. Clin Case Rep. 2018; 6(11):2111-6. [DOI:10.1002/ccr3.1800] [PMID] [PMCID]
4. Hashemi SB, Ashraf MJ, Saboori M, Azarpira N, Darai M. Prevalence of GJB2 (CX26) gene mutations in South Iranian patients with autosomal recessive nonsyndromic sensorineural hearing loss. Mol Biol Rep. 2012; 39(12):10481-7. [DOI:10.1007/s11033-012-1929-9] [PMID]
5. Missoum A. The role of gene GJB2 and connexin 26 in hearing impairment. Ukr Biochem J. 2018; 90(6):5-11. [DOI:10.15407/ubj90.06.005]
6. Zytsar MV, Barashkov NA, Bady-Khoo MS, Shubina-Olejnik OA, Danilenko NG, Bondar AA, et al. Updated carrier rates for c.35delG (GJB2) associated with hearing loss in Russia and common c.35delG haplotypes in Siberia. BMC Med Genet. 2018; 19:138. [DOI:10.1186/s12881-018-0650-5] [PMID] [PMCID]
7. Bahrami T, Jalilian N, Karbasi G, Noori-Daloii MR. Specific distribution of GJB2 mutations in Kurdistan province of Iran; Report of a relatively isolated population. J Sci Islam Repub Iran. 2017; 28(1):5-11. https://jsciences.ut.ac.ir/article_59397.html
8. Bonyadi M, Esmaeili M, Abhari M, Lotfi A. Mutation analysis of familial GJB2-related deafness in Iranian Azeri Turkish patients. Genet Test Mol Biomarkers. 2009; 13(5):689-92. [DOI:10.1089/gtmb.2009.0026] [PMID]
9. Mahmoodi H, Mohamadiari S, Sohrab jaidari M, Kordi S, Bakhtiari S, Mahdieh N. [The frequency of mutations in GJB2 gene in deaf subjects referring to the welfare center of Ilam: lack of 35delG mutation (Persian)]. J ilam Univ Med Sci. 2014; 22(3):41-5. https://www.magiran.com/paper/1299261
10. Ahangari N, Masoudi M, Poursadegh A, Nejatizadeh A. [Investigating the relationship of genetic mutations in GJB2 and linkage analysis of DFNB4 Locus in a group of non-syndromic hearing impaired people with autosomal recessive inheritance in Hormozgan (Persian)]. Jorjani Biomed J. 2014; 2(2):11-8. http://goums.ac.ir/jorjanijournal/article-1-311-en.html
11. Jahangiri S, Onsori H. [Investigation of the GJB2 gene mutations among subjects with non-syndromic sensorineural hearing loss (Persian)]. Razi J Med Sci. 2017; 24(155):33-9. http://rjms.iums.ac.ir/browse.php?a_id=4475&sid=1.&slc_lang=en
12. Kalay E, Caylan R, Kremer H, de Brouwer AP, Karaguzel A. GJB2 mutations in Turkish patients with ARNSHL: Prevalence and two novel mutations. Hear Res. 2005; 203(1-2):88-93. [DOI:10.1016/j.heares.2004.11.022] [PMID]
13. Minarik G, Ferakova E, Ficek A, Polakova H, Kadasi L. GJB2 gene mutations in Slovak hearing-impaired patients of Caucasian origin: Spectrum, frequencies and SNP analysis. Clin Genet. 2005; 68(6):554-7. [DOI:10.1111/j.1399-0004.2005.00529.x] [PMID]
14. Azadegan-Dehkordi F, Bahrami T, Shirzad M, Karbasi G, Yazdanpanahi N, Farrokhi E, et al. Mutations in GJB2 as major causes of autosomal recessive non-syndromic hearing loss: First report of c.299-300delAT mutation in Kurdish population of Iran. J Audiol Otol. 2019; 23(1):20-6. [DOI:10.7874/jao.2018.00185] [PMID] [PMCID]
15. Naddafnia H, Noormohammadi Z, Irani S, Salahshoorifar I. Frequency of GJB2 mutations, GJB6-D13S1830 and GJB6-D13S1854 deletions among patients with non-syndromic hearing loss from the central region of Iran. Mol Genet Genomic Med. 2019; 7(7):e00780. [DOI:10.1002/mgg3.780] [PMID] [PMCID]
16. Sepahvand M, Kahrizi K, Daneshi A, Riaz Alhosseini Y, Mohseni Marzieh BN. [Relative frequency of GJB2 gene mutations in autosomal recessive non-syndromic hearing loss (ARNSHL) patients in Lorestan population (Persian)]. Yafte. 2007; 8(2):89-95. http://yafte.lums.ac.ir/article-1-1018-en.html
17. Bazazzadegan N, Nikzat N, Fattahi Z, Nishimura C, Meyer N, Sahraian S, et al. The spectrum of GJB2 mutations in the Iranian population with non-syndromic hearing loss--a twelve year study. Int J Pediatr Otorhinolaryngol. 2012; 76(8):1164-74. [DOI:10.1016/j.ijporl.2012.04.026] [PMID]
18. Eyerci N, Altaş E, Pirim I. GJB2 mutations in Turkish patients with nonsyndromic hearing loss. Meta Gene. 2016; 10:56-60. [DOI:10.1016/j.mgene.2016.10.006]
19. Yu X, Lin Y, Xu J, Che T, Li L, Yang T, et al. Molecular epidemiology of Chinese Han deaf patients with bi-allelic and mono-allelic GJB2 mutations. Orphanet J Rare Dis. 2020; 15:29. [DOI:10.1186/s13023-020-1311-2] [PMID] [PMCID]
20. Hashemzadeh Chaleshtori M, Hoghooghi Rad H, Dolati M, Sasanfar R, Hoseinipour A, Montazer Zohour M, et al. Frequencies of mutations in the connexin 26 Gene (GJB2) in two populations of Iran (Tehran and Tabriz). Iran J Publ Health. 2005; 34(1):1-7. https://pesquisa.bvsalud.org/portal/resource/pt/emr-71101
21. Bakhchane A, Bousfiha A, Charoute H, Salime S, Detsouli M, Snoussi K, et al. Update of the spectrum of GJB2 gene mutations in 152 Moroccan families with autosomal recessive nonsyndromic hearing loss. Eur J Med Genet. 2016; 59(6-7):325-9. [DOI:10.1016/j.ejmg.2016.05.002] [P;D]
22. Mahdieh N, Rabbani B, Wiley S, Akbari MT, Zeinali S. Genetic causes of nonsyndromic hearing loss in Iran in comparison with other populations. J Hum Genet. 2010; 55(10):639-48. [DOI:10.1038/jhg.2010.96] [PMID]