GJB2 mutations in Iranian Azeri population with autosomal recessive nonsyndromic hearing loss (ARNSHL): First report of c.238 C>A mutation in Iran

Abstract Objective Autosomal‐recessive nonsyndromic hearing loss (ARNSHL) is a heterogeneous genetic disorder. Mutations in the gap junction protein beta 2 (GJB2) gene, encoding connexin 26, are a significant cause of ARNSHL in different ethnic groups. This study aimed to identify the frequency and type of GJB2 mutations in the Iranian Azeri population. Methods Fifty unrelated families presenting ARNSHL in Ardabil Province, the northwest of Iran, were studied to determine the frequency and type of GJB2 mutations leading to ARNSHL. ARMS‐PCR screened all DNA samples to detect c.35delG; p. Gly12Val mutation. In addition, normal samples for c.35delG; p. Gly12Val were analyzed by direct sequencing for other GJB2 mutations. Result Of the fifty families, 13 (26%) showed a GJB2 gene mutation, with c.35delG; p. Gly12Val mutation was the most prevalent one that occurred in eight (61.5%) out of the 13 families. Of the families, two were homozygous for c.358‐360delGAC; p. Glu120del mutation, and one was homozygous for c.290dupA; p. Tyr97Ter and c.299–300delAT; p. His100Arg mutations. Also, we detected a novel mutation, c.238C>A; p. Gln80lys, in one of the families. Conclusion Our findings are comparable to previous studies, indicating c.35d3lG; p. Gly12Val mutation in the GJB2 gene is the most common cause of GJB2‐related hearing loss in the Iranian Azeri population. Furthermore, our study highlights the significance of ARNSHL screening programs of live births based on local population data in Iran.


| INTRODUC TI ON
Hearing loss (HL) is a common sensory disorder that affects millions of people worldwide, with an incidence of one in every 500 newborns (http://heari ng.scree ning.nhs.uk/natio nalprog). There are two different genetic forms of HS, including syndromic and nonsyndromic forms. There is a preponderance of nonsyndromic hearing loss (NSHL) in different populations, in which 70% represents an autosomal recessive pattern of inheritance. 1 Autosomal recessive nonsyndromic hearing loss (ARNSHL) is highly heterogeneous, with about 100 mapped loci and over 70 causative genes (http://hered itary heari ngloss.org). 2 In different populations, especially Asian and European descent, a mutation in GJB2 and GJB6 is a major cause of ARNSHL. GJB2 is located on the locus of DFNB1 (chromosome 13q12), which encodes the connexin 26 protein as a particular gap junction belonging to the families of intercellular channels for cell-to-cell interaction and small molecule distribution.
GJB2 protein has been present in the human cochlea during the development of the embryo in the 22nd week, and it has an essential function in the homeostasis of the inner ear by recycling potassium ions. 3 Meanwhile, several studies have shown in Iran that no mutation in the GJB6 gene is involved in congenital hearing loss.
In other words, this probably suggests that mutations in GJB6 gene, especially deletions, clearly do not have a distinct effect on the etiology of ARNSHL among Iranian population. Therefore, the diagnostic value of genetic tests to detect mutations in the GJB6 gene as a first step would not be as valuable as determining mutations in the GJB2 gene. These findings demonstrated that GJB6 deletions were restricted to certain areas and populations, showing a funder effect regarding these mutations as well. [4][5][6][7][8][9] Different studies performed during the last decade indicate an ethnic bias in GJB2 mutation. For example, c.35delG; p.
Gly12Val is highly the prevalent in Whites (about 85%), c.167delT; p.Leu56fs in Ashkenazi Jews, c.235delC; p.Leu79fs in Japanese, Chinese, and Korean people, c.109G>A; p.Val37Ile in Thai people, c.71G>A c.Trp24X in Indians, and c.427C > T; p.Arg143Trp in Ghanaian people. 10 Since there are many different ethnic groups in Iran, it is necessary to produce ethnic-organized data regarding GJB2 mutation. During the last decade, several studies have been carried out on different Iranian ethnic groups to identify the frequency and spectrum of GJB2 mutations. According to different scientific reports, GJB2-related ARNSHL occurs in 16%-18% of Iranian populations, and c.35delG; p. Gly12Val is the most frequent mutation, leading to premature termination of the protein and GJB2-related deafness. 11,12 This study aimed to determine the frequency and type of some GJB2 mutations (including the coding region of GJB2) in the Azeri people of Ardabil province in the northwest of Iran, a province with high consanguinity mating rate. 13 2 | MATERIAL S AND ME THOD

| Subjects
In total, 50 non-related families with ARNSHL from Ardabil were investigated. ARNSHL patients were selected by screening the pedigree of the families and their medical records, the patients' audiologic testing, and the information of two or more patients in the families. There are no symptoms and signs other than HL. Patients were excluded if HL was due to environmental factors, such as intrauterine (eg, rubella virus) infections, ototoxic drugs (eg, aminoglycoside antibiotics), and noise exposure. Only ARNSHL individuals were recruited from families with segregated hearing impairment, including two or more patients. Before beginning the research, written informed consent was obtained from the patients and their families. Then, an analysis of DNA extraction and amplification was conducted.

| Clinical evaluation of proband with new mutation c.238C>A; p. Gln80lys
The affected proband is the second offspring of the family in which the parents have a more distant relationship according to their relatives. He was 61 years old with ARNSHL, and his mother and father had passed away ( Figure 3).

| DNA extraction
About 6 ml of peripheral blood was obtained from the patients and their parents and siblings at any time possible and collected in EDTAcontaining tubes (0.5 M); subsequently, genomic DNA was extracted from peripheral leukocytes using the standard salting-out method. 14 Finally, the purity and integrity of extracted DNA samples were assessed with the Nano drop spectrophotometer (Thermo Fisher Scientific) and agarose gel electrophoresis, respectively. 15

| The genetic analysis of the gap junction protein beta 2 (GJB2)
Mutation in the GJB2 (exon2) coding region was amplified and screened using PCR and direct sequencing, respectively. At first, allele-specific PCR amplification (ARMS PCR) was used with spe- Genetic Analyzer) were applied to sequence PCR products.

| Computational analysis
We used some bioinformatics tools, including SIFT, Mutation Taster, and ClinVar, to predict mutations' effect on the connexin 26 protein structure. The pathogenicity of the detected variant was evaluated through SIFT, Mutation Taster, and the ClinVar webserver. According to the analysis of mutations using Mutation Taster, all the mutations were disease-causing and based on ClinVar analysis, all of them were pathogenic. SIFT webserver analysis showed that some of the mutations were damaging (Table 1).

| RE SULTS
Data from 50 families with ARNSHL in Ardabil were analyzed, and GJB2 mutations were detected in 13 (26%) of the families ( Table 1).
The patients had an age range between 5-74 years. Of the 13 patients, 11 (84.6%) were males and three (23.07%) were females. In total, five homozygous variants were identified, with homozygous c.35delGl; p. Gly12Val being the most common mutation observed in eight (61.5%) of the families. Moreover, c.358-360delGAG; p.
Tyr97Ter, were found in three of the patients, and c.238C>A among them was reported in Iran for the first time. This mutation was categorized as a pathogenic and missense mutation, leading to an aminoacid exchange of glutamine 80 to lysine ( Figure 1).

| DISCUSS ION
Hearing loss (HL) is the most common human-inherited sensory abnormality that has become a worldwide public health concern. At least 1 in 500 newborns is affected by congenital hearing loss. 17 Studies on various ethnic groups in several countries and nations have indicated that different genes associated with HL, especially mutations in the connexin 26 gene (GJB2), which is located in the DFNB1 locus (13q12), are the most common cause of ARNSHL. 18 There are variations in the spectrum of GJB2 mutations in diverse societies, ranging from 87.7% in Finland, 19 57.5% in Lithuania, 20 45.6% in Slovakia, 21 33.3% in Croatia, 22 and 25% in Turkey 23 to 3.7% in Pakistan 24 and 0% in Oman. 25 Research on GJB2 mutations in Iran has indicated that the frequency of GJB2 mutations ranges between 0% and 35% across different ethnicities and regions of the country ( Figure 2). 26 This study as a continuation of a previous study conducted by Davarnia  (75% of mutant alleles) as the most common GJB2-related mutation among Sistani and Baluchi ethnic groups. 28 This mutation has also been found to be the most detected variant in India. 32 Surprisingly, Galehdari et al. 33  The Iran, it has no effect on other ethnicities.
The novel variant identified in this study was c.238C>A; p.
Gln80Lys that has not been reported in Iranian population so far.
This mutation was identified as homozygous in a patient with severe HL (Figure 3). Kalay

| CON CLUS ION
No c.238C>A; p. Gln80Lys variant in the GJB2 gene has been detected in the Iranian population, which is in line with the results of this study and indicates that it is likely to have a significant effect on the ARNSHL etiology in Iranian population. Also, our findings support the idea that GJB2-related HL has various profiles in different populations of Iran with c.35delG variant that is the most common mutation in GJB2-related HL in the Iranian population, especially in the Azeri population, which is higher than the average.

ACK N OWLED G M ENT
We would like to thank all the patients and their family members for their participation in this research.

AUTH O R CO NTR I B UTI O N S
Ehsan Abbaspour, Mohammad Panahi, and Bahareh Rahimi contributed to sequence analysis, bioinformatics analysis, and patient data interpretation regarding sequencing. These authors also contributed to this work equally and together wrote the manuscript.
Haleh Mokaber and Reza Farajollahi wrote some sections of the manuscript. Behzad Davarnia supervised, reviewed, and edited the manuscript. All the authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
Input data for the analyses are available from the corresponding authors on request.