Introduction

Hearing loss (HL) is a complex disorder and accounts for 1.86 of 1000 newborns worldwide. The frequency of the disorder increases by age, that is, it affects 2.7 of 1000 before the age of 5 years and 3.5 of 1000 in adolescents.1 Many environmental factors, such as drug exposure, bacterial or viral infections and trauma, can cause HL; however, a significant proportion of cases is due to genetic factors. Different criteria are applied for the classification of HL (Table 1). Considering clinical phenotypes, nonsyndromic hearing loss (NSHL) is responsible for nearly two-third of hereditary congenital cases and the remaining accounts for syndromic forms of HL. The only impairment in the former group is HL, whereas HL is a part of broader clinical symptoms in the syndromic form.

Table 1 Various criteria for the classification of hearing loss
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The cochlea is an important part of the inner ear, containing the organ of Corti. Sound vibrations are converted into electrobiochemical impulses in this organ and sent to the brain through the auditory nerve. Numerous genes have been described to be expressed in this pathway, including genes encoding cytoskeleton proteins, gap junction channels, membrane transport proteins, ion channels, regulatory elements and microRNAs (miRNAs).2, 3, 4

Monogenic inheritance (NSHL) is responsible for 70% of all congenital HL. All Mendelian inheritance patterns have been observed for prelingual HL. Different distributions of each pattern have been delineated in many studies: autosomal recessive (DFNB loci) in 80%, autosomal dominant (DFNA loci) in 20%, X-linked (DFN loci) in 1% and mitochondrial inheritance in 1% of cases.2, 3, 4, 5, 6, 7 Thus, autosomal recessive NSHL (ARNSHL) has a high prevalence among these patterns of inheritance. In traditional communities such as the Middle East, consanguineous or intragroup marriages may even increase the proportion of ARNSHL among all forms of HL. More than 80 loci have been identified for ARNSHL, of which a significant percentage is attributed to mutations in the DFNB1 locus (especially GJB2 mutations) in Caucasians.

Iran's population is ∼75 million, with HL affecting an estimated 450 000 individuals.8 In other words, 1 in 166 individuals is affected. Accurate genetic counseling is desired for early intervention of late-onset forms. Significant amounts of health-care expenditures are targeted for its maintenance each year in Iran; hence, preliminary molecular diagnosis would help to reduce the socioeconomical and mental disturbances of the affected individuals and their families.

HL encompasses a broad field of research, including understanding mechanisms underlying the disease, genes involved in different pathways, gene therapy (as a new treatment modality) and molecular diagnosis, for example, preimplantation genetic diagnosis. Considerable assistance in care is achieved through collection of mutations for diagnostic laboratories, clinicians and researchers.9 Recently, the genetic causes of NSHL in Jews, Japanese and Chinese populations have been described.10, 11, 12 In this survey, we emphasized the importance of identifying the genetic bases of NSHL and summarized the published data on the frequency of gene mutations in various loci studied in Iran; we provided counseling criteria for the patients. The distribution of the gene mutations was also shown.

Molecular genetics

Many genes and loci pinpoint prelingual HL, including genes encoding cytoskeletal proteins such as myosin VI (MYO6), VIIA (MYO7A), XV (MYO15) and IIIA (MYO3A), structural proteins such as α-tectorin (TECTA), stereocilin (STRC), collagen type 11 (COL11A2) and otoancorin (OTOA), regulatory elements such as POU3F4 (POU3F4), POU4F3 (POU4F3) and EYA4 (EYA4), ion channel and transport proteins such as connexin-26, -30, -31, -40 proteins and pendrein (SLC26A4) and miRNAs.2, 3, 4 Clinical diagnostic and research laboratories are widely available for conducting research on these genes. Studies on the characterization of these genes and the impact of the different combination of mutations are still an emerging field. It is widely believed that there are further unknown genes to be recognized.

At present, 10 genes have been examined in the country (Table 2). Whereas some genes have been analyzed in just one family, GJB2 mutations have been investigated in at least 1934 families throughout Iran.

Table 2 Studied genes involved in congenital hearing loss in Iranian population
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GJB2 gene structure and mutations

DFNB1 locus for ARNSHL was mapped to chromosome 13q11–12.13 This locus contains two genes, GJB2 (MIM 121011, GeneID 2706) and GJB6 (MIM 604418, GeneID 10804); GJB2 (gap junction subunit β2) encodes connexin (26 kDa) protein, which has two exons of which exon 1 is untranslated. GJB2 is expressed as a 2.5-Kb protein, containing 226 amino acids in cochlea-supporting cells, suggesting a potential role in endolymph potassium recycling. This protein is arranged into a four-transmembrane (TM)-spanning fragment (Figure 1). Mutations in this single gene are the main reason of prelingual HL in Caucasians and in many ethnic groups worldwide. Six subunits of connexin proteins are gathered to form a hemiconnexon localizing in the cell membrane. Two hemiconnexons from adjacent cells form a channel to transport small metabolites and ions such as potassium. Connexin-26, expressed in hair cells, is implicated in reclaiming K+ in the cochlear duct. Deterioration of K+ recycling caused by GJB2 mutations may give rise to HL.14, 15

Figure 1
figure 1

Schematic structure, domains and distribution of mutations of Cx26 protein in Iranian patients. Novel mutations reported for the first time are indicated in bold. The most common mutations in specific populations (35delG, 167delT, 235delC, R143W and W24X mutations in Caucasian, Ashkenazi Jewish, Japanese, Ghanian and Indian populations, respectively) are underlined. 35delG, W24X, 167delT, 235delC and R143W are located on NT, TM1, EC1, TM2 and TM3 domains, respectively. Other depicted mutations have also been reported in other populations. TM1–TM4 denotes transmembrane domains, EC1-2 denotes extracellular domains, IC denotes cytoplasmic domain, NT denotes amino (NH2) terminus and CT denotes carboxyl (COOH) terminus.

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As of June 2010, more than 200 different allelic variants in the GJB2 gene have been ascertained (http://www.hgmd.cf.ac.uk/ac/index.php). Among these variations, one truncating mutation named 35delG (deletion of guanine in position 30–35; rs80338939) is the most prevalent mutation and responsible for up to 70% of GJB2 variants. This mutation leads to a frameshift change and synthesis of a premature truncated protein. The mean carrier frequencies of the 35delG mutation are 1.89, 1.52, 0.64, 1 and 0.64 for European, American, Asian, Oceanic and African populations, respectively.16 The 167delT, 235delC, R143W and W24X mutations are the most common mutations in the Ashkenazi Jewish,17 Japanese,18 Ghanian19, 20 and Indian populations,21, 22 respectively (Figure 1). Thus, an ethnic-specific pattern is noted for some mutations of this gene,16, 21 which is different from that reported among various cohorts of Iran.

GJB2 mutations could result in NSHL. Genotype–phenotype analyses have not completely established a specific correlation between different combinations of genotypes and clinical features. There are many inter- and even intrafamilial phenotypic variations, ranging from mild to profound HL.23 However, the role of modifier genes is proposed, which may give rise to more phenotypic variations.

Approximately 50 mutations in the GJB2 gene have been detailed in deaf Iranian families (Table 3 and Figure 1). At least 1934 affected families have been investigated so far. In a study, GJB2-related HL was reported to occur in 16.7% of cases (111 of 664 different nuclear families).24 In this survey, 35 simplex cases were also analyzed. In 2007, Hashemzadeh Chaleshtori et al.,25 stated that the frequency of GJB2-related HL is 19.1% of deaf families (170 of 890 families). In addition, 209 unrelated Azeri patients were reviewed, with a frequency of 28.22% for GJB2 gene mutations.26 The mean frequency of GJB2 mutations among the Iranian deaf groups is ∼18.17%, whereas GJB2 mutations are responsible for 50% of HL in Caucasian populations. We also examined 50 unrelated deaf families (data not shown). Briefly, medical history was taken, and audiometric examinations and pedigree analyses were carried out; genomic DNAs were extracted according to standard methodology. The 35delG homozygous subjects were determined using allele-specific PCR.27 We sequenced the remaining samples by ABI 3130 sequencer (Applied BioSystems, Foster City, CA, USA).24, 27

Table 3 Frequencies of GJB2, SLC26A4, PJVK, TECTA and TMC1 gene mutations in the previous studies in Iranian populations2, 24, 25, 26, 60, 66, 67, 69, 76, 77, 78, 113, 114, 115
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On the basis of these results, it can be concluded that the frequency of GJB2 mutations decreases gradually both west to east and north to south. A northwest-to-southeast GJB2-related HL gradient is suggested across Iran, drawing the migration pathway of the initial founders. The 35delG mutation is the most prevalent mutation in deaf Iranian individuals, accounting for 66.26% of all GJB2 mutations (466 of 703) and 12.05% of all studied alleles (466 of 3868) (Table 3). As known, this mutation provides 20% of all hereditary HL among American-Caucasian populations.28 The eight most frequent mutations of the GJB2 gene, namely, 35delG, R127H, delE120, W24X, V27I+ E114G, R184P, -3170G>A and 235delC, are responsible for 86.34% (607 of 703) of all pathogenic alleles in Iran (Table 3 and Figure 1). The 235delC mutation, which is the most common (up to 80%) among East Asian countries,18, 29, 30, 31, 32 makes up for 1.85% (13 of 703) of GJB2 mutations in Iran. The R127H, delE120 and W24X are the second, third and fourth common mutations, with a sum of 11.52% (81 of 703) of all pathogenic alleles. The R127H mutation is due to a G-to-A transition at codon 127, substituting arginine with histidine. The pathogenic effect of this amino-acid substitution is controversial and remains to be confirmed. Combined genotypes with variants such as V37I, delE120 and/or R127H could show a variable phenotypic expression modulated by modifier agents.23, 33 Interestingly, R127H is distributed throughout Iran with the same frequency;25 thus, arginine 127 may be a mutational hotspot. The delE120 (deletion of glutamic acid in position 120) and W24X (tryptophan to stop codon) may arise from ancestral founders in western and southwestern parts of Iran, respectively. However, further investigations are required to clarify this assumption.

More detailed studies on GJB2 showed new mutations of the gene. So far, 12 novel mutations have been identified in deaf Iranian individuals, which are as follows: 167del16, H16R, Q80L, 507insAACG, K102Q, 329delA, 327delGGinsA, 363deC, G130V, K112N, −3517G>A and G200R (Table 3 and Figure 1). One novel mutation (−3517G>A) is found in regulatory regions. Five of these mutations are located on intracytoplasmic domain 2. GJB2 polymorphisms are observed in 3.83% (148 of 3868) of deaf patients; V153I, valine to isoleucine substitution, accounts for 62.84% of polymorphic alleles (93 of 148). Gene polymorphisms could alter an amino acid, thereby, causing changes in the properties of the given isoformic protein. Thus, it could be postulated that GJB2 gene polymorphisms among deaf patients, may in association with other gene polymorphisms, lead to some degree of HL. In other words, specific isoforms of two different proteins, for example, connexin-26 and other proteins such as connexin-30, may not have a functional interaction. However, structural and functional analyses on different polymorphisms of various interacting proteins are needed to confirm this hypothesis.

More recently, researchers have shown that mutations of GJB2 can function in a digenic manner with the GJB3 gene.34 Hence, mutation analysis of this gene should be considered in GJB2 heterozygotes. Because of the high frequency of GJB2 mutations in many populations, GJB2 analysis should be depicted for sensorineural NSHL, although the frequency of GJB2-related HL in Iran is lower than that in European countries.

Molecular genetic testing for GJB2 in Iran includes an amplification refractory mutation system for common mutations, followed by a sequencing procedure.

GJB6 gene

GJB6 (MIM 604418, GeneID 10804), in the vicinity of GJB2 located on 13q12, encodes a connexin (30 kDa) protein that participates in the formation of hemiconnexon structures in the membrane of hair cells. As in GJB2, each connexin protein has four TM segments, two extracellular loops, a cytoplasmic loop formed between the two inner TM segments and the amino and carboxy termini, both facing the cytoplasmic side. Connexin-30 has 261 amino acids having 76% homology to human connexin-26. A few point mutations in the GJB6 gene have been found to associate with autosomal dominant HL and hidrotic ectodermal dysplasia.35

Four GJB6 large deletions, a 140 kb deletion36 or a 150 kb deletion,37 named del(GJB6-D13S1830) 342 kb,38 del(GJB6-D13S1854) 232 kb,39 a 920 kb deletion40 and del(chr13:19,837,344–19,968,698),41 have been described to bring about HL in association with a single mutation in the GJB2 gene. Existence of several breakpoints within this region suggests that other unknown deletions may be responsible for GJB2 heterozygotes. Up to 50% of GJB2 heterozygotes have a large deletion around the GJB6 gene.42 The del(GJB6-D13S1830), the most frequent mutation in Spain,42 France,42, 43 Argentina,44 Brazil45 and the United Kingdom,42 has not been found in Iranian populations,24, 26, 27, 46, 47 suggesting the existence of point mutations in regulatory elements of the gene, and possibly different breakpoints around the gene and/or another gene(s). Moreover, these deletions have not been reported in Turkey,48 India,49 China and Croatia.50

Genetic testing for GJB6 large deletions may be different on the basis of the deletion that is investigated, and includes gap-PCR, quantitative-PCR and DNA sequencing for point mutations.

SLC26A4 gene

Solute carrier gene (SLC26A4 gene, MIM 605646, GeneID 5172), located on DFNB4 (chromosome 7q31), encodes pendrin protein, which functions as a transmembrane chloride/iodide transporter in cochlea and in a few other tissues.51, 52 An ∼5 Kb transcript is expressed into 780 amino-acid (86 kDa) protein. The real structure of pendrin has not been defined yet; on the basis of protein structure prediction programs, two models have been proposed for pendrin protein: 12TM and 15TM models.53, 54 This ion transporter also exchanges other anions such as HCO−, OH−, I− or formate.55 Variations in this gene, as a second prevalent cause of HL, can contribute to both syndromic (Pendred syndrome, PS) and ARNSHL (DFNB4).56, 57 PS is associated with severe sensorineural HL and thyroid symptoms ranging from small changes in thyroid size to a large extent goiter. An enlarged vestibular aqueduct determined by a computed tomographic image is also present in PS patients.58 DFNB4 and PS explain ∼1–8% of prelingual HL.59

Approximately 100 mutations have been designated in the SLC26A4 gene.58, 60 A total of 80 Iranian families were investigated for mutations in this gene using the autozygosity by descent approach using short tandem repeat markers.60 Autozygosity by descent of the SLC26A4 gene was observed in 12 of them. Enlarged vestibular aqueduct and an impaired function of the thyroid were also found in all families.60 In their work, Kahrizi et al. found the following mutations in eight families: T420I, 1197delT, G334V, R409H, T721M, R79X, S448L, L597S, 965insA and L445W. R79X and R409H mutations (Figure 2a) were the most common among other mutations in this population, whereas L236P, T416P, E384G and IVS8+1G>A are the common mutations in other populations that are responsible for 75% of all mutated alleles.61 The IVS7-2A>G and H732R mutations are more prevalent in East Asia.62, 63, 64, 65 Thus, ethnic specificity of mutations in this gene is also observed and it should be considered in screening programs in Iranian subpopulations. On the basis of preliminary data, SLC26A4 mutations are responsible for up to 10% of prelingual HL. However, further studies are needed to elucidate the exact frequencies of SLC26A4 mutations in these subpopulations.

Figure 2
figure 2

Novel mutations reported for the first time are indicated in bold at the top of the SLC26A4, PJVK and TECTA genes in Iranian patients. (a) Schematic representation exons of the SLC26A4 gene and mutations found in Iranian families. Only mutations reported in the Iranian population are shown in the figure. (b) Schematic exons of the PJVK gene and distribution of its mutations. Mutations reported in other populations are shown below. (c) Schematic exons of the TECTA gene and distribution of its mutations. Mutations reported in other populations are shown below.

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DFNB59 gene

DFNB59 gene (PJVK, MIM 610219, GeneID 494513), located on chromosome 2q31.1–q31.3, encodes pejvakin, which functions as a member of the gasdermin protein family in the cochlea.66 Pejvakin possibly has an important role in the cell signaling of hair cells and sensory neurons.67, 68 The 352-amino-acid pejvakin protein is translated from a 5 kb cDNA.

The gene has seven exons spanning 9.8 kb of the genomic sequence, of which the first exon is noncoding. PJVK mutations were clarified as the cause of sensorineural HL for the first time in Iranian families.66 So far, four mutations (T54I, R183W, 726delT and c.988delG) in PJVK gene have been found in Iran (Figure 2b). The frequency of PJVK mutations has been reported to be 4 of 60 chromosomes in Iran.69 Further studies are required to determine other mutations and carrier frequencies of mutations in the PJVK gene in our different ethnic groups. Mutations of this gene have been also documented in Turkish and Dutch populations.70

TECTA gene

The TECTA gene (MIM 602574, GeneID 7007), located on chromosome 11q22–q24, encodes α-tectorin, which is one of the major noncollagenous parts of the tectorial membrane in the inner ear. This membrane as an extracellular matrix covers the neuroepithelium of the cochlea and contacts the stereocilia of hair cells transducing sounds into electrical signals.71

The TECTA gene encodes a precursor protein of 2155 amino acids, determined to contain 23 exons. Mutations in the TECTA gene have been found in both autosomal dominant and autosomal recessive HL (DFNA8/12 and DFNB21).71, 72, 73, 74 All presented missense mutations lead to the dominant form, whereas inactivating mutations bring about recessive HL.75 TECTA mutations have been detected in Austrian, Belgian, French and Swedish families.7 Three studies have been recorded on TECTA mutations in Iranian population; totally, TECTA mutations were demonstrated in 5 of 121 studied patients.76, 77, 78 The five mutations with the same frequency (2 of 242 alleles for each) reported are as follows: c.649insC, 266delT, c.5211C>A, 9.6 kb deletion and c.6203–6218del16 (Figure 2c).76, 77, 78 Altogether, the TECTA gene was involved in 10 of 242 alleles (4.13%) in our population. α-Tectorin has three types of functional domains: entactin-like domain at N-terminus, four von Willebrand type D domains in the central part of the protein and zona pellucida domain at the C-terminal end.79 Mutations of various parts of α-tectorin lead to HL at different frequencies and lead to specific genotype–phenotype correlations.71, 80, 81, 82

TMC1 gene

Mutations in the transmembrane cochlear-expressed gene 1 (TMC1), located on chromosome 9q13–21, can also result in both progressive autosomal dominant and autosomal recessive HL (DFNA36 and DFNB7/DFNB11).83, 84 There are eight vertebrate TMC genes on the basis of their sequence homology.85 This gene (MIM 606706, GeneID 117531) is considered as a transmembrane protein, and its mutations are associated with prelingual and postlingual HL. A total of 24 exons of TMC1 encode the mRNA. TMC1 mutations have been found in at least 6% (4 of 65) of GJB2-unrelated ARNSHL patients from eastern Turkish populations. Mutation prevalence of the TMC1 gene is ∼5% of GJB2-negative ARNSHL in Tunisian, Pakistani and Indian families.86, 87, 88 One mutation, named 100C>T (R34X), is responsible for 47% of all TMC1 mutations in ARNSHL families.2 On the basis of the linkage analysis between the mutation and polymorphic markers, it may arise from a common ancestor dating back to 1075–1900 years.89 This mutation was found only in one family from Iran.2 Another mutation, 77611 G>A, with the same frequency was also determined in Iranian families.2

COL11A2 gene

The COL11A2 gene (MIM 120290, GeneID 1302) is located on chromosome 6p21.3, encoding the α-2 chain of minor fibrillar collagen XI. Collagens are the major components of bones and cartilages. Several cases of syndromic HL due to mutations of collagens have been reported. Collagen type XI comprises <10% of cartilage collagens. This gene contains at least 62 exons spanning 30.5 Kb.90 The gene lies within the MHC region and is only 1.1 kb from the retinoid X receptor-β and ∼40 kb from DPB2. COL11A2 mutations can generate both autosomal recessive and progressive autosomal dominant HL (DFNB53 and DFNA13).91, 92, 93 Mutations in this gene have illustrated to cause DFNA13 HL in one Dutch family.94 In one Iranian family, homozygosity for a COL11A2 mutation, P621T, has also been reported to result in ARNSHL.92

Genetic testing includes PCR–restriction fragment length polymorphism for known missense mutations and DNA sequencing.

MYO15A gene

MYO15A (MIM 602666, GeneID 51168), located on 17p11.2, encodes an unconventional myosin XVA (myosin heavy-chain-15A) that functions as a motor molecule moving along actin filaments in the stereocilia of hair cells.95, 96 At least 39 myosin genes, grouped into 12 classes, have been found within the human genome.97

It contains 66 exons encoding an 11.9 Kb transcript. Mutations in this large gene can cause autosomal recessive HL (DFNB3) and account for 5% of ARNSHL in Pakistan.7, 95, 98, 99 Two MYO15A mutations have also been reported in 66 ARNSHI Turkish families.100 More recently, two homozygous missense mutations were exemplified in two consanguineous Iranian families; c.6371G>A and c.6555C>T lead to p.R2124Q and p.P2073S amino-acid substitutions.101

RDX gene

RDX gene (MIM 179410, GeneID 5962) has been mapped on chromosome 11q23, encoding a cytoskeletal protein named radixin, which possibly functions as linking molecule between actin and plasma membrane.102

Mutations in this gene can lead to autosomal recessive HL (DFNB24).103, 104 It contains 14 exons. There are two RDX psuedogenes in the human genome; a truncated version of the gene named RDXP2 was mapped to Xp21.3 and another one, RDXP1, was mapped on chromosome 11p. A homozygous splice site mutation (c.698+1G>A) in intron 7 of the RDX gene has been reported in an Iranian ARNSHL family.104

microRNA

HL has been ascribed to variations in miRNA genes.4 miRNAs binding to their complementary domains in mRNAs affect posttranscriptional repression.105 Mutations in miR-96 genes belonging to the miR-183 miRNA family in two ARNSHL DFNA50 families have been determined by Mencía et al. Hildebrand et al. screened 192 unmapped Iranian ARNSHL families and identified a homozygous c.*95C>A variation in the miR-96/182 binding site in the RDX 3′ untranslated region. This nucleotide substitution disrupts the binding of miR-96 and miR-182, and/or creates a new binding site for miR-507 and miR-557. However, c.*95C>A has not been associated with HL in humans.106 They also studied miR-183 genes in 576 families and found no potential pathogenic variants.

Molecular testing for syndromic and NSHL

Molecular diagnosis of syndromic and NSHL has a significant benefit for affected children and their parents because of prevention of unessential diagnostic tests and undesirable clinical consequences of psychological and socioeconomical effects. Planning for therapy approaches such as cochlear implant also demands an accurate diagnosis.107

Attributed to extreme heterogeneity and the relatively small contribution of different genes, it is very expensive and really impractical to perform a comprehensive analysis of all known and unknown genes involving in inherited HL. Syndromic bases can be diagnosed by an accurate clinical evaluation and by molecular tests for the known syndromes; hence, a multidisciplinary team is needed for this goal.

It is generally accepted to screen the mutations of some genes that are more prevalent than others. Two genes, GJB2 and GJB6, are analyzed for patients affected by prelingual NSHL in Iranian genetic laboratories. The procedure of genetic testing includes DNA extraction from peripheral blood cells, buccal cells, a part of blood spots or tissues and mutation analysis by molecular methods such as allele-specific PCR for common mutations and sequencing of the exonic region of the gene.

GBJ2 has only a single coding exon; hence, it is simply sequenced. There are many difficulties in the interpretation of the GJB2 analysis; first, many families show a heterozygous genotype for the GJB2 gene, that is, having only a single mutant allele. Second, inter and intrafamilial phenotypic variability caused by even the same mutation complicates the prediction of the degree of HL.

Regarding the phenotypic expression of individuals being PS or enlarged vestibular aqueduct, the homozygosity mapping of the affected is implemented for the SLLC26A4 gene, which is carried out by short tandem repeat markers.

However, some potential difficulties may occur in establishing a genetic cause for HL in patients; genetic heterogeneity, uncertainty about pathogenicity of the mutation, reduced penetrance of some mutations, phenotypically affected heterozygotes and a negative family history of the patient are the common reasons for such difficulties. Here, affected homozygotes for GJB2 mutations are ruled out by molecular study. Heterozygote patients for the GJB2 gene are subjected to analysis to investigate GJB6 large deletions. GJB2-unrelated affected individuals (GJB2 negatives) are clinically examined in detail. On the basis of previous studies, there are specific clinical indications for the genes to be examined first; for example, if sensorineural HL is accompanied by enlarged vestibular aqueduct and goitrous changes, SLC26A4 is analyzed. This procedure for the genetic diagnosis of HL would be beneficial to developing countries such as Iran as it is cost-effective and diminishes the fees spent on health practice. Moreover, a multiplex screening system for the analysis of ARNHL by the use of short tandem repeat markers could be an advantageous way to determine the involved genes in these countries; in this approach, affected families should be investigated for the prevalent genes by autozygosity by decent and subsequent sequencing of the related gene. However, many clinical and research laboratories are available for genetic testing around the world. Genetic investigation of HL genes is feasible by microarray and genome-wide scanning.108, 109

Ethnicity

Genetic and molecular testing for diagnosis of the major cause of HL should be designed considering the ethnicity of patients, especially in Iran with various cohorts and different cultures. Ethnicity should be considered even for other genetic diseases. The critical and specific position of Iran and the existence of various ethnic groups with different cultures (for example, Persian, Azeri, Gilaki and Mazandarani, Kurd, Lur, Turkmen, Arab, balooch and so on) suggest the high heterogeneity throughout Iran,23, 110 but specific intraethnic traditions such as intragroup marriages may give rise to a high homogeneity in some loci and mutations within groups and not among groups.

GJB2 mutations are responsible for 22.2% of deaf families in northwest Iran, that is, in west and east Azerbaijan, Ardebil and Zanjan; a significant proportion of population in these provinces are Azeri. GJB2-related HL has the highest frequency (38%) in north Iran, that is, in Gilan, Mazandaran and Golestan provinces, and the lowest frequency (0–4.3%) of GJB2 mutations has been observed in south and southeast Iran, that is, in Booshehr, Hormazgan and Sistan and Baloochestan.24, 25 GJB2-related HL in western Iran (15.7% including Kermanshah, Kordestan, Hamedan, Lorestan and Ilam) is more than that in central and northeast Iran: 15.3 and 13%, respectively (Figure 3).

Figure 3
figure 3

Distribution of GJB2 mutations in different regions of Iran. Azeri, Lur, Kurd, Arab, Gilaki and Baloochi populations are distributed in the northwest (22–27%), west (15–16%), southwest (6–15%), south (0–4%), north (27–38%) and southeast (0–4%) regions of the country. Data are adapted from references Najmabadi et al.,24 Hashemzadeh Chaleshtori et al.,25 Bonyadi et al.26 and Mahdieh et al.27

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Ethnic background has an important role in the frequencies of GJB2 mutations; for example, 35delG is the most common mutation that allocates a large proportion of GJB2 mutations in nearly all populations, especially in Caucasians, as well as 167delT, 235delC and R143W in Ashkenazi Jews, East Asians and Ghana, respectively. The 35delG mutation is also the most common (71.6%) allele of GJB2 in deaf Iranian patients;24 its carrier frequency (2.8%) in the Gilan province is even more than that in some Caucasian populations.111 Further and more comprehensive studies are needed to define other genes and their mutations on the basis of ethnicity in Iran. However, ethnicity should be an important factor for determining the most appropriate type of assay.

Genetic counseling

HL is a sensory impairment that can be treated by cochlear implant, especially in children before the age of 5 years. Genetic counseling and genetic testing for families with HL can be helpful, effective and appropriate for preimplantation genetic diagnosis, newborn screening, detection of carrier mutations and preventing the recurrence of the condition in affected families.

HL affects 0.22–3.61 per 1000 neonates in the United States,112 whereas 1 out of 166 individuals suffers from HL. Thus, the frequency of HL in Iran is 1.7–27 higher than in the United States. In addition, this condition may show up higher rates compared with other populations of the world by two to three times.

A complete family history is taken. It includes age of onset of HL, environmental agents that may bring about HL, mother's complications during pregnancy, careful and detailed physical examinations and pedigree taking. For establishing HL, audiological tests are required. Recurrent risk is calculated on the basis of inheritance pattern. Regarding the GJB2 mutations, 35delG is the most common mutation that is checked first. In Iranian ethnic groups, specific mutations are common, which are investigated in each group; for example, the frequency of the IVS1þ+1G>A mutation and W24X shows a high frequency in Kurdish and Baloochi groups and accounts for 9.4 and 6% of the mutant alleles in these populations, respectively.23, 24, 27

In conclusion, unraveling gene mutations of HL would help to improve health care and affect our treatment of the condition. Executing appropriate screening programs may lead to designing therapeutic and treatment strategies in Iran.