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GJB2 gene mutations in Syrians with sensorineural hearing loss

Mahayri, Zeina N.; Monem, Fawza S.

Middle East Journal of Medical Genetics: July 2012 - Volume 1 - Issue 2 - p 80–84
doi: 10.1097/01.MXE.0000414811.94762.bb
Original articles
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Purpose Hearing loss affects one in every 500 newborns. Approximately 50% of these cases have a genetic cause. Mutations in GJB2 are responsible for most cases of nonsyndromic recessive deafness in many populations. The aim of this study is to determine the prevalence of GJB2 gene mutations in a sample of patients with hearing loss from Syria.

Patients and methods Forty-one patients with sensorineural hearing loss were included in the study. Direct DNA sequencing was used to analyze exon 2 of the GJB2 gene.

Results Seven patients (17.07%) had at least one mutation. Five of these (71.43%) had the 35delG mutation, of which three were homozygous, whereas the other two cases were heterozygous. The other detected mutations were W77R in one case and 167delT with R184P mutations that together occurred as compound heterozygous in another case.

Conclusion Screening of the GJB2 gene must be considered in the evaluation of patients with hearing loss in Syria. The variable mutation types found may indicate the genetic heterogeneity of the Syrian population.

Cytogenetics Unit, Clinical Laboratories, Assad University Hospital, Damascus, Syria

Correspondence to Zeina N. Mahayri, Cytogenetics Unit, Clinical Laboratories, Assad University Hospital, 17th Nissan Street, Damascus, Syria Tel: +963 212 6500 x2329; fax: +963 213 9441; e-mail: zeina.mahayri@gmail.com

Received July 25, 2011

Accepted October 26, 2011

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Introduction

Hearing loss is the most common sensory defect in humans. One of every 500 newborns has a degree of sensorineural hearing loss (Hilgert et al., 2009), most of which are nonsyndromic hearing loss (NSHL). Approximately 50% of hearing loss cases have a genetic cause and most of these are inherited in an autosomal recessive manner (Smith et al., 2010). The 1990s witnessed mapping of many genes for NSHL (Willems, 2000), and the number of these genes has been increasing since then. This provided some families with the opportunity to receive proper genetic counseling services (Battey, 2000).

Mutations in GJB2, the gene encoding connexin 26, are responsible for most cases of NSHL in many populations (Denoyelle et al., 1997; Zhao et al., 2006; Hilgert et al., 2009). This gene is located in the long arm of chromosome 13 (13q11), and contains two exons, of which one is a protein coding exon (Kemperman et al., 2002). The prevalence of mutations in GJB2 as a major cause of congenital NSHL requires that its screening be a starting point of any molecular investigations of these cases (Kemperman et al., 2002).

The 35delG mutation is the most common mutation in GJB2 in many populations. The high prevalence of this mutation makes it one of the most frequent disease mutations ever described (Denoyelle et al., 1997).

This study was designed as a cross-sectional survey to analyze the relation between hearing loss and GJB2 gene mutations in a sample of patients with hearing loss from Syria.

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Patients and methods

Participants were recruited from private ENT clinics and medical caring facilities in Damascus during the years 2009–2010. Families attending the collaborating centers were invited to participate and patients who provided consent to participate were interviewed.

Inclusion criteria included that hearing loss had to be congenital, bilateral, and sensorineural, besides the absence of any medical history related to acquired hearing loss, and absence of syndromic features.

Forty-one unrelated Syrian families from different regions were included in this study. Thirty-six of 41 families had two or more affected siblings, but one proband from each family was included. All participants were subjected to a series of auditory examinations, and were found to have sensorineural hearing impairments ranging in severity from mild to profound.

Informed consent was obtained from all individuals or from the parents of minors.

Detailed medical history was taken for each patient, according to the guidelines of the ‘American Collage for Medical Genetics’ (ACMG statement 2002). The pedigree of three generations was constructed for each family.

Blood samples were collected from selected probands (i.e. 41 samples). DNA was extracted from patients’ blood samples then exon 2 of the GJB2 gene was amplified by conventional PCR using the following primers: F: 5′-TCTTTTCCAGAGCAAACCGCC-3′and R: 5′-TGAGCACGGGTTGCCTCATC-3′ (Denoyelle et al., 1997) from TIB Molbiol (Berlin, Germany). The reaction mix was prepared in a final volume of 50 µl containing MgCl2 and the primers at final concentrations of 5 and 2.5 mmol/l, respectively. The following program was used: one cycle at 95°C for 5 min, followed by 45 cycles at 95°C for 15 s, 58°C for 15 s, and 72°C for 1 min, and then 72°C for 7 min once. A PCR product of 738 bp was visualized by electrophoresis on an agarose gel (2%).

Sequencing of the PCR products was carried out with the same primers used for PCR, using the following program: one cycle at 96°C for 1 min, followed by 25 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. The labeled DNA fragments produced from the sequencing reaction were run on an ABI PRISM 3100 Avant Genetic Analyzer (Applied Biosystems, California, USA).

Sequencing data were aligned by the NCB–blast interface (http://ncbi.nlm.nih.gov/blast).

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Results

Forty-one samples were obtained from Syrian patients with hearing loss. Their mean age was 12.56 years (2–37 years). Twenty-one of 41 were women and 20/41 were men. All the patients studied were from Syrian families and none were from mixed families.

Out of the 41 sequencing results, seven samples (17.07%) had at least one mutation, whereas 34 samples did not have any pathological mutation in exon 2 of the GJB2 gene.

From the seven cases detected with a mutation, five (71.43%) with severe to profound hearing loss had the 35delG mutation, three of which were homozygous (Fig. 1), whereas the other two cases were heterozygous (Fig. 2).

Figure 1

Figure 1

Figure 2

Figure 2

The second detected mutation was W77R, in which a substitution of the thymine residue (T) by a cytosine (C) from the position 229 (Fig. 3) occurs. This was detected in one case patient who had profound hearing loss.

Figure 3

Figure 3

167delT and R184P mutations were found as compound heterozygous in the same case with severe hearing loss. The former was a deletion of a thymine residue (T) from the position 167 (Fig. 4), whereas the latter as a replacement of a guanine (G) by cytosine (C) in the position 551 (Fig. 5).

Figure 4

Figure 4

Figure 5

Figure 5

All mutations were detected in familial cases, except one heterozygous 35delG mutation that presented in a sporadic case. A summary of all the detected mutations is shown in Table 1.

Table 1

Table 1

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Discussion

Mutations in the GJB2 gene have been proved repeatedly as the most common genetic factor in congenital NSHL. Our GJB2 gene sequencing results detected four mutations.

35delG, detected in five cases (12.2%), was the most prevalent mutation in our sample. Three cases were homozygous, including 42.86% of all detected GJB2 mutations here. This percentage increased to 71.4% when the heterozygous cases were included. Both were lower than those found in a study carried out in Jordan, in which the 35delG mutation was found in 100% of the detected mutations (Medlej-Hashim et al., 2002). In another study carried out by Mustapha et al. (2001) in Lebanon, the 35delG mutation was detected in 15 of 48 studied cases and included 94% of detected mutations in GJB2; this was also significantly higher than the prevalence found in the current study. In contrast, we found a higher prevalence of the 35delG mutation than the study carried out by Al-Qahtani et al. (2010) in Saudi Arabia, in which it was 63.63%. The variable mutation prevalences reported in different populations could be attributed to ethnic diversity, heterogeneity of inhabitants or to different methodologies used (Bayazit et al., 2003).

Table 2 illustrates a comparison of 35delG mutation prevalences across different countries.

Table 2

Table 2

Although heterozygous 35delG does not usually exert a phenotypic effect, two patients with severe hearing loss were found to have this mutation in a heterozygous state. This could be attributed to the presence of another heterozygous unidentified mutation in the GJB6 gene, which was not studied here. Several studies have shown that GJB2 and GJB6 have a digenic pattern of inheritance in which the loss of two alleles from the four alleles of both genes results in the pathogenic phenotype (Del Castillo et al., 2002). Another factor that may explain hearing loss in these heterozygous patients is the presence of another heterozygous mutation in the nontranslated region at exon 1. This mutation may affect gene splicing, and can cause, with heterozygous 35delG, hearing loss (Shahin et al., 2002), even though we cannot exclude that the presence of heterozygous 35delG is due to high carrier rate of this mutation while the pathogenic mutation for these two cases is in another locus.

More studies of these heterozygous cases are required to obtain more valid conclusions.

W77R detected as a homozygous mutation in one case, a replacement of a T in the position 229 by a C, was first found in Palestine (Carrasquillo et al., 1997). However, the current patient was Syrian and did not have Palestinian ancestors.

167delT and R184P were found together as compound heterozygous in the same patient. The former is a frameshift mutation that is highly common in Ashkenazi Jews (Zelante et al., 1997), whereas the latter was first reported in Australia (Denoyelle et al., 1997). To our knowledge, the occurrence of these two mutations in the same patient has not been reported before in the medical literature; hence, this is the first reported case of such compound heterozygous. Examination of more members from the same family may help clarify this case more.

In general, the prevalence of detected GJB2 mutations in the current study (17%) is consistent with what was found in Egypt (Meguid et al., 2008), Jordan (Medlej-Hashim et al., 2002), and Saudi Arabia (Al-Qahtani et al., 2010), but less than what was found in Lebanon (Mustapha et al., 2001). However, our results are consistent with a prevalence ranging from 10 to 33% for GJB2 mutations reported in different populations. A summary of some of these studies carried out in different populations is presented in Table 2.

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Conclusion

The prevalence of GJB2 mutations reported in this study makes GJB2 a good candidate as the first-step molecular screening of patients with NSHL in Syria.

This study showed 4 different mutations although it covered only one exon of one gene in a relatively small sample. These findings may indicate the genetic heterogeneity of the Syrian population, and highlights the importance of carrying out further genetic studies in this population.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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Keywords:

connexin 26; 35delG; GJB2; hearing loss; Syria

© 2012 Middle East Journal of Medical Genetics