Contribution of GJB2 Mutations and Four Common DFNB Loci in Autosomal Recessive Non-Syndromic Hearing Impairment in Markazi and Qom Provinces of Iran

Document Type: Brief Report

Authors

1 Department of Genetics, Faculty of Basic Sciences, Tarbiat Modarres University, P.O. Box 1411-5317, Tehran, I.R. Iran

2 Department of Medical Genetics, National Research Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran

3 Cellular and Molecular Research Center, Shahrekord University of Medical Sciences, P.O. Box 571, Shahrekord, I.R. Iran

4 Research Center of ENT-HNS, Iran University of Medical Sciences, P.O. Box 14455-364, Tehran, I.R. Iran

Abstract

This study aimed to investigate the contribution of four common DFNB (“DFN” for deafness and “B” for autosomal resessive locus) loci and GJB2 gene mutations (exon 2) in hearing impairment in individuals living in Markazi and Qom provinces of Iran. Forty consanguineous Iranian families with at least three affected individuals in family or pedigree who suffer from an autosomal recessive non-syndromic congenital hearing impairment were the subjects of this study. Blood samples were taken from both hearing and non-hearing individuals, DNA was extracted and amplified by using specific primers for the coding region of GJB2 gene (exon 2). The PCR product of GJB2 gene was then sequenced.  Also short tandem repeat (STR) markers amplified by using specific primers for loci DFNB2, DFNB3, DFNB4 and DFNB21. At least 2 microsatellite markers (STR) for each DFNB locus exceeding to 4-6 markers for the linked families were used. The amplified markers were analyzed by conventional Polyacrylamide Gel Electrophoresis followed by silver staining. Six families were homozygous or compound heterozygous for GJB2 mutations and were excluded from further studies. Linkage analysis was carried out for the remaining 34 families by genotyping the flanked STR markers of DFNB2, DFNB3, DFNB4 and DFNB21 loci. Six families showed linkage; including  one family to DFNB2, two families to DFNB3 and three families to DFNB4 locus while no family showed linkage to DFNB21 locus. Undoubtedly, the best understanding of the genetic basis of hearing loss in Iranian population will be achieved by performing similar experiments in other provinces and also by analyzing more loci.

Keywords


Congenital deafness is the most prevalent sensorineural disorder that affects one in 1000 neonates with 50% genetic basis (Kalay et al., 2005; Ramshankar et al., 2003). Hereditary deafness is a genetically heterogeneous disorder that is classified as non-syndromic (70%) and syndromic. Non-syndromic hearing impairment can be further subdivided by the mode of inheritance. The majority of the non-syndromic cases (77%) show autosomal recessive inheritance while 22% are autosomal dominant and only 1% is X-linked or due to mitochondrial mutations (Petersena  and  Willemsb 2006; Mukherjee  et al., 2003). More than 100 genes are estimated to be involved in hearing impairment and to date about 130 loci have been described in previous studies and 47 relative genes have so far been mapped (http://webh01.ua.ac.be/hhh/). The different loci for numerous forms of deafness have been called DFN (for deafness) and are numbered in chronological order of discovery. Autosomal dominant forms have been designated as DFNA, autosomal recessive loci as DFNB and X-linked as DFN (Mukherjee et al., 2003).
Despite this heterogeneity, up to 50% of prelingual recessive non-syndromic hearing loss (ARNSHL) can be attributed to mutations in GJB2 in many populations (Petersena and Willems, 2006).  Consanguinity in the Iranian population is highly prevalent (Saadat et al., 2004), so research on ARNSHL is much recommended (Hashemzadeh Chaleshtori et al., 2007; Najmabadi et al., 2005). The main purpose of this study was to identify the contribution of 4 common DFNB loci (FDNB2, DFNB3, DFNB4 and DFNB21) and GJB2 gene mutations (exon 2) in hearing impairment of the Markazi and Qom provinces of Iran.
Questionnaires were distributed in deaf schools, hearing loss centers and rehabilitation centers in Qom and Markazi provinces and were collected after being filled by the parents. Forty families were selected for this study. All of the studied families in this research have consanguineous marriages with the autosomal recessive mode of inheritance. These families have at least 3 deaf individuals in their pedigree. All affected individuals of this study suffer from a prelingual autosomal recessive hearing impairment with no other associated clinical findings and no history suggestive of any other possible etiology.
Informed forms were provided for each adult individual of this study (for the individuals under 18 years old, permission was taken from their parents). DNA was extracted from peripheral blood samples using the phenol/chloroform standard procedure (John et al., 1991).
The coding exon of GJB2 (Genbank accession no. M86849) was amplified using the following primer pair; Forward: 5´-GTCTCCCTGTTCTGTCCTA-3´and Reverse: 5´-TCTAACAACTGGGCAATG-3´. The amplified DNA fragment of 743 bp contains the coding sequence (681bp). The PCR products of the GJB2 gene were quality controlled on the 1.5% Agarose gel and then sequenced. Sequencing the whole coding region of this gene was carried out using a capillary automated system 3700 ABI sequencer (Macrogen, South Korea). One affected sample in each family was sequenced for both strands initially and then mutations were confirmed by sequencing another affected member of these families. DNA samples from the families without a mutation in the coding region of GJB2 were amplified by using specific primers (Table 1). At least 2 microsatellite markers were used for each locus (Alasti et al., 2008). The PCR products of the STR markers were analyzed by 12% (29:1, acryl:bis) polyacrylamide gel electrophoresis (PAGE) followed by silver staining (Fig. 1).
Altogether 354, questionnaires containing completed information about hearing loss families were collected. The families studied were the result of consanguineous (59.3%) and non-consanguineous marriages (36.7%). The marriage type was not determined in 4% of the families. Mean of deaf individuals among these families was 1.8 ± 1.4. By assessing the filled questionnaires and pedigrees and by talking to the parents, the deafness etiology in the studied population was categorized as genetic (70.9%), environmental (9%) and unknown (20.1%). Forty families with ARNSHL were selected for this study. At first, these families were analyzed for GJB2 gene mutations (exon 2). Six families were homozygous or compound heterozygous for a mutation in GJB2 and were excluded from linkage analysis of the four common loci. The mutations found in GJB2 gene are represented in Table 2.
Linkage analysis using highly polymorphic microsatellite markers was performed on 34 remaining consanguineous families. The families were genotyped for microsatellite markers close to DFNB2, DFNB3, DFNB4 and DFNB21 loci. A linkage was identified in 6 families, including one to DFNB2, two families to DFNB3 and three others to DFNB4 loci, while none of the families showed linkage to the DFNB21 locus (Table 3).
Mutations in GJB2 have been shown to be the major cause of ARNSHL in many populations (Mustafa, 2004; Bayazit et al., 2003). The results of this study also show that the contribution of GJB2 mutations is significant in familial deafness in Iran (Hashemzadeh Chaleshtori et al., 2007; Najmabadi et al., 2005). Deafness in 15% of these families is due to mutations in GJB2, which is consistent with other studies in the Iranian population (Hashemzadeh Chaleshtori et al., 2007; Najmabadi et al., 2005). However, this needs further assessment of the other known loci as well as searching for factors related to hereditary deafness in the Iranian population.
ARNSHL is the most prevalent type of hereditary hearing loss in non-Caucasian populations with a high frequency of consanguinity (Petersena and Willemsb, 2006; Kalay et al., 2005; Mukherjee et al., 2003). The consanguinity in the Iranian population is highly prevalent (Saadat et al., 2004), so further research on ARNSHL is highly recommended in Iran (Hashemzadeh Chaleshtori et al., 2007; Najmabadi et al., 2005; Sadeghi et al., 2005).
Fifteen percent of these families showed linkage to 3 ARNSHL loci. Hearing loss in the linked families is likely due to mutation in the known deafness genes in these 3 loci (Myosin 7A, Myosin 15A, SLC26A). Undoubtedly, the best results on the contribution of different loci to Iranian hearing loss will be achieved by arranging these experiments in other provinces and also analyzing some other loci.
Mutation in the GJB2 gene and linkage to 3 analyzed common loci were found in 30% of studied families. Because, 59 loci for ARNSHL have already been reported (http://webh01.ua.ac.be/hhh/), it is likely that the linkage to one of these known loci as well as the involvement of a new genomic region can contribute to deafness in the remaining families. The next step of this work would be sequencing the known deafness genes which have already been mapped in those loci in the linked families, so as to identify the pathogenic variants in these families.

Acknowledgments

We would like to thank all the families, students and personnel of the schools for hearing-impairment in Qom and Arak provinces for their cooperation. Also we wish to thank other hearing impairment centers for their contribution to this study. This work was supported by Tarbiat Modarres University and the National Institute of Genetic Engineering and Biotechnology (NIGEB), Grant No. 164, Tehran, I.R. Iran.

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