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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 25  |  Issue : 2  |  Page : 97-102

Mutation analysis of GJB2 and GJB6 genes and screening of nine common dfnb loci in iranian pedigrees with autosomal recessive nonsyndromic hearing loss


1 Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran
2 Medical Plants Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran
3 Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
4 Department of Genetics, Islamic Azad University, Falavarjan Branch, Tehran, Iran
5 Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
6 Department of Medical Genetics, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran

Date of Web Publication16-Aug-2019

Correspondence Address:
Dr. Effat Farrokhi
Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord
Iran
Prof. Morteza Hashemzadeh-Chaleshtori
Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/indianjotol.INDIANJOTOL_113_18

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  Abstract 


Background: Hearing loss (HL) is one of the most common sensory disorders (1/1000). Various studies have shown that a large proportion of autosomal recessive nonsyndromic HL (ARNSHL) in Iranian populations is caused by defects in a certain number of genes; new detection methods such as sequencing of the exome (exome-sequencing) can increase the frequency identification of responsible mutations for HL. The aim of this study was to screen for ARNSHL pedigrees and to find the responsible genes. Materials and Methods: Fifty big ARNSHL pedigrees including 130 Iranian patients with ARNSHL took part in this study. Direct sequencing and multiplex polymerase chain reaction were, respectively, used for the presence of GJB2 and GJB6 (del D13S1830 and del D13S1854) mutations in all of the families. The negative pedigrees for these genes were tested to homozygosity mapping for the linkage to nine known loci, then the negative pedigrees for them were sent for exome-sequencing. Results: Nine of 50 pedigrees had GJB2 ( 18%) mutations. No GJB6 mutation was found. Totally, 11 of 50 pedigrees (22%) showed linkage to 6 known loci DFNB, including 2 pedigrees to DFNB2, 2 pedigrees to DFNB3, 2 pedigrees to DFNB4, 2 pedigrees to DFNB7/11, 3 pedigrees to DFNB12, and 2 pedigrees to DFNB104. An individual from a large pedigree was examined by exome-sequencing and was observed no changes. Conclusion: In this study, DFNB1 and DFNB12 are the main causes of ARNSHL. GJB6 mutations, DFNB6, and DFNB59 were absent. Therefore, 40% of the ARNSHL etiology was found in this study.

Keywords: Autosomal recessive nonsyndromic hearing loss, gap junction protein beta 2, hearing loss


How to cite this article:
Azadegan-Dehkordi F, Koohiyan M, Shirzad M, Bahrami T, Yazdanpanahi N, Tabatabaiefar MA, Pourpaknia R, Farrokhi E, Hashemzadeh-Chaleshtori M. Mutation analysis of GJB2 and GJB6 genes and screening of nine common dfnb loci in iranian pedigrees with autosomal recessive nonsyndromic hearing loss. Indian J Otol 2019;25:97-102

How to cite this URL:
Azadegan-Dehkordi F, Koohiyan M, Shirzad M, Bahrami T, Yazdanpanahi N, Tabatabaiefar MA, Pourpaknia R, Farrokhi E, Hashemzadeh-Chaleshtori M. Mutation analysis of GJB2 and GJB6 genes and screening of nine common dfnb loci in iranian pedigrees with autosomal recessive nonsyndromic hearing loss. Indian J Otol [serial online] 2019 [cited 2019 Sep 19];25:97-102. Available from: http://www.indianjotol.org/text.asp?2019/25/2/97/264668




  Introduction Top


To understand the mechanism of a disease and to find a way to treat it, first the genetic loci involved in the disease should be identified.[1]

Linkage analysis-based approaches have identified a number of genes with large effect size.[2] Nowadays, more attention is paid to diseases that cannot be studied by these approaches since collecting a large number of samples and finding a sizeable pedigree with the disease is a very difficult task.[3] The homozygosity mapping (HM) method was developed to identify a disease-causing gene including autosomal recessive nonsyndromic hearing loss (ARNSHL) through analyses of patients from large consanguineous families.[4] A powerful technology known as next-generation sequencing (NGS) is capable of sequencing millions of small fragments covering the whole genome or large regions of interest, such as the entire coding portion of the genome (exome), at a reasonable cost and deleted runtime compared to Sanger sequencing.[5] Clarification of the genetic basis of human diseases is vital for understanding the basic pathology, this makes it easier to diagnose and improves treatment and genetic counseling.[6]

This method has greatly been able to solve problems such as the necessity of large families, and the existence of reduced penetrance, variable expressivity, and locus heterogeneity for geneticists, NGS can also be used to analyze genomic functions through transcriptome, methylome, and chromatin structure studies.[7]

Autosomal recessive is the most common form of ARNSHL that occurs approximately in more than 80% of inherited nonsyndromic hearing loss (NSHL) cases and can be caused by a variety of genetic and environmental factors.[8] To date, more than 150 genetic loci and 89 different genes have been identified for NSHL, and the most common form of NSHL is the autosomal recessive form (DNFB, 75%–80%).[9],[10]

Hereditary HL is a genetically heterogeneous disease, exhibiting different patterns of inheritance that includes autosomal recessive, autosomal dominant, mitochondrial inheritance, and X-linked.[11]GJB2 gene is involved in the ARNSHL that responsible for 16% and 18% of mutations in Iran.[12] Two GJB2 and GJB6 genes encode the proteins connexin 26 and connexin 30, respectively. A transmembrane connexin protein is oligomerized with five other connexin molecules to form homo- or hetero-hexameric hemichannels. These hemichannels are called “connexons.” Connexons can bind with other ones from adjacent cells to form functional gap junctions.[13] More than 100 different mutations in the GJB2 gene have been found, and the most common GJB2 mutation is c. 35delG. This mutation has been reported among whites (carrier frequency of 2%–4%). Gene and mutation frequencies vary according to the various populations being studied, suggesting an ethnical diversity for genetic causes of deafness. The c. 35delG mutation, responsible for the majority of the mutant alleles (at least >60%) in the Caucasian population,[14] causes the deletion of a guanine base in a series of 6Gs (guanine), which extends from nucleotide position 30–35 in the coding region of the GJB2 gene, leading to a frameshift mutation of codon 38 which causes a premature termination of the connexin 26 protein at amino acid 13. This deletion is lead to the production of a 12 amino acids truncated polypeptide instead of the 226 amino acids normal protein.[15] In many European countries, the prevalence of heterozygotes for GJB2- c. 35delG has been determined approximately 2%–4% of the healthy population.[16]

A study conducted in Iran found heterozygote prevalence for this mutation (4 families out of 45 families, 8.8%) (accepted).

Different investigations have determined that the contribution of GJB2 mutations in ARNSHL individuals is about 16%–18% in Iran.[12] Since the last decade, a series of studies have been conducted on the Iranian population to detect the prevalence and spectrum of GJB2 mutations. The various ethnicities, associated with a high proportion of kinship marriages (with the average of 38%),[8] result in various mutation frequencies among ethnic groups. Therefore, for a useful and effective genetic counseling, various ethnic groups must be checked.[12] Studies on the main loci involved in the Iranian ARNSHL patients seem to be essential to clarify their roles. The results of such studies could be applied to a more efficient genetic screening of the disease and the concomitant genetic counseling. The present study was aimed to detect the contribution of several genetic factors in 50 pedigrees with ARNSHL. The first was screened from the ARNSHL pedigrees for two main genes including GJB2 and GJB6 (del D13S1830 and del D13S1854) and then research associated to screening of eight known DFNB loci, including DFNB2 (MYO7A), DFNB3 (MYO15A), DFNB4 (SLC26A4), DFNB6 (TMIE) DFNB7/11 (TMC1), DFNB12 (CDH23), DFNB59 (PJVK), and DFNB104 (FAM65B) by the linkage analysis in the negative pedigrees for mutations in the two GJB2 and GJB6 genes. Then, a relatively large pedigree with three deaf people that was not connected to any of the loci was selected and sent for exome sequencing, which did not change.


  Materials and Methods Top


Patients and samples

The fifty Iranian pedigrees with the ARNSHL were selected to take part in a clinical and hearing evaluation to delete environmental causes of HL. The family pedigrees, the patients' audiograms, the medical records, and also the individual information were evaluated in each probe and in each pedigree. Each pedigree included the least three families and two patients with ARNSHL. Furthermore, each probe had moderate to profound bilateral sensory HL. This research was approved by the Ethics Committee of Shahrekord University of Medical Sciences, identification number: IR.SKUMS.REC.1395.105. Genomic DNA was extracted from 5 ml peripheral blood of each ARNSHL family member using a DNA extraction kit (DNPTM, CinnaGen, and Tehran, Iran). After the quality and quantity of DNA samples by nano-drop were checked, they were prepared for polymerase chain reaction (PCR).

Genotyping short tandem repeat markers, SLINK, and linkage analysis

We initially analyzed a part of a pedigree, which included one consanguinity loop including parents, two affected individuals, and two healthy individuals by five short tandem repeat (STR) markers. All the patients were homozygous, and different patterns were seen for the healthy individuals. In the following, all the parts remained from the pedigree were analyzed to confirm that the linkage was genotyped. Meanwhile, in the beginning, the screening was with intragenic markers. SLINK was estimated by pedigrees option of easy linkage (version 5.05) to evaluate the power of the 50 pedigrees for linkage strategy.[17] STR markers and primers were selected based on NCBI map viewer and UniSTS data. LOD scores (two-point and multi-point) were calculated using Super link (version 1.6) and Gene hunter (version 2.91). For LOD calculation, complete penetrance, autosomal recessive inheritance, the disease-allele frequency of 0.001, no phenocopy, and equal recombination frequencies in both male and female were assumed. Furthermore, reconstruction of haplotypes was done by HaploPainter software (version 029.5) [Figure 1].[18]
Figure 1: A sample of the haplotype analysis of the pedigree IF50 linked to DFNB2

Click here to view


Mutation screening

The GJB2 gene mutation analyzing was done for exons 1 and 2. Genomic DNA was PCR amplified for sequencing using the following primer pairs: F2: 5'CTC CCT GTT CTG TCC TAG CT3', R2: 5'CTC ATC CCT CTC ATG CTG TC3' for exon 2 (coding) of GJB2. To amplify exon 1 (noncoding) of GJB2 gene, the following primers were used: F1: 5'CGT CTT TGG GGG TGT TGC TT3', R1: 5'CAT GAA GAG GGC GTA CAA GTT AGA A3'. The patients with heterozygous pathogenic mutations in exon 1 and/or 2 of the GJB2 gene were screened for mutations detection in the GJB6 gene. Therefore, the GJB6 gene mutations detection were performed by a multiplex PCR analysis with the following primers: F1830: 5'CAC CAT GCG TAG CCT TAA CCA TTT T3' and R1830: 5'TTT AGG GCA TGA TTG GGG TGA TTT3' for the detection of del (GJB6/D13S1830), F1854: 5'CAG CGG CTA CCC TAG TTG TGG T3' and R1854: 5'TCA TAG TGA AGA ACT CGA TGC TGT TT3' for detection del (GJB6/D13S1854), and at the end for screening exon 1 of the gene GJB6 F1: 5'CAT GAA GAG GGC GTA CAA GTT AGA A3' and R1:5'CGT CTT TGG GGG TGT TGC TT3'.

Whole exome sequencing

Nowadays, despite the high ability of sequencing the whole genome, whole exome sequencing is more useful and it has more fans. Because of its cost and the amount of data produced, it was not practical until recently. To determine the genes causing Mendelian diseases, especially genetic heterogeneity, a comparatively easy to use the method in the research area is shown.[5]

While the coding regains of the human genome including exons of genes, includes only about 1% of the entire human genome, 85% of mutations known to cause Mendelian disorders are located in the coding region or in splice site regions. However, the scientists have discovered that DNA changes out of the exons can affect the activity of the gene and the production of the protein and eventually lead to genetic disorders and whole exome sequencing may be lost.[7] Although whole exome sequencing is a very useful tool in the identification of Mendelian diseases, it increases read coverage and length but these problems can be solved.


  Results Top


Screening of GJB2 and GJB6 genes

Of the 50 big ARNSHL pedigrees which were screened for the following GJB2 and GJB6 (del D13S1830 and del D13S1854) mutations, 9 (18%) out of 50 Iranian pedigrees had GJB2 mutations. In the remaining 41 pedigrees, GJB6 deletions were investigated. No GJB6 mutation was found.


  Clinical Results Top


Clinical characterizations of families have been reviewed. All the patients displayed bilateral HL had a spectrum of moderate-to-severe, severe, and severe to profound sensorineural prelingual HL. Pendred syndrome (enlarged vestibular aqueduct) and other syndromes were seen in any of the pedigrees tested. None of the pedigrees with mutations had the goiter. The phenotype of the thyroid was normal among the patients.

Results of genotyping, SLINK, and linkage analysis

Forty-one pedigrees with no GJB6 and GJB2 mutations were analyzed by the genetic linkage analysis for eight DFNB loci including DFNB2, DFNB3, DFNB4, DFNB6, DFNB7/11, DFNB12, DFNB59, and DFNB104. Two pedigrees showed linkage to DFNB2. We also found 3 pedigrees linked to DFNB3, 2 pedigrees linked to DFNB4, 2 pedigrees linked to DFNB7/11, 3 pedigrees linked to DFNB12, and 2 pedigrees linked to DFNB104. None of the families were linked to DFNB6 and DFNB59. Different markers for screening every eight loci were shown in [Table 1]. Ten pedigrees had SLINK values 2, twenty pedigrees had SLINK values of 1, and twenty pedigrees had SLINK values of <1.
Table 1: Different markers for screening each nine loci in this study

Click here to view


Results of whole exome sequencing

In this study, a large pedigree with three deaf individuals that did not show any linkage to loci was selected, and one patient was examined by whole exome sequencing. Unfortunately, no change was observed, which indicates that the changes may be in a place other than genome, except the genome coding parts.


  Discussion Top


Studies demonstrated that about half of the ARNSHL is originated from the mutations within the GJB2 gene.[8] The ARNSHL is the most prevalent form of HL. Although over 100 genes have been identified for ARNSHL, GJB2 mutations are the most prevalent causes of ARNSHL in the Mediterranean.[19]

The genetic heterogeneity of NSHL, due to a large number of mutations described in genes related to hearing impairment, makes molecular diagnosis a difficult task. Furthermore, epidemiological data indicate that differences exist as to the spectrum of GJB2 mutations in different populations.

Ethnic mutation diversity in the GJB2 gene can be explained by the “founder effect,” which predicts that for every population, the original mutation would have occurred in one or a few individuals and was perpetuated through time mainly by inheritance and not by de novo mutations.[8] First, the locus DFNB1 (GJB2 and GJB6) was screened by linkage analysis for 50 big ARNSHL pedigrees that GJB6 deletions were not seen in any of the families, and rate of GJB2 involvement in ARNSHL in previous studies on the Iranian population was 9 of 50 pedigrees and would confirm the variation of the Iranian population and the fact that the contribution of other loci should be studied. In addition, three mutations 35delG (4 pedigrees), c.-23 + 1G> A (4 pedigrees), and c. 299-300delAT (1 pedigree) in the previous study were reported in Iran (accepted).

Previous studies on the GJB2 mutations in Iran have shown that the mutation frequency of GJB2 varies between 0% and 35% among different regions of this country. GJB2 mutations were detected in 9 ARNSHL (18%) patients which was similar to the results of the previous investigations in other regions of Iran.[20] As a result, GJB2 gene-related HL has a low portion of ARNSHL in our population comparing to other ones.[8] The high percentage in the northwest and the center of Iran may be due to immigration patterns in some cities of Iran or may be due to the consanguinity or ascertainment among different ethnic groups.[8],[12] The observations support the presence of a founder effect in northern Iran (compared to 43% in Anatolia).[8] The GJB2 mutations are the most common variants found in Mediterranean families, different European populations, and in North America.[21],[22]

In this study, 2 of 50 pedigrees showed linkage to DFNB2. This gene is a member of the myosin gene family. This gene encodes an unconventional myosin with a short tail. Defects in this gene are associated with the human Usher syndrome 1B (HL) and the mouse shaker-1 phenotype.[20] Based on previous reports, various MYO7A mutations are also associated with HL severity and could cause progressive or profound congenital HL. Reported mutations among Iranian HL patients were in myosin motor.[20]

Furthermore, 2 of the 50 pedigrees were linked to DFNB3. Considering the relatively high mutation frequency of MYO15A-related HL in neighboring countries of Iran (Pakistan 5% in 7 families and Turkey 9.9% in 104 families)[23],[24] and in Iran, 5.71% in 140 families.[25] In another study, 302 Iranian families with MYO15A mutation frequency 9.6% accounted for the HL. Therefore this gene in these populations for HL is important.[26]

Our present data showed that 2 of 50 pedigrees were linked to DFNB4. Based on several investigations, it is observed that after the GJB2 mutation, the variants of the SLC26A4 gene are the most common cause of ARNSHL in the world and also in Iran.[9]

To date, over 170 different alterations have been identified in SLC26A4 (healthcare.uiowa.edu/labs/pendredandbor/slcMutations.htm). The variant profile and allele frequency may vary among different populations. The hotspot mutation in HL genes is mainly single-gene biallelic mutation.[20] In our study, 2 of 50 pedigrees were linked to DFNB7/11. TMC1 mutations are the sixth-most common causes of ARNSHL in North Africa, the Middle East, and India.[27] Frequencies of TMC1 mutations in different studies on Iranian deaf participants reported about 2.2%–3.2%.[20] Three of 50 pedigrees were linked to DFNB12. CDH23-encoded protein was involved in calcium-dependent cell-cell adhesion. Mutations in the CDH23 gene are associated with both Usher syndrome 1D (USH1D) and ARNSHL (DFNB12). Most of the mutations in the CDH23 gene which have been reported from Iranian patients were missense and caused ARNSHL.[20] Two out of 50 pedigrees were linked to DFNB104. This gene FAM65B (MIM611410) encodes plasma membrane-associated protein of hair cell stereocilia, in a relatively large family of Turkey; the absence of exon 3 of this gene was observed in all deaf people.[28]

In the present study, none of the families were linked to DFNB59 and DFNB6. Furthermore, for the sample, exome sequencing was performed which did not change.

In the present study, in spite of high heterogeneity of ARNSHL, we could detect 35 of 50 (70%) ARNSHL etiology.

The results of the present study confirm those of previous studies in Iran. The results would also give an overview of the most frequent loci: DFNB1, DFNB2, DFNB3, DFNB4, DFNB2, DFNB12, DFNB7/11, and DFNB104. GJB2 is the most common gene responsible for ARNSHL worldwide.[8] The majority of the responsible loci in ARNSHL are limited to a few loci; however, the remaining are rare or unknown. HM is a powerful tool with which to detect such loci causing ARNSHL in large consanguineous families.


  Conclusion Top


We performed HM analysis and detected 40% of the genetic etiology of ARNSHL in Iran. This dataset will be a powerful tool with which to detect causing gene mutations in a highly heterogeneous monogenic disorder and shows the genetic epidemiology of ARNSHL in Iran.

Financial support and sponsorship

This study was .nancially supported by the research deputy of the Shahrekord University of Medical Sciences (grant: 2165). The research also was approved by the Ethics Committee of Shahrekord University of Medical Sciences with a number: IR.SKUMS.REC.1395.105.

Conflicts of interest

There are no conflicts of interest.



 
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