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PEDOT-7374; No. of Pages 3 International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Finding mutation within non-coding region of GJB2 reveals its importance in genetic testing of Hearing Loss in Iranian population Atie Kashef a,h, Nooshin Nikzat a, Niloofar Bazzazadegan a, Zohreh Fattahi a, Farahnaz Sabbagh-Kermani b, Maryam Taghdiri c, Batool Azadeh d, Faezeh Mojahedi e, Atefeh Khoshaeen f, Haleh Habibi g, Hossein Najmabadi a, Kimia Kahrizi a,* a
Genetic Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran Department of Genetic, Kerman Welfare Organization, Kerman, Iran c Genetic Counseling Center, Shiraz Welfare Organization, Fars, Iran d Welfare Counseling Center, Isfahan, Iran e Mashhad Genetic Counseling Center, Mashhad, Khorasan, Iran f Welfare Counseling Center, Mazandaran, Iran g Genetic Counseling Center, Family Health Clinic, Mobasher Hospital, Hamedan, Iran h Deputy of Student Research, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 August 2014 Received in revised form 17 November 2014 Accepted 21 November 2014 Available online xxx
Objective: Hereditary hearing loss is the most common neurosensory disorder in humans. Half of the cases have genetic etiology with extraordinary genetic heterogeneity. Mutations in one gene, GJB2, are the most common cause for autosomal recessive non-syndromic hearing loss (ARNSHL) in many different populations. GJB2 encodes a gap junction channel protein (connexin 26), and is located on DFNB1 locus on chromosome 13q12.11 which also involve another connexin gene, GJB6. Mutation screening of GJB2 revealed that a high number of patients with deaf phenotype have heterozygous genotype and carry only one mutant allele. As the first comprehensive study in Iran, we have targeted GJB2-related Iranian heterozygotes, looking for second mutant allele which leads to hearing impairment. They bear first mutation in their coding exon of GJB2. Method: Using PCR-based direct sequencing, we assessed 103 patients with ARNSHL for variants in noncoding exon and promoter region of this gene, for the first time in Iran. Result: We have identified the second mutant allele in splice site of exon-1 of GJB2 which is known as IVS1 + 1G > A in 17 probands. We found no mutation in promoter region of GJB2. Conclusion: Our findings reveal that IVS1 + 1G > A mutation in noncoding exon of GJB2 is the most common mutation after 35delG within multi ethnical Iranian heterozygote samples. It emphasizes to approach exon1 of GJB2 in case of ARNSHL genetic diagnosis. ß 2014 Published by Elsevier Ireland Ltd.
Keywords: Autosomal recessive non-syndromic hearing loss GJB2 Heterozygote Noncoding mutation IVS1 + 1G > A
1. Introduction GJB2, the most prevalent responsible gene in hereditary hearing loss (HHL), encodes a gap junction protein called connexin 26 (cx26) [1]. In the inner ear connexin proteins included cx26 and cx30 (coded by GJB6 gene), construct heteromeric connexons in intracellular gap junctions which have an important role in
* Corresponding author at: Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Koodakyar St., Daneshjoo Blvd., Evin, Tehran 19834, Iran. Tel.: +98 21 240 78 14; fax: +98 21 240 78 14. E-mail address: [email protected] (K. Kahrizi).
potassium ions homeostasis. It is known that recycling potassium ions plays role as a part of signal transduction in auditory system [2,3]. Mutations in GJB2 cause nonsyndromic autosomal recessive (DFNB1), autosomal dominant (DFNA3), and even syndromic pattern of HHL [4]. As such, DFNB1, a locus which is located on chromosome 13q11, is introduced responsible for about 70% of HHL patients and GJB2 stands at top of the list [5]. GJB2 gene structurally contains two exons; one coding (exon2) and another non-coding one (exon1). To date, over 200 mutations, polymorphism and unclassified variants has known in relationship to this gene, and it causally accounts for 10–80% of autosomal recessive nonsyndromic hearing loss (ARNSHL) cases in different populations. In Iran, a comprehensive study revealed that GJB2
http://dx.doi.org/10.1016/j.ijporl.2014.11.024 0165-5876/ß 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: A. Kashef, et al., Finding mutation within non-coding region of GJB2 reveals its importance in genetic testing of Hearing Loss in Iranian population, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.11.024
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PEDOT-7374; No. of Pages 3 A. Kashef et al. / International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx
2
2.2. Genetic testing
mutations are detectable in about 16% of ARNSHL patients [6,7]. Although, the majority of screened GJB2 mutants bears a homo or compound heterozygote (biallelic) mutation in coding exon of GJB2, a minority fraction of ARNSHL has detected as monoallelic mutated in coding region of this gene. Some studies through European population have reported a percentage of 10–42 monoallelic deaf patients with one mutation in coding GJB2 [8]. Regarding last studies in Iran, GJB6 mutations and two large deletions through DFNB1, have no significant contribution in Iranian deaf population [7,9]. Such being the case, in this study for the first time, we have assessed exon1 and promoter region as the prominent noncoding regions of GJB2, to find out the second defected allele in 103 GJB2-related heterozygous patients out of total 2322 Iranian deaf patients. It presents about four percent of this population which is not insignificant. Splice site mutation which disrupts functional GJB2 mRNA was first reported by Denoyelle et al. [10]. Considering the importance of this type of mutations, it has been investigated through many different populations [11–14] and here we have assessed its contribution in a pool of Iranian genotypes.
DNA direct sequencing performed to assay promoter region and noncoding exon of GJB2. Primarily, Touch-down PCR done by specific pair primers, Forward (ACT CAT GGG GGC TCA AAG GA) and Reverse (GCA ACC GCT CTG GGT CTC GCG GTC CCT) which amplify 458 bp sequence from upstream of basal promoter to downstream of first exon. Primers were designed using Primer3 algorithm. PCR products qualified by agarose (2%) gel electrophoresis, then, sequenced using BigDye Terminator Cycle Sequencing kit and ABI 3130 automated DNA sequencing machine. Sequencing data analyzed with Applied Biosystems DNA sequencing Analysis software v.5.3.1, and aligned against reference sequence (downloaded from UCSC Genome Browser) using CodonCode Aligner software. 3. Results Direct sequencing analysis on DNA samples through 103 autosomal recessive non-syndromic heterozygote deaf patients showed that in 17 probands, the second heterozygous mutant allele exist in noncoding exon of GJB2. This mutation is called IVS1 + 1 G > A and occurs at donor splice site of first exon. Considering genotype–phenotype correlation, all the patients were exhibited early onset (six month to one year in age) deafness with severe to profound degrees, equally in both male and female genders. Moreover, sequencing analysis on the basal promoter region of GJB2 gene revealed no variant in our studied patients.
2. Materials and methods 2.1. Patients and DNA samples During a twelve year study, 2322 Iranian families with ARNS deaf probands have been referred to Genetic Research Center of Social Welfare & Rehabilitation University, from different provinces of Iran. According to precise clinical examinations and audiogram analysis, the pattern of deafness and its inheritance confirmed as nonsyndromic autosomal recessive. As the first step of genetic analysis, these patients have been screened for prevalent variants of GJB2 by allele specific PCR (AS PCR) and Sanger sequencing methods. Furthermore, two large GJB6 related deletions (GJB6D13S1830 and GJB6-D13S1854) in DFNB1 locus have been studied by the method described by Del Castillo and colleagues [8]. Notwithstanding, many samples identified with homo or compoundheterozygous mutations, a fraction of samples were shown to be heterozygote with no evidence of another mutant allele in GJB2 gene or DFNB1 large deletions. Our target was genomic DNA samples of 100 GJB2-related heterozygous patients excluded from such a broad ethnical population. All the patients were shown early onset deaf phenotype with severe to profound degree. All procedures were approved by the human research institutional review boards of the Welfare Science & Rehabilitation University and Iran University of Medical Sciences, Tehran, Iran.
4. Discussion Nowadays, in almost populations GJB2 is the first candidate gene to screen in the case of HHL [15]. This gene as the most prevalent cause of ARNSHL – the most prevalent form of HHL – is construct of two exons separated by an intron, and an active basal promoter located at proximal 5P-flanking region from 128 to +2 (relative to the transcription initiation site) [16]. To date, over 200 different variants have been identified through coding exon (exon2), noncoding exon (exon1) and even in basal promoter of GJB2 [17]. Benign GJB2 mutations are detected in the homozygous, compound heterozygous, and also in heterozygous patients with an unknown second defective allele. According to studies on European populations, a large fraction (10–42%) of GJB2-related deaf patients have only one recessive mutant allele at DFNB1 locus, with the accompanying mutation unidentified [8]. According to available studies in Iranian population [6], as a
Table 1 Distribution of 17 compound heterozygous patients considering number of affected individuals within core family as well as whole pedigree, genotype, and ethnicity. Family ID
Mutation within coding exon
Mutation within noncoding region
Affected in core family
Affected in pedigree
HL severity
Ethnicity
373 8600249 8600294 8600324 8600338 8600432 8600447 8600186 8700067 9100005 8600398 357 1856 3042 1258 305 328
35delG 35delG 35delG 35delG 35delG 35delG 35delG 35delG 35delG 35delG R32C E47X 35delG 290-291insA 35delG R184P 35delG
IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A
1 1 1 1 1 1 1 2 3 2 1 2 1 3 2 2 4
3 5 1 1 2 2 2 5 6 12 2 4 2 4 2 4 5
Severe Profound Profound Profound Profound Profound Profound Profound Profound Moderate Severe Severe Profound Moderate Severe Severe Severe
Fars Fars Kurd Turk Turk Turk Gilak Fars Turk Mazani Turk Fars Lur Fars Fars Fars Fars
Please cite this article in press as: A. Kashef, et al., Finding mutation within non-coding region of GJB2 reveals its importance in genetic testing of Hearing Loss in Iranian population, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.11.024
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PEDOT-7374; No. of Pages 3 A. Kashef et al. / International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx Table 2 Distribution of total 103 probands in respect of GJB2 genotyping results. Coding exon mutations
Deletion 35delG 120delE 167delT 312del 329delA 300delE2(AT) 312-326delN Insertion 290-291insA 507insAACG
Noncoding region mutations
Proband frequency
Splice site
Proband frequency
29 5 3 1 1 1 1
IVS1 + 1G > A
13
1 1
IVS1 + 1G > A
Missense R128H V27I A171T M163V R124P R184P V153I K221N S139N M93I G59S R32C G130V V37I
25 10 3 2 2 2 2 2 1 1 1 1 1 1
Nonsense W24X E47X
2 1
3
we suggest screening all of them using this interval, efficiently. (3) In post genomic era, it is identified that noncoding sequences have more effective role than protein coding exons in gene expression and regulation. Thus, regulatory elements on unknown sites of DFNB1 may be considered. (4) Other ARNSHL related loci, may cause the impairment and the presented patients are normal carrier for one mutation in GJB2 exon2. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgment
1
We wish to thank patients and their families for their participation in this study. This study was supported by Elite Foundation and Iranian National Science Foundation. (grant number of 88002310) References
IVS1 + 1G > A
1
IVS1 + 1G > A
1
IVS1 + 1G > A
1
multi ethnic population, this fraction composes about 4.5% of deaf patients. In present study, we targeted these monoallelic patients to identify the second allele if it is exist in noncoding regions of GJB2. For the first time, Denoyelle and colleagues (1999) showed that One out of ten GJB2 heterozygote patients showed IVS1 + 1G > A mutation (at donor splice site of GJB2 noncoding exon1) in European families [10]. Afterward, E. Green et and colleagues (2000) reported that one out of 41 deaf patients showed IVS1 + 1G > A mutation in Midwestern United States [13]. Testing for the GJB2 IVS1 + 1G > A mutation in Chinese population explained deafness in 1.89% of Chinese GJB2 monoallelic patients [11]. Also in Turkish population in vicinity of Iran, this mutation is identified in eight out of 16 GJB2 heterozygote patients [12]. Our results reveal that in Iranian samples IVS1 + 1G > A mutation can explain hearing impairment in about 17% of heterozygous GJB2 mutants. As such, noncoding exon1 is prominent to check routinely in our clinical genetic testing. There was no significant difference in severity of HL in our families, and we included patients with wide variety of ethnicity. Moreover, the most frequent first mutant allele was 35delG in coding exon2 (Table 1). As it is shown in Table 1, each proband has affected siblings in his/ her core family as well as more affected individuals in a large consanguineus pedigree. So, our results are applicable in genetic testing and counseling for many more patients. We found no variation in 120 bp basal promoter of GJB2, hence, promoter region seems to have no contribution in our studied population. Considering 83 monoallelic patients that we could not detect second allele variation in GJB2 gene (Table 2), four possible causes touch the mind; (1) GJB6 as the second important gene on DFNB1 locus in HHL would be looked for recessive mutations, although it seems not to be highly probable according to present studies in Iranian population. (2) To date, four gross DFNB1 deletions are known in GJB2-related deaf patients with European origins [18]. As these four deletions have a common large interval,
[1] N. Hilgert, R.J.H. Smith, G. Van Camp, Forty-six genes causing nonsyndromic hearing impairment: which ones should be analyzed in DNA diagnostics? Mutat. Res. 681 (2) (2009) 189–196. [2] X. Estivill, P. Fortina, S. Surrey, R. Rabionet, S. Melchionda, L. D’Agruma, et al., Connexin-26 mutations in sporadic and inherited sensorineural deafness, Lancet 351 (9100) (1998) 394–398. [3] N. Pallares-Ruiz, P. Blanchet, M. Mondain, M. Claustres, A.F. Roux, A large deletion including most of GJB6 in recessive non syndromic deafness: a digenic effect? Eur. J. Hum. Genet.: EJHG 10 (1) (2002) 72. [4] S. Iossa, E. Marciano, A. Franze´, GJB2 gene mutations in syndromic skin diseases with sensorineural hearing loss, Curr. Genomics 12 (7) (2011) 475. [5] X.Z. Liu, X.J. Xia, L.R. Xu, A. Pandya, C.Y. Liang, S.H. Blanton, et al., Mutations in connexin31 underlie recessive as well as dominant non-syndromic hearing loss, Hum. Mol. Genet. 9 (1) (2000) 63–67. [6] N. Bazazzadegan, N. Nikzat, Z. Fattahi, C. Nishimura, N. Meyer, Sh. Sahraian, et al., The spectrum of GJB2 mutations in the Iranian population with non-syndromic hearing loss—a twelve year study, Int. J. Pediatr. Otorhinolaryngol. 76 (2012) 1164–1174. [7] H. Najmabadi, C. Nishimura, K. Kahrizi, Y. Riazalhosseini, M. Malekpour, A. Daneshi, et al., GJB2 mutations: passage through Iran, Am. J. Med. Genet. A 133 (2) (2005) 132–137. [8] F. Del Castillo, M. Rodrı´guez-Ballesteros, A. Alvarez, T. Hutchin, E. Leonardi, C.A. de Oliveira, et al., A novel deletion involving the connexin-30 gene: del (GJB6-d13s1854), found in trans with mutations in the GJB2 gene (connexin-26) in subjects with DFNB1 non-syndromic hearing impairment, J. Med. Genet. 42 (7) (2005) 588–594. [9] N. Mahdieh, C. Nishimura, K. Ali-Madadi, Y. Riazalhosseini, H. Yazdan, S. Arzhangi, et al., The frequency of GJB2 mutations and the D (GJB6-D13S1830) deletion as a cause of autosomal recessive non-syndromic deafness in the Kurdish population, Clin. Genet. 65 (6) (2004) 506–508. [10] F. Denoyelle, S. Marlin, D. Weil, L. Moatti, P. Chauvin, E.N. Garabe´dian, et al., Clinical features of the prevalent form of childhood deafness, DFNB1, due to a connexin-26 gene defect: implications for genetic counselling, Lancet 353 (9161) (1999) 1298–1303. [11] Y. Yuan, F. Yu, G. Wang, Sh. Huang, R. Yu, X. Zhang, et al., Prevalence of the GJB2 IVS1 + 1G > A mutation in Chinese hearing loss patients with monoallelic pathogenic mutation in the coding region of GJB2, J. Transl. Med. 8 (1) (2010) 127. [12] A. Sirmaci, D. Akcayoz-Duman, M. Tekin, The c. IVS1 + 1G > A mutation intheGJB2 gene is prevalent and large deletions involving theGJB6 gene are not present in the Turkish population, J. Genet. 85 (3) (2006) 213–216. [13] G.E. Green, A. Scott, M. McDonald, G. Woodworth, C. Sheffield, R.J. Smith, et al., Carrier rates in the midwestern United States for GJB2 mutations causing inherited deafness, JAMA: J. Am. Med. Assoc. 281 (23) (1999) 2211. [14] M. Bonyadi, N. Fotouhi, M. Esmaeili, Prevalence of IVS1 + 1G > A mutation among Iranian Azeri Turkish patients with autosomal recessive non-syndromic hearing loss (ARNSHL), Int. J. Pediatr. Otorhinolaryngol. 75 (2011) 1612–1615. [15] N. Hilgert, R.J.H. Smith, G. Van Camp, Forty-six genes causing nonsyndromic hearing impairment: which ones should be analyzed in DNA diagnostics? Mutat. Res. 681 (2–3) (2009) 189–196. [16] Z.J. Tu, D.T. Kiang, Mapping and characterization of the basal promoter of the human connexin26 gene, Biochim. Biophys. Acta (BBA) 1443 (1) (1998) 169–181. [17] T.D. Matos, H. Caria, H. Simo˜es - Teixeira, T. Aasen, R. Nickel, D.J. Jagger, et al., A novel hearing loss-related mutation occurring in the GJB2 basal promoter, J. Med. Genet. 44 (11) (2007) 721–725. [18] E. Wilch, H. Azaiez, R.A. Fisher, J. Elfenbein, A. Murgia, R. Birkenha¨ger, et al., A novel DFNB1 deletion allele supports the existence of a distant cis-regulatory region that controls GJB2 and GJB6 expression, Clin. Genet. 78 (3) (2010) 267–274.
Web references http://hereditaryhearingloss.org http://davinci.crg.es/deafness http://genome.ucsc.edu/ primer3.wi.mit.edu/
http://
Please cite this article in press as: A. Kashef, et al., Finding mutation within non-coding region of GJB2 reveals its importance in genetic testing of Hearing Loss in Iranian population, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.11.024
PEDOT-7374; No. of Pages 3 International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Finding mutation within non-coding region of GJB2 reveals its importance in genetic testing of Hearing Loss in Iranian population Atie Kashef a,h, Nooshin Nikzat a, Niloofar Bazzazadegan a, Zohreh Fattahi a, Farahnaz Sabbagh-Kermani b, Maryam Taghdiri c, Batool Azadeh d, Faezeh Mojahedi e, Atefeh Khoshaeen f, Haleh Habibi g, Hossein Najmabadi a, Kimia Kahrizi a,* a
Genetic Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran Department of Genetic, Kerman Welfare Organization, Kerman, Iran c Genetic Counseling Center, Shiraz Welfare Organization, Fars, Iran d Welfare Counseling Center, Isfahan, Iran e Mashhad Genetic Counseling Center, Mashhad, Khorasan, Iran f Welfare Counseling Center, Mazandaran, Iran g Genetic Counseling Center, Family Health Clinic, Mobasher Hospital, Hamedan, Iran h Deputy of Student Research, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 August 2014 Received in revised form 17 November 2014 Accepted 21 November 2014 Available online xxx
Objective: Hereditary hearing loss is the most common neurosensory disorder in humans. Half of the cases have genetic etiology with extraordinary genetic heterogeneity. Mutations in one gene, GJB2, are the most common cause for autosomal recessive non-syndromic hearing loss (ARNSHL) in many different populations. GJB2 encodes a gap junction channel protein (connexin 26), and is located on DFNB1 locus on chromosome 13q12.11 which also involve another connexin gene, GJB6. Mutation screening of GJB2 revealed that a high number of patients with deaf phenotype have heterozygous genotype and carry only one mutant allele. As the first comprehensive study in Iran, we have targeted GJB2-related Iranian heterozygotes, looking for second mutant allele which leads to hearing impairment. They bear first mutation in their coding exon of GJB2. Method: Using PCR-based direct sequencing, we assessed 103 patients with ARNSHL for variants in noncoding exon and promoter region of this gene, for the first time in Iran. Result: We have identified the second mutant allele in splice site of exon-1 of GJB2 which is known as IVS1 + 1G > A in 17 probands. We found no mutation in promoter region of GJB2. Conclusion: Our findings reveal that IVS1 + 1G > A mutation in noncoding exon of GJB2 is the most common mutation after 35delG within multi ethnical Iranian heterozygote samples. It emphasizes to approach exon1 of GJB2 in case of ARNSHL genetic diagnosis. ß 2014 Published by Elsevier Ireland Ltd.
Keywords: Autosomal recessive non-syndromic hearing loss GJB2 Heterozygote Noncoding mutation IVS1 + 1G > A
1. Introduction GJB2, the most prevalent responsible gene in hereditary hearing loss (HHL), encodes a gap junction protein called connexin 26 (cx26) [1]. In the inner ear connexin proteins included cx26 and cx30 (coded by GJB6 gene), construct heteromeric connexons in intracellular gap junctions which have an important role in
* Corresponding author at: Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Koodakyar St., Daneshjoo Blvd., Evin, Tehran 19834, Iran. Tel.: +98 21 240 78 14; fax: +98 21 240 78 14. E-mail address: [email protected] (K. Kahrizi).
potassium ions homeostasis. It is known that recycling potassium ions plays role as a part of signal transduction in auditory system [2,3]. Mutations in GJB2 cause nonsyndromic autosomal recessive (DFNB1), autosomal dominant (DFNA3), and even syndromic pattern of HHL [4]. As such, DFNB1, a locus which is located on chromosome 13q11, is introduced responsible for about 70% of HHL patients and GJB2 stands at top of the list [5]. GJB2 gene structurally contains two exons; one coding (exon2) and another non-coding one (exon1). To date, over 200 mutations, polymorphism and unclassified variants has known in relationship to this gene, and it causally accounts for 10–80% of autosomal recessive nonsyndromic hearing loss (ARNSHL) cases in different populations. In Iran, a comprehensive study revealed that GJB2
http://dx.doi.org/10.1016/j.ijporl.2014.11.024 0165-5876/ß 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: A. Kashef, et al., Finding mutation within non-coding region of GJB2 reveals its importance in genetic testing of Hearing Loss in Iranian population, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.11.024
G Model
PEDOT-7374; No. of Pages 3 A. Kashef et al. / International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx
2
2.2. Genetic testing
mutations are detectable in about 16% of ARNSHL patients [6,7]. Although, the majority of screened GJB2 mutants bears a homo or compound heterozygote (biallelic) mutation in coding exon of GJB2, a minority fraction of ARNSHL has detected as monoallelic mutated in coding region of this gene. Some studies through European population have reported a percentage of 10–42 monoallelic deaf patients with one mutation in coding GJB2 [8]. Regarding last studies in Iran, GJB6 mutations and two large deletions through DFNB1, have no significant contribution in Iranian deaf population [7,9]. Such being the case, in this study for the first time, we have assessed exon1 and promoter region as the prominent noncoding regions of GJB2, to find out the second defected allele in 103 GJB2-related heterozygous patients out of total 2322 Iranian deaf patients. It presents about four percent of this population which is not insignificant. Splice site mutation which disrupts functional GJB2 mRNA was first reported by Denoyelle et al. [10]. Considering the importance of this type of mutations, it has been investigated through many different populations [11–14] and here we have assessed its contribution in a pool of Iranian genotypes.
DNA direct sequencing performed to assay promoter region and noncoding exon of GJB2. Primarily, Touch-down PCR done by specific pair primers, Forward (ACT CAT GGG GGC TCA AAG GA) and Reverse (GCA ACC GCT CTG GGT CTC GCG GTC CCT) which amplify 458 bp sequence from upstream of basal promoter to downstream of first exon. Primers were designed using Primer3 algorithm. PCR products qualified by agarose (2%) gel electrophoresis, then, sequenced using BigDye Terminator Cycle Sequencing kit and ABI 3130 automated DNA sequencing machine. Sequencing data analyzed with Applied Biosystems DNA sequencing Analysis software v.5.3.1, and aligned against reference sequence (downloaded from UCSC Genome Browser) using CodonCode Aligner software. 3. Results Direct sequencing analysis on DNA samples through 103 autosomal recessive non-syndromic heterozygote deaf patients showed that in 17 probands, the second heterozygous mutant allele exist in noncoding exon of GJB2. This mutation is called IVS1 + 1 G > A and occurs at donor splice site of first exon. Considering genotype–phenotype correlation, all the patients were exhibited early onset (six month to one year in age) deafness with severe to profound degrees, equally in both male and female genders. Moreover, sequencing analysis on the basal promoter region of GJB2 gene revealed no variant in our studied patients.
2. Materials and methods 2.1. Patients and DNA samples During a twelve year study, 2322 Iranian families with ARNS deaf probands have been referred to Genetic Research Center of Social Welfare & Rehabilitation University, from different provinces of Iran. According to precise clinical examinations and audiogram analysis, the pattern of deafness and its inheritance confirmed as nonsyndromic autosomal recessive. As the first step of genetic analysis, these patients have been screened for prevalent variants of GJB2 by allele specific PCR (AS PCR) and Sanger sequencing methods. Furthermore, two large GJB6 related deletions (GJB6D13S1830 and GJB6-D13S1854) in DFNB1 locus have been studied by the method described by Del Castillo and colleagues [8]. Notwithstanding, many samples identified with homo or compoundheterozygous mutations, a fraction of samples were shown to be heterozygote with no evidence of another mutant allele in GJB2 gene or DFNB1 large deletions. Our target was genomic DNA samples of 100 GJB2-related heterozygous patients excluded from such a broad ethnical population. All the patients were shown early onset deaf phenotype with severe to profound degree. All procedures were approved by the human research institutional review boards of the Welfare Science & Rehabilitation University and Iran University of Medical Sciences, Tehran, Iran.
4. Discussion Nowadays, in almost populations GJB2 is the first candidate gene to screen in the case of HHL [15]. This gene as the most prevalent cause of ARNSHL – the most prevalent form of HHL – is construct of two exons separated by an intron, and an active basal promoter located at proximal 5P-flanking region from 128 to +2 (relative to the transcription initiation site) [16]. To date, over 200 different variants have been identified through coding exon (exon2), noncoding exon (exon1) and even in basal promoter of GJB2 [17]. Benign GJB2 mutations are detected in the homozygous, compound heterozygous, and also in heterozygous patients with an unknown second defective allele. According to studies on European populations, a large fraction (10–42%) of GJB2-related deaf patients have only one recessive mutant allele at DFNB1 locus, with the accompanying mutation unidentified [8]. According to available studies in Iranian population [6], as a
Table 1 Distribution of 17 compound heterozygous patients considering number of affected individuals within core family as well as whole pedigree, genotype, and ethnicity. Family ID
Mutation within coding exon
Mutation within noncoding region
Affected in core family
Affected in pedigree
HL severity
Ethnicity
373 8600249 8600294 8600324 8600338 8600432 8600447 8600186 8700067 9100005 8600398 357 1856 3042 1258 305 328
35delG 35delG 35delG 35delG 35delG 35delG 35delG 35delG 35delG 35delG R32C E47X 35delG 290-291insA 35delG R184P 35delG
IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A IVS1 + 1G > A
1 1 1 1 1 1 1 2 3 2 1 2 1 3 2 2 4
3 5 1 1 2 2 2 5 6 12 2 4 2 4 2 4 5
Severe Profound Profound Profound Profound Profound Profound Profound Profound Moderate Severe Severe Profound Moderate Severe Severe Severe
Fars Fars Kurd Turk Turk Turk Gilak Fars Turk Mazani Turk Fars Lur Fars Fars Fars Fars
Please cite this article in press as: A. Kashef, et al., Finding mutation within non-coding region of GJB2 reveals its importance in genetic testing of Hearing Loss in Iranian population, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.11.024
G Model
PEDOT-7374; No. of Pages 3 A. Kashef et al. / International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx Table 2 Distribution of total 103 probands in respect of GJB2 genotyping results. Coding exon mutations
Deletion 35delG 120delE 167delT 312del 329delA 300delE2(AT) 312-326delN Insertion 290-291insA 507insAACG
Noncoding region mutations
Proband frequency
Splice site
Proband frequency
29 5 3 1 1 1 1
IVS1 + 1G > A
13
1 1
IVS1 + 1G > A
Missense R128H V27I A171T M163V R124P R184P V153I K221N S139N M93I G59S R32C G130V V37I
25 10 3 2 2 2 2 2 1 1 1 1 1 1
Nonsense W24X E47X
2 1
3
we suggest screening all of them using this interval, efficiently. (3) In post genomic era, it is identified that noncoding sequences have more effective role than protein coding exons in gene expression and regulation. Thus, regulatory elements on unknown sites of DFNB1 may be considered. (4) Other ARNSHL related loci, may cause the impairment and the presented patients are normal carrier for one mutation in GJB2 exon2. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgment
1
We wish to thank patients and their families for their participation in this study. This study was supported by Elite Foundation and Iranian National Science Foundation. (grant number of 88002310) References
IVS1 + 1G > A
1
IVS1 + 1G > A
1
IVS1 + 1G > A
1
multi ethnic population, this fraction composes about 4.5% of deaf patients. In present study, we targeted these monoallelic patients to identify the second allele if it is exist in noncoding regions of GJB2. For the first time, Denoyelle and colleagues (1999) showed that One out of ten GJB2 heterozygote patients showed IVS1 + 1G > A mutation (at donor splice site of GJB2 noncoding exon1) in European families [10]. Afterward, E. Green et and colleagues (2000) reported that one out of 41 deaf patients showed IVS1 + 1G > A mutation in Midwestern United States [13]. Testing for the GJB2 IVS1 + 1G > A mutation in Chinese population explained deafness in 1.89% of Chinese GJB2 monoallelic patients [11]. Also in Turkish population in vicinity of Iran, this mutation is identified in eight out of 16 GJB2 heterozygote patients [12]. Our results reveal that in Iranian samples IVS1 + 1G > A mutation can explain hearing impairment in about 17% of heterozygous GJB2 mutants. As such, noncoding exon1 is prominent to check routinely in our clinical genetic testing. There was no significant difference in severity of HL in our families, and we included patients with wide variety of ethnicity. Moreover, the most frequent first mutant allele was 35delG in coding exon2 (Table 1). As it is shown in Table 1, each proband has affected siblings in his/ her core family as well as more affected individuals in a large consanguineus pedigree. So, our results are applicable in genetic testing and counseling for many more patients. We found no variation in 120 bp basal promoter of GJB2, hence, promoter region seems to have no contribution in our studied population. Considering 83 monoallelic patients that we could not detect second allele variation in GJB2 gene (Table 2), four possible causes touch the mind; (1) GJB6 as the second important gene on DFNB1 locus in HHL would be looked for recessive mutations, although it seems not to be highly probable according to present studies in Iranian population. (2) To date, four gross DFNB1 deletions are known in GJB2-related deaf patients with European origins [18]. As these four deletions have a common large interval,
[1] N. Hilgert, R.J.H. Smith, G. Van Camp, Forty-six genes causing nonsyndromic hearing impairment: which ones should be analyzed in DNA diagnostics? Mutat. Res. 681 (2) (2009) 189–196. [2] X. Estivill, P. Fortina, S. Surrey, R. Rabionet, S. Melchionda, L. D’Agruma, et al., Connexin-26 mutations in sporadic and inherited sensorineural deafness, Lancet 351 (9100) (1998) 394–398. [3] N. Pallares-Ruiz, P. Blanchet, M. Mondain, M. Claustres, A.F. Roux, A large deletion including most of GJB6 in recessive non syndromic deafness: a digenic effect? Eur. J. Hum. Genet.: EJHG 10 (1) (2002) 72. [4] S. Iossa, E. Marciano, A. Franze´, GJB2 gene mutations in syndromic skin diseases with sensorineural hearing loss, Curr. Genomics 12 (7) (2011) 475. [5] X.Z. Liu, X.J. Xia, L.R. Xu, A. Pandya, C.Y. Liang, S.H. Blanton, et al., Mutations in connexin31 underlie recessive as well as dominant non-syndromic hearing loss, Hum. Mol. Genet. 9 (1) (2000) 63–67. [6] N. Bazazzadegan, N. Nikzat, Z. Fattahi, C. Nishimura, N. Meyer, Sh. Sahraian, et al., The spectrum of GJB2 mutations in the Iranian population with non-syndromic hearing loss—a twelve year study, Int. J. Pediatr. Otorhinolaryngol. 76 (2012) 1164–1174. [7] H. Najmabadi, C. Nishimura, K. Kahrizi, Y. Riazalhosseini, M. Malekpour, A. Daneshi, et al., GJB2 mutations: passage through Iran, Am. J. Med. Genet. A 133 (2) (2005) 132–137. [8] F. Del Castillo, M. Rodrı´guez-Ballesteros, A. Alvarez, T. Hutchin, E. Leonardi, C.A. de Oliveira, et al., A novel deletion involving the connexin-30 gene: del (GJB6-d13s1854), found in trans with mutations in the GJB2 gene (connexin-26) in subjects with DFNB1 non-syndromic hearing impairment, J. Med. Genet. 42 (7) (2005) 588–594. [9] N. Mahdieh, C. Nishimura, K. Ali-Madadi, Y. Riazalhosseini, H. Yazdan, S. Arzhangi, et al., The frequency of GJB2 mutations and the D (GJB6-D13S1830) deletion as a cause of autosomal recessive non-syndromic deafness in the Kurdish population, Clin. Genet. 65 (6) (2004) 506–508. [10] F. Denoyelle, S. Marlin, D. Weil, L. Moatti, P. Chauvin, E.N. Garabe´dian, et al., Clinical features of the prevalent form of childhood deafness, DFNB1, due to a connexin-26 gene defect: implications for genetic counselling, Lancet 353 (9161) (1999) 1298–1303. [11] Y. Yuan, F. Yu, G. Wang, Sh. Huang, R. Yu, X. Zhang, et al., Prevalence of the GJB2 IVS1 + 1G > A mutation in Chinese hearing loss patients with monoallelic pathogenic mutation in the coding region of GJB2, J. Transl. Med. 8 (1) (2010) 127. [12] A. Sirmaci, D. Akcayoz-Duman, M. Tekin, The c. IVS1 + 1G > A mutation intheGJB2 gene is prevalent and large deletions involving theGJB6 gene are not present in the Turkish population, J. Genet. 85 (3) (2006) 213–216. [13] G.E. Green, A. Scott, M. McDonald, G. Woodworth, C. Sheffield, R.J. Smith, et al., Carrier rates in the midwestern United States for GJB2 mutations causing inherited deafness, JAMA: J. Am. Med. Assoc. 281 (23) (1999) 2211. [14] M. Bonyadi, N. Fotouhi, M. Esmaeili, Prevalence of IVS1 + 1G > A mutation among Iranian Azeri Turkish patients with autosomal recessive non-syndromic hearing loss (ARNSHL), Int. J. Pediatr. Otorhinolaryngol. 75 (2011) 1612–1615. [15] N. Hilgert, R.J.H. Smith, G. Van Camp, Forty-six genes causing nonsyndromic hearing impairment: which ones should be analyzed in DNA diagnostics? Mutat. Res. 681 (2–3) (2009) 189–196. [16] Z.J. Tu, D.T. Kiang, Mapping and characterization of the basal promoter of the human connexin26 gene, Biochim. Biophys. Acta (BBA) 1443 (1) (1998) 169–181. [17] T.D. Matos, H. Caria, H. Simo˜es - Teixeira, T. Aasen, R. Nickel, D.J. Jagger, et al., A novel hearing loss-related mutation occurring in the GJB2 basal promoter, J. Med. Genet. 44 (11) (2007) 721–725. [18] E. Wilch, H. Azaiez, R.A. Fisher, J. Elfenbein, A. Murgia, R. Birkenha¨ger, et al., A novel DFNB1 deletion allele supports the existence of a distant cis-regulatory region that controls GJB2 and GJB6 expression, Clin. Genet. 78 (3) (2010) 267–274.
Web references http://hereditaryhearingloss.org http://davinci.crg.es/deafness http://genome.ucsc.edu/ primer3.wi.mit.edu/
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Please cite this article in press as: A. Kashef, et al., Finding mutation within non-coding region of GJB2 reveals its importance in genetic testing of Hearing Loss in Iranian population, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.11.024
