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Prevalence of IVS1+1G>A mutation among Iranian Azeri Turkish patients with autosomal recessive non-syndromic hearing loss (ARNSHL)
International Journal of Pediatric Otorhinolaryngology 75 (2011) 1612–1615
Contents lists available at SciVerse ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Prevalence of IVS1+1G>A mutation among Iranian Azeri Turkish patients with autosomal recessive non-syndromic hearing loss (ARNSHL) Mortaza Bonyadi *, Nikou Fotouhi, Mohsen Esmaeili Center of Excellence for Biodiversity, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 June 2011 Received in revised form 17 September 2011 Accepted 20 September 2011 Available online 14 October 2011
Objective: Mutations in the GJB2 gene has been reported as a main cause for autosomal recessive nonsyndromic hearing loss (ARNSHL) all over the world. IVS1+1G>A which is splice site mutation have been detected in several populations as disruptive mutation. This study has intended to assess the significance of this mutation, IVS1+1G>A, to the autosomal recessive non-syndromic genetic load among Iranian Azeri Turkish patients. Methods: Following our previous study, one hundred and seventy four unrelated patients with prelingual ARNSL were included in this study. Thirty nine patients had only one identified mutated allele, whereas hundred and thirty five patients were negative for coding region of GJB2. All these patients were screened for IVS1+1G>A by applying PCR-RFLP assay. Results: Among studied patients nine compound heterozygote with 35delG, delE120, 235delC were identified. Additionally, six patients were detected with only one IVS1+1G>A mutated allele. In these patients, the other mutated allele was left unidentified. One patient was identified to be homozygous for IVS1+1G>A. Further studies carried out on parents of positive cases, showed that one of the healthy parents (mother) to be homozygous for IVS1+1G>A mutation. By self-report, this person had no hearing impairment, although it is possible that she has mild or moderate hearing loss, which she is unable to detect. Her child was compound heterozygous (IVS1+1G>A/35delG) with profound deafness. The frequency of IVS1+1G>A was found to be about 4.9%, however the parental consanguinity was observed in 37.5% of IVS1+1G>A-mutated families. Conclusions: Our results support founder effect regarding these mutations and the presence of an ancient ancestor is strengthened in comparison to hot spot hypothesis. Also the results suggest variable expressivity of IVS1+1G>A mutation with respect to hearing loss. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: ARNSHL Connexin 26 IVS1+1G>A Iranian Azeri Turkish GJB2 Deafness
1. Introduction Hearing loss is the most common inherited sensory disorder in humans affecting one in 1000 live birth [1]. It comprises a broad spectrum of clinical presentations and mild to profound phenotype [2]. Hearing loss is very heterogeneous and happens due to genetic and environmental causes or both [3]. Approximately half of the cases can be attributed to genetic factors [1]. Autosomal recessive non-syndromic hearing loss (ARNSHL) is the most prevalent form of deafness with contribution of 23 different genes in its etiology. However, GJB2 (gap junction beta 2) mutations (homozygous or compound heterozygous) are main causes of ARNSHL in many populations [4,5], hence, it is normally the first gene to be tested routinely in patients with ARNSHL [5].
* Corresponding author. Tel.: +98 4113357622; fax: +98 4113357622. E-mail addresses: [email protected] (M. Bonyadi), [email protected] (N. Fotouhi), [email protected] (M. Esmaeili). 0165-5876/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2011.09.024
The GJB2 gene located on chromosome 13q12-13 encodes the structurally highly related protein connexin 26. This transmembrane protein is the important component of gap junctions which are necessary for potassium homeostasis to maintain inner ear function [2,3,6–9]. More than 110 mutations in GJB2 gene have so far been reported (http://davinci.crg.es/deafness). GJB2 mutation spectrum differs among various populations or even within a particular country [1,10–12]. Therefore, it is necessary to establish the frequency of GJB2 mutations in any population. Our previous study showed that coding region (exon 2) mutations of the GJB2 gene are responsible for about 28% of ARNSHL in Iranian Azeri Turkish ethnic group [7]. Majority of these patients had only one mutation in the GJB2 gene. Searching neighboring gene GJB6 for common deletion (del(GJB6-D13S1830)) was negative for this mutation in the cohort studied [7]. Therefore, there may be mutations in other region of the GJB2 gene or other unidentified gene(s) responsible for ARNSHL [13]. The splice site mutation IVS1+1G>A, also called -3172G>A, in the splice donor site of intron 1 in GJB2 was originally considered by Denoyelle et al. (1999) as a disruptive mutation yielding no detectable mRNA [13].
M. Bonyadi et al. / International Journal of Pediatric Otorhinolaryngology 75 (2011) 1612–1615
In recent years IVS1+1G>A mutation have been investigated all over the world by genetic laboratories and this splice site have been found in several populations [3,5,9,13–19]. The present study was planned to study the significance of the IVS1+1G>A mutation among Iranian Azeri Turkish patients with ARNSHL. 2. Materials and methods A total of 174 unrelated patients with ARNSHL were included in the study. All patients were affected by prelingual (early) nonsyndromic hearing loss and 39 patients (22.5%) had only one identified mutated allele, whereas 135 patients (77.5%) were negative for coding region of GJB2 [4]. All patients were of Azeri Turk origin from northwestern Iran. Medical history and pedigree information were obtained by questionnaire to ensure that the hearing loss was not a result of acquired environmental factors like infection, trauma, acoustic trauma or ototoxic drugs. Written informed consent was obtained from all participants or their parents. Genomic DNA was extracted from peripheral blood leukocytes according to standard protocols [20]. Molecular testing for c.IVS1+1G>A mutation (also called -3172 G>A) was performed using polymerase chain reaction restrictiondigestion testing procedure (PCR-RFLP). In this procedure restriction enzyme digestion was done on PCR products amplified with primers flanking exon 1 and its exon-intron boundaries. The presence of c.IVS1+1G>A mutation destroys HphI recognition site [21]. The amplified 360 bp product was digested into two fragments of 242 bp and 118 bp for wild-type allele while mutant allele remains uncut (Fig. 1). PCR products and restriction enzymedigested fragments were electrophoresed in a 2% agarose gel and visualized by ethidium bromide staining.
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the frequency of c.IVS1+1G>A in this cohort is estimated 4.9% (17/ 348). Additionally, this mutation was also identified as compound heterozygous with 35delG in one girl whose parents were healthy (with non-consanguinity) and there were 4 affected patients in this family. Further analysis of the family demonstrated that her father was healthy but heterozygote for 35delG mutation. Interestingly her mother was homozygous IVS1+1G>A with no effect on her hearing ability by self-report. Unfortunately this mother was not available for further studies (Fig. 2). Table 1 describes the IVS1+1G>A genotypes found in Iranian Azeri Turkish patients. Also the effect of the parental consanguinity among this population (Iranian Azeri Turkish) is demonstrated in Table 2.
3. Results In this study, there were 86 (49%) males and 88 (51%) females with presumed ARNSHL. The age range of the patients was 2 months to 45 years (mean 23 years) and consanguinity was revealed in about 56.5% of the families. Sixteen (1 homozygous and 15 heterozygous) families out of the 174 families had c.IVS1+1G>A mutation. In the other words not only we found fifteen patients carrying the c.IVS1+1G>A mutation in the heterozygous state in addition to their already known 35delG, del E120 and 235delC mutations but did we also find one homozygote patient, therefore
Fig. 1. Poly acrylamide gel electrophoresis for separation of the RFLP-PCR products. Lane 1: a positive control case showing IVS1+1G>A mutation with heterozygosity for this transition. 242 bp and 118 bp bands show wild type and 360 bp mutant alleles respectively. Lanes 2 and 5: deaf patients with heterozygous IVS1+1G>A mutation in GJB2 gene. Lane 3: a patient with homozygous IVS1+1G>A mutation. Lane 10: a negative control case. Other Lanes are deaf individuals with ARNSHL whom did not show this mutation.
Fig. 2. sequence of the homozygote individual with less effect on her hearing. The arrow indicates the location of base change.
M. Bonyadi et al. / International Journal of Pediatric Otorhinolaryngology 75 (2011) 1612–1615
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Table 1 Genotypes of the patients in terms of connexin 26. Genotype
Deafness
IVS I/IVS I IVS I/? 35delG/IVS I del E120/IVS I 235delC/IVS I
1 6 7 1 1
Table 2 Distribution of IVS I G>A in respect of parental consanguinity. Parental consanguinity
IVS I/IVS I
IVS I/?
35delG/IVS I
del E120/IVS I
235del C/IVS I
+
0 1 0%
2 4 33%
3 4 43%
1 0 100%
0 1 0%
Rate
4. Discussion Hearing loss at DFNB1 locus is the prevalent cause of ARNSHL in most of the countries [22]. In all populations which have been studied, connexin 26 mutations were found to be responsible for considerable proportion of hereditary hearing loss. Also the high heterogeneity of ARNSHL was reported in these mutations’ spectrum [6]. Two exons of GJB2 are separated by an intron, and the coding region is restricted to exon 2. The most of the mutations in this gene take place in coding region, like c.35delG mutation, but one GJB2 mutation, the IVS1+1G>A(-3172G>A), is approximately common in some populations [4]. Iran is consisted of various ethnics and around 15–20 million Azeri Turks live in northwest of Iran, which are ethnically similar to Azeris and closely related to Turks. Mutational screening performed to date has usually focused on coding region of GJB2 gene and few studies have been conducted on the non-coding region and exon 1. In the other words incidence in exon 1 of GJB2 gene is hardly reported. c.IVS1+1G>A mutation which lies outside the GJB2 coding region, is a splice site mutation [21,23]. Seeman and Sakmoryova found c.IVS1+1G>A as a frequent mutation in Czech population (45%) [18]. Other study showed that this mutation is the third common mutation after 35delG and del (GJB6-D13S1830) mutations in Dutch patients [18]. In these studies the mutation was found in heterozygous subjects carrying one pathogenic mutation in coding region of the GJB2 gene, so it was suggested to investigate this mutation in those who were heterozygous for only one pathogenic mutation and after that those for whom that no pathogenic mutation was found previously [7]. Recently it is found that the IVS1+1G>A is the main mutated allele for GJB2 in Yakut Population, located in Eastern Siberia (Russian federation), where the large number of the Yakut population were homozygous for IVS1+1G>A mutation [24]. It should be noted that different results have been reported in various population, i.e. the frequency of this mutation in Turkey, Croatia, China, India, Palestine, Brazil, Yakut population in Sakha Republic (Russian Federation), Hungry and Argentina 1.4%, 0.9%, 1.89%, 5%, 1%, 4.6%, 11.7%, 23.4%, 3% is reported respectively [5,13,14,16,17,19,24–26]. In Iran few studies have been reported but the reported results are noticeable and the spectrum varies between 0.2% and 9.4% [1,22]. Our previous study for GJB2 in Iranian Azeri Turkish revealed that the most common mutation is 35delG. The first non-coding exon including the splice sites was analyzed for DNA changes. Fifteen heterozygous and one homozygous patient (16/174) showed the already described splice site mutation in non-coding region of GJB2 (9.2%).
A compound heterozygote with 35delG mutation was detected in one deaf girl. Her parents were healthy (with non-consanguinity) and there were 4 affected patients with similar genotype in this family. Further molecular analysis of the family showed that her father was a carrier for the 35delG mutation while her mother was homozygous for IVS1+1G>A. By self- report, she had no hearing impairment, although it is possible that she has only a mild or moderate hearing loss, which she is unable to detect. In general, inactivating mutations were associated with more hearing loss than non-inactivating mutations. In some arrangements IVS1+1G>A have been classified as non-inactivating mutation, those mutations potentially allow the synthesis of some proteins, hence the presence of a minute amount of mRNA cannot be ignored [2,16]. Therefore our results suggest variable expressivity of IVS1+1G>A mutation with respect to hearing loss. Apart from genetic and inheritance problem, consanguinity is a common pattern of marriage all over the studied cohort. This study revealed 56% of consanguinity in the deaf families. Observing that 62.5% of the studied families with IVS1+1G>A mutation belongs to non-consanguinity marriages may indicate that rate of the IVS1+1G>A mutation in hearing population is relatively high. Further molecular studies are required to evaluate these findings. Although the frequency of IVS1+1G>A mutation in this cohort is higher than the rate of reported from Turkey (4.9% against 1.4%) [25], but further screening in Iran and Turkey may reveal identical results. Of course our study indicating that a founder effect concerning this mutation is more strengthened instead of the hot spot theory. Our findings besides Indian and Palestinian frequencies suggest some kind of gradient in Middle East [16].
Acknowledgments Authors would like to thank all families of the patients. This project was financially supported by the Center of Excellence for Biodiversity (University of Tabriz).
References [1] N. Mahdieh, C. Nishimura, K. Ali-Madadi, Y. Riazailhosseini, H. Yazdan, S. Arzhangi, et al., The frequency of GJB2 mutations and the (GJB6-D13S1830) deletion as a cause of autosomal recessive non-syndromic deafness in the Kurdish population, Clin. Genet. 65 (2004) 506–508. [2] Y. Bajaj, T. Sirimanna, D.M. Albert, P. Qadir, L. Jenkins, M. Bitner-Glindzicz, Spectrum of GJB2 mutations causing deafness in the British Bangladeshi population, Clin. Otolaryngol. 33 (2008) 313–318. [3] M. Hashemzadeh Chaleshtori, D.D. Farhud, M.A. Patton, Familial and sporadic GJB2-related deafness in Iran: review of gene mutations, Iran. J. Publ. Health 36 (2007) 1–14. [4] M. Bonyadi, M. Esmaeili, M. Abhari, A. Lotfi, Mutation analysis of familial GJB2related deafness in Iranian Azeri Turkish patients, Genet. Test. Mol. Biomarkers 13 (2009) 689–692. [5] Y. Yuan, F. Yu, G. Wang, S. 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. Trans. Med. (2010). [6] M. Esmaeili, M. Bonyadi, M. Nejadkazem, Common mutation analysis of GJB2 and GJB6 genes in affected families with autosomal recessive non-syndromic hearing loss from Iran: Simultaneous detection of two common mutations (35delG/ del(GJB6-D13S1830)) in the DFNB1-related deafness, Int. J. Pediatr. Otorhi. 71 (2007) 869–873. [7] M. Hashemzadeh Chaleshtori, D.D. Farhud, R. Taylor, V. Hadavi, M.A. Patton, A.R. Afzal, Deafness – associated connexin 26 gene (GJB2) mutations in Iranian population, Iran. J. Publ. Health 31 (2002) 75–79. [8] K. Cryns, E. Orzan, A. Murgia, P.L.M. Huygen, F. Moreno, I. del Castillo, et al., A genotype-phenotype correlation for GJB2 (connexin 26) deafness, J. Med. Genet. 41 (2004) 147–154. [9] E. Baysal, Y.A. Bayazit, S.R. Ceylaner, N.T. Alatas, B.T. Donmez, G.Y. Ceylaner, et al., GJB2 and mitochondrial A1555G gene mutations in nonsyndromic profound hearing loss and carrier frequencies in healthy individuals, J. Genet. 87 (2008) 53–57. [10] T.D. Matos, H. Caria, H. Simo˜es-Teixeira, et al., A novel hearing loss-related mutation occurring in the GJB2 basal promoter, J. Med. Genet. 44 (2007) 721–725.
M. Bonyadi et al. / International Journal of Pediatric Otorhinolaryngology 75 (2011) 1612–1615 [11] T.D. Matos, H. Caria, H. Simo˜es-Teixeira, T. Aasen, O. Dias, M. Andrea, et al., A novel M163L mutation in connexin 26 causing cell death and associated with autosomal dominant hearing loss, Hearing Res. 240 (2008) 87–92. [12] E. Kalay, R. Caylan, H. Kremer, A.P.M. de Brouwer, A. Karaguzel, GJB2 mutations in Turkish patients with ARNSHL: prevalence and two novel mutations, Hearing Res. 203 (2005) 88–93. [13] S.M. da Silva-Costa, F.B. Coeli, C.R. Lincoln-de-Carvalho, A.P. Marques-de-Faria, M. Kurc, T. Pereira, et al., Screening for the GJB2 c-3170 G>A (IVS 1þ1 G>A) mutation in Brazilian deaf individuals using multiplex ligation – dependent probe amplification, Genet. Test. Mol. Biomarkers 13 (2009) 701–704. [14] G. Padma, P.V. Ramchander, U.V. Nandur, T. Padma, GJB2 and GJB6 gene mutations found in Indian probands with congenital hearing impairment, J. Genet. 88 (2009) 267–272. [15] M. Tekin, X.-J. Xia, R. Erdenetungalag, F.B. Cengiz, T.W. White, J. Radnaabazar, et al., GJB2 mutations in Mongolia: complex alleles low frequency, and reduced fitness of the deaf, Ann. Hum. Genet. 74 (2010) 155–164. [16] H.S. Tom Walsh, T. Sobe, E. Lynch, M.-C. King, K.B. Avraham, et al., Genetics of congenital deafness in the Palestinian population: multiple connexin 26 alleles with shared origins in the Middle East, Hum. Genet. 10 (2002) 284–289. [17] I. Sansovic´, J. Knezev ic´, V. Musani, P. Seeman, I. Barisˇ ic´, J. F Pavelic´, GJB2 mutations in patients with nonsyndromic hearing loss from Croatia, Genet. Test. Mol. Biomarkers 13 (2009) 693–699. [18] P. Seeman, I. Sakmaryova, High prevalence of the IVS 1+1 G to A/GJB2 mutation among Czech hearing impaired patients with monoallelic mutation in the coding region of GJB2, Clin. Genet. 69 (2006) 410–413.
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[19] T.A. To´th, S.N. Kupka, B.T. Haack, F.C. Fazakas, L. Muszbek, N. Blin, et al., Coincidence of mutations in different connexin genes in Hungarian patients, Int. J. Mol. Med. 20 (2007) 315–321. [20] S.A. Miller, D.D. Dykes, H.F. Polesky, A simple salting out procedure for extracting DNA from human nucleated cells, Nucl. Acids Res. 16 (1988) 1215. [21] 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 (1999) 1298–1303. [22] H. Najmabadi, C. Nishimura, K. Kahrizi, Y. Riazalhosseini, M. Malekpour, A. Daneshi, et al., GJB2 mutations: passage through Iran, Am. J. Med. Genet. 133A (2005) 132–137. [23] R.L. Snoeckx, P.L.M. Huygen, D. Feldmann, S. Marlin, F. Denoyelle, J. Waligora, GJB2 mutations and degree of hearing loss: a multicenter study, J. Hum. Genet. 77 (2005) 945–957. [24] N.A. Barashkov, L.U. Dzhemileva, S.A. Fedorova, F.M. Teryutin, O.L. Posukh, E.E. Fedotova, et al., Autosomal recessive deafness 1A (DFNB1A) in Yakut population isolate in Eastern Siberia: extensive accumulation of the splice site mutation IVS1+1G>A in GJB2 gene as a result of founder effect, J. Hum. Genet. (2011) 72. [25] A. Sirmaci, D. Akcayoz-Duman, T. Mustafa, The c.IVS1+1G>A mutation in the GJB2 gene is prevalent and large deletions involving the GJB6 gene are not present in the Turkish population, J. Genet. 85 (2006) 213–216. [26] L.P. Gravina, M.E. Foncuberta, M.E. Prieto, J. Garrido, C. Barreiro, L. Chertkoff, Prevalence of DFNB1 mutations in Argentinean children with non-syndromic deafness report of a novel mutation in GJB2, Int. J. Pediatr. Otorhi. 74 (2010) 250–254.
Contents lists available at SciVerse ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Prevalence of IVS1+1G>A mutation among Iranian Azeri Turkish patients with autosomal recessive non-syndromic hearing loss (ARNSHL) Mortaza Bonyadi *, Nikou Fotouhi, Mohsen Esmaeili Center of Excellence for Biodiversity, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 June 2011 Received in revised form 17 September 2011 Accepted 20 September 2011 Available online 14 October 2011
Objective: Mutations in the GJB2 gene has been reported as a main cause for autosomal recessive nonsyndromic hearing loss (ARNSHL) all over the world. IVS1+1G>A which is splice site mutation have been detected in several populations as disruptive mutation. This study has intended to assess the significance of this mutation, IVS1+1G>A, to the autosomal recessive non-syndromic genetic load among Iranian Azeri Turkish patients. Methods: Following our previous study, one hundred and seventy four unrelated patients with prelingual ARNSL were included in this study. Thirty nine patients had only one identified mutated allele, whereas hundred and thirty five patients were negative for coding region of GJB2. All these patients were screened for IVS1+1G>A by applying PCR-RFLP assay. Results: Among studied patients nine compound heterozygote with 35delG, delE120, 235delC were identified. Additionally, six patients were detected with only one IVS1+1G>A mutated allele. In these patients, the other mutated allele was left unidentified. One patient was identified to be homozygous for IVS1+1G>A. Further studies carried out on parents of positive cases, showed that one of the healthy parents (mother) to be homozygous for IVS1+1G>A mutation. By self-report, this person had no hearing impairment, although it is possible that she has mild or moderate hearing loss, which she is unable to detect. Her child was compound heterozygous (IVS1+1G>A/35delG) with profound deafness. The frequency of IVS1+1G>A was found to be about 4.9%, however the parental consanguinity was observed in 37.5% of IVS1+1G>A-mutated families. Conclusions: Our results support founder effect regarding these mutations and the presence of an ancient ancestor is strengthened in comparison to hot spot hypothesis. Also the results suggest variable expressivity of IVS1+1G>A mutation with respect to hearing loss. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: ARNSHL Connexin 26 IVS1+1G>A Iranian Azeri Turkish GJB2 Deafness
1. Introduction Hearing loss is the most common inherited sensory disorder in humans affecting one in 1000 live birth [1]. It comprises a broad spectrum of clinical presentations and mild to profound phenotype [2]. Hearing loss is very heterogeneous and happens due to genetic and environmental causes or both [3]. Approximately half of the cases can be attributed to genetic factors [1]. Autosomal recessive non-syndromic hearing loss (ARNSHL) is the most prevalent form of deafness with contribution of 23 different genes in its etiology. However, GJB2 (gap junction beta 2) mutations (homozygous or compound heterozygous) are main causes of ARNSHL in many populations [4,5], hence, it is normally the first gene to be tested routinely in patients with ARNSHL [5].
* Corresponding author. Tel.: +98 4113357622; fax: +98 4113357622. E-mail addresses: [email protected] (M. Bonyadi), [email protected] (N. Fotouhi), [email protected] (M. Esmaeili). 0165-5876/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2011.09.024
The GJB2 gene located on chromosome 13q12-13 encodes the structurally highly related protein connexin 26. This transmembrane protein is the important component of gap junctions which are necessary for potassium homeostasis to maintain inner ear function [2,3,6–9]. More than 110 mutations in GJB2 gene have so far been reported (http://davinci.crg.es/deafness). GJB2 mutation spectrum differs among various populations or even within a particular country [1,10–12]. Therefore, it is necessary to establish the frequency of GJB2 mutations in any population. Our previous study showed that coding region (exon 2) mutations of the GJB2 gene are responsible for about 28% of ARNSHL in Iranian Azeri Turkish ethnic group [7]. Majority of these patients had only one mutation in the GJB2 gene. Searching neighboring gene GJB6 for common deletion (del(GJB6-D13S1830)) was negative for this mutation in the cohort studied [7]. Therefore, there may be mutations in other region of the GJB2 gene or other unidentified gene(s) responsible for ARNSHL [13]. The splice site mutation IVS1+1G>A, also called -3172G>A, in the splice donor site of intron 1 in GJB2 was originally considered by Denoyelle et al. (1999) as a disruptive mutation yielding no detectable mRNA [13].
M. Bonyadi et al. / International Journal of Pediatric Otorhinolaryngology 75 (2011) 1612–1615
In recent years IVS1+1G>A mutation have been investigated all over the world by genetic laboratories and this splice site have been found in several populations [3,5,9,13–19]. The present study was planned to study the significance of the IVS1+1G>A mutation among Iranian Azeri Turkish patients with ARNSHL. 2. Materials and methods A total of 174 unrelated patients with ARNSHL were included in the study. All patients were affected by prelingual (early) nonsyndromic hearing loss and 39 patients (22.5%) had only one identified mutated allele, whereas 135 patients (77.5%) were negative for coding region of GJB2 [4]. All patients were of Azeri Turk origin from northwestern Iran. Medical history and pedigree information were obtained by questionnaire to ensure that the hearing loss was not a result of acquired environmental factors like infection, trauma, acoustic trauma or ototoxic drugs. Written informed consent was obtained from all participants or their parents. Genomic DNA was extracted from peripheral blood leukocytes according to standard protocols [20]. Molecular testing for c.IVS1+1G>A mutation (also called -3172 G>A) was performed using polymerase chain reaction restrictiondigestion testing procedure (PCR-RFLP). In this procedure restriction enzyme digestion was done on PCR products amplified with primers flanking exon 1 and its exon-intron boundaries. The presence of c.IVS1+1G>A mutation destroys HphI recognition site [21]. The amplified 360 bp product was digested into two fragments of 242 bp and 118 bp for wild-type allele while mutant allele remains uncut (Fig. 1). PCR products and restriction enzymedigested fragments were electrophoresed in a 2% agarose gel and visualized by ethidium bromide staining.
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the frequency of c.IVS1+1G>A in this cohort is estimated 4.9% (17/ 348). Additionally, this mutation was also identified as compound heterozygous with 35delG in one girl whose parents were healthy (with non-consanguinity) and there were 4 affected patients in this family. Further analysis of the family demonstrated that her father was healthy but heterozygote for 35delG mutation. Interestingly her mother was homozygous IVS1+1G>A with no effect on her hearing ability by self-report. Unfortunately this mother was not available for further studies (Fig. 2). Table 1 describes the IVS1+1G>A genotypes found in Iranian Azeri Turkish patients. Also the effect of the parental consanguinity among this population (Iranian Azeri Turkish) is demonstrated in Table 2.
3. Results In this study, there were 86 (49%) males and 88 (51%) females with presumed ARNSHL. The age range of the patients was 2 months to 45 years (mean 23 years) and consanguinity was revealed in about 56.5% of the families. Sixteen (1 homozygous and 15 heterozygous) families out of the 174 families had c.IVS1+1G>A mutation. In the other words not only we found fifteen patients carrying the c.IVS1+1G>A mutation in the heterozygous state in addition to their already known 35delG, del E120 and 235delC mutations but did we also find one homozygote patient, therefore
Fig. 1. Poly acrylamide gel electrophoresis for separation of the RFLP-PCR products. Lane 1: a positive control case showing IVS1+1G>A mutation with heterozygosity for this transition. 242 bp and 118 bp bands show wild type and 360 bp mutant alleles respectively. Lanes 2 and 5: deaf patients with heterozygous IVS1+1G>A mutation in GJB2 gene. Lane 3: a patient with homozygous IVS1+1G>A mutation. Lane 10: a negative control case. Other Lanes are deaf individuals with ARNSHL whom did not show this mutation.
Fig. 2. sequence of the homozygote individual with less effect on her hearing. The arrow indicates the location of base change.
M. Bonyadi et al. / International Journal of Pediatric Otorhinolaryngology 75 (2011) 1612–1615
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Table 1 Genotypes of the patients in terms of connexin 26. Genotype
Deafness
IVS I/IVS I IVS I/? 35delG/IVS I del E120/IVS I 235delC/IVS I
1 6 7 1 1
Table 2 Distribution of IVS I G>A in respect of parental consanguinity. Parental consanguinity
IVS I/IVS I
IVS I/?
35delG/IVS I
del E120/IVS I
235del C/IVS I
+
0 1 0%
2 4 33%
3 4 43%
1 0 100%
0 1 0%
Rate
4. Discussion Hearing loss at DFNB1 locus is the prevalent cause of ARNSHL in most of the countries [22]. In all populations which have been studied, connexin 26 mutations were found to be responsible for considerable proportion of hereditary hearing loss. Also the high heterogeneity of ARNSHL was reported in these mutations’ spectrum [6]. Two exons of GJB2 are separated by an intron, and the coding region is restricted to exon 2. The most of the mutations in this gene take place in coding region, like c.35delG mutation, but one GJB2 mutation, the IVS1+1G>A(-3172G>A), is approximately common in some populations [4]. Iran is consisted of various ethnics and around 15–20 million Azeri Turks live in northwest of Iran, which are ethnically similar to Azeris and closely related to Turks. Mutational screening performed to date has usually focused on coding region of GJB2 gene and few studies have been conducted on the non-coding region and exon 1. In the other words incidence in exon 1 of GJB2 gene is hardly reported. c.IVS1+1G>A mutation which lies outside the GJB2 coding region, is a splice site mutation [21,23]. Seeman and Sakmoryova found c.IVS1+1G>A as a frequent mutation in Czech population (45%) [18]. Other study showed that this mutation is the third common mutation after 35delG and del (GJB6-D13S1830) mutations in Dutch patients [18]. In these studies the mutation was found in heterozygous subjects carrying one pathogenic mutation in coding region of the GJB2 gene, so it was suggested to investigate this mutation in those who were heterozygous for only one pathogenic mutation and after that those for whom that no pathogenic mutation was found previously [7]. Recently it is found that the IVS1+1G>A is the main mutated allele for GJB2 in Yakut Population, located in Eastern Siberia (Russian federation), where the large number of the Yakut population were homozygous for IVS1+1G>A mutation [24]. It should be noted that different results have been reported in various population, i.e. the frequency of this mutation in Turkey, Croatia, China, India, Palestine, Brazil, Yakut population in Sakha Republic (Russian Federation), Hungry and Argentina 1.4%, 0.9%, 1.89%, 5%, 1%, 4.6%, 11.7%, 23.4%, 3% is reported respectively [5,13,14,16,17,19,24–26]. In Iran few studies have been reported but the reported results are noticeable and the spectrum varies between 0.2% and 9.4% [1,22]. Our previous study for GJB2 in Iranian Azeri Turkish revealed that the most common mutation is 35delG. The first non-coding exon including the splice sites was analyzed for DNA changes. Fifteen heterozygous and one homozygous patient (16/174) showed the already described splice site mutation in non-coding region of GJB2 (9.2%).
A compound heterozygote with 35delG mutation was detected in one deaf girl. Her parents were healthy (with non-consanguinity) and there were 4 affected patients with similar genotype in this family. Further molecular analysis of the family showed that her father was a carrier for the 35delG mutation while her mother was homozygous for IVS1+1G>A. By self- report, she had no hearing impairment, although it is possible that she has only a mild or moderate hearing loss, which she is unable to detect. In general, inactivating mutations were associated with more hearing loss than non-inactivating mutations. In some arrangements IVS1+1G>A have been classified as non-inactivating mutation, those mutations potentially allow the synthesis of some proteins, hence the presence of a minute amount of mRNA cannot be ignored [2,16]. Therefore our results suggest variable expressivity of IVS1+1G>A mutation with respect to hearing loss. Apart from genetic and inheritance problem, consanguinity is a common pattern of marriage all over the studied cohort. This study revealed 56% of consanguinity in the deaf families. Observing that 62.5% of the studied families with IVS1+1G>A mutation belongs to non-consanguinity marriages may indicate that rate of the IVS1+1G>A mutation in hearing population is relatively high. Further molecular studies are required to evaluate these findings. Although the frequency of IVS1+1G>A mutation in this cohort is higher than the rate of reported from Turkey (4.9% against 1.4%) [25], but further screening in Iran and Turkey may reveal identical results. Of course our study indicating that a founder effect concerning this mutation is more strengthened instead of the hot spot theory. Our findings besides Indian and Palestinian frequencies suggest some kind of gradient in Middle East [16].
Acknowledgments Authors would like to thank all families of the patients. This project was financially supported by the Center of Excellence for Biodiversity (University of Tabriz).
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