Genetics Research International

Genetics Research International / 2011 / Article
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Genetics of Deafness

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Research Article | Open Access

Volume 2011 |Article ID 827469 |

T. D. Matos, H. Simões-Teixeira, H. Caria, R. Cascão, H. Rosa, A. O'Neill, Ó. Dias, M. E. Andrea, D. P. Kelsell, G. Fialho, "Assessing Noncoding Sequence Variants of GJB2 for Hearing Loss Association", Genetics Research International, vol. 2011, Article ID 827469, 8 pages, 2011.

Assessing Noncoding Sequence Variants of GJB2 for Hearing Loss Association

Academic Editor: Ignacio Del Castillo
Received30 Apr 2011
Revised19 Jul 2011
Accepted27 Jul 2011
Published05 Oct 2011


Involvement of GJB2 noncoding regions in hearing loss (HL) has not been extensively investigated. However, three noncoding mutations, c.-259C>T, c.-23G>T, and c.-23+1G>A, were reported. Also, c.-684_-675del, of uncertain pathogenicity, was found upstream of the basal promoter. We performed a detailed analysis of GJB2 noncoding regions in Portuguese HL patients (previously screened for GJB2 coding mutations and the common GJB6 deletions) and in control subjects, by sequencing the basal promoter and flanking upstream region, exon 1, and 3'UTR. All individuals were genotyped for c.-684_-675del and 14 SNPs. Novel variants (c.-731C>T, c.-26G>T, c.*45G>A, and c.*985A>T) were found in controls. A hearing individual homozygous for c.-684_-675del was for the first time identified, supporting the nonpathogenicity of this deletion. Our data indicate linkage disequilibrium (LD) between SNPs rs55704559 (c.*168A>G) and rs5030700 (c.*931C>T) and suggest the association of c.[*168G;*931T] allele with HL. The c.*168A>G change, predicted to alter mRNA folding, might be involved in HL.

1. Introduction

About two hundred GJB2 mutations causing nonsyndromic hearing loss (NSHL) have been reported ( [1]. Most GJB2 mutations described so far localize to the coding region (totally included within exon 2), which is routinely analysed upon the study of GJB2 in HL patients. Also involved in HL, two deletions, del(GJB6-D13S1830) [24] and del(GJB6-D13S1854) [5] disrupt the GJB6 gene which codes for connexin-30, and it is thought that they may ablate a GJB2 cis-regulatory sequence [57]. This putative element is likely to be ablated by a third deletion, del(chr13 : 19,837,344–19,968,698), localized upstream of GJB2 and GJB6 [8, 9].

Along with GJB2 coding region, the noncoding first exon and donor splice site have been analysed in several studies, and two pathogenic mutations, c.-23G>T (exon 1) [10] and c.-23+1G>A (intron) [11], both in the donor splice site, have been identified. The c.-23+1G>A mutation (commonly known as IVS1+1G>A), shown to impair splicing [12], has been identified in several cases, being particularly frequent in Czech Republic, Turkey, and Hungary [1315].

A few studies have investigated, in addition to exon 1, the noncoding region immediately upstream of this exon, including the basal promoter [14, 1621].

Houseman and coworkers [16] analysed HL patients heterozygous for c.101T>C (p.Met34Thr), in which no second GJB2 coding mutation had been detected, and identified a monoallelic 10 bp deletion, c.-684_-675del (firstly designated -493del10), upstream of the basal promoter. The deletion was also present in other hearing impaired individuals as well as in control individuals, with or without c.101T>C. However, c.-684_-675del homozygosity was only observed in c.101T>C homozygous patients. The fact that in the control population 22 of the 25 (88%) c.101T>C heterozygotes carried the deletion suggested the existence of LD between c.101T>C and c.-684_-675del, later demonstrated by Zoll and coworkers [22]. Transcription was observed from alleles harbouring in cis the deletion and the variant c.101T>C, derived from keratinocytes and cell lines. However, eventual subtle differences would not have been detected, since this was not a quantitative analysis [16]. To date, the role of c.-684_-675del in HL has remained uncertain.

More recently, a pathogenic basal promoter mutation, c.-259C>T (firstly designated -3438C>T) was identified, in trans with c.250G>A (p.Val84Met), in a Portuguese HL patient, highlighting the relevance of screening GJB2 noncoding regions in nonelucidated cases [18].

In the present study, we have analysed the basal promoter and the flanking upstream region, as well as the exon 1 and the 3′UTR of the GJB2 gene in 89 Portuguese HL patients. The same analysis was conducted on 91 normal hearing control individuals from the Portuguese population.

2. Methods

2.1. Subjects

Eighty-nine Portuguese HL patients previously screened for mutations in the GJB2 coding region and acceptor splice site (by SSCP and/or sequencing) and for the del(GJB6-D13S1830) and del(GJB6-D13S1854) GJB6 deletions (using the methodology described in [5]) were enrolled in this study. Eight patients were heterozygous for a GJB2 coding mutation: c.71G>A (p.Trp24X; ), c.35delG ( ), c.109G>A (p.Val37Ile; ), c.380G>A (p.Arg127His; ), c.457G>A (p.Val153Ile; ), and one patient was heterozygous for the c.-22-12C>T variant (apparently a polymorphism; dbSNP accession number rs9578260). No patient harboured either of the known GJB6 deletions. The HL was nonsyndromic in all patients, except for one of them, who presented with Waardenburg syndrome. The patient was heterozygous for the controversial c.457G>A mutation and was thus included in the study. The patients presented with bilateral, mild to profound HL, and were either familial or sporadic cases. The familial cases predominantly showed a recessive pattern of inheritance. All patients were audiologically evaluated by pure tone audiometry.

The control sample was composed of 91 Portuguese individuals with apparent normal hearing. The status regarding c.101T>C GJB2 variant of those control individuals harbouring the c.-684_-675del, here referred, had been previously investigated, by sequencing, as part of an unpublished work. The status of the entire GJB2 coding region is not known for the vast majority of the 91 control individuals, which were blindly included in this study (and not based on their eventually available GJB2 coding region status).

Informed consent was obtained from all the participants.

2.2. Genetic Analysis

In all individuals, we have sequenced a region of about 0.7 kb immediately upstream of the exon 1 (which includes the basal promoter), the exon 1, and the whole 3′UTR. The region upstream of the exon 1, plus exon 1 and donor splice site, was amplified in a 1009 bp amplicon, using the pair of primers PF2 5′-CgTTCgTTCggATTggTgAg-3′ and PR1 5′-CAgAAACgCCCgCTCCAgAA-3′, as previously described [18]. The amplicons were sequenced using the primers PF2 and PF1 5′-ggCTCAAAggAACTAggAgATCg-3′. When necessary, primers PR1 and PR2 5′-ggAgACTgggAAAgTTACgg-3′ were used for sequencing. The 3′UTR (plus the last 90 nucleotides and stop codon) was amplified in 3 overlapping fragments using the following three pairs of primers: 3UTRaF 5′-gCAgTgTCTggAATTTgCATC-3′, 3UTRaR 5′-AggCACTggTAACTTTgTCC-3′, 3UTRbF 5′-CACgTTAAAggTgAACATTgg-3′, 3UTRbR 5′-CgACAgAAACTTCTCCCTC-3′, 3UTRcF 5′-gTAgCCAgCATCggAAAgAAC-3′, 3UTRcR 5′-ACTCTggCAACTTACCCATTg-3′. The 3′UTR PCR products were sequenced using the respective amplification forward primers.

2.3. DNA Sequence Variants and SNPs Description

Description of variants follows the HGVS recommendations, and is based on the GJB2 reference sequences accessed through the following links:(1);(2);(3);(4)

These sequences show 100% identity with the NM_004004.5 (link 1) and NG_008358.1 (links 2, 3, and 4) NCBI reference sequences.

Novel variants were submitted to dbSNP and the respective reference SNP (rs) accession numbers are provided within the text.

SNPs are referred to by the dbSNP reference SNP (rs) accession number whenever it was available, and by the HGVS recommended designation, relative to the forementioned reference sequences.

2.4. Genotyping and Statistical Analysis

We have genotyped all individuals for the c.-684_-675del deletion; three SNPs in the promoter (rs9550621 (c.-484T>C), rs73431557 (c.-410T>C), rs9552101 (c.-369A>G)); ten SNPs in the 3′UTR (c.*1C>T, rs3751385 (c.*84T>C), rs7337074 (c.*104A>T), rs7329857 (c.*111C>T), rs55704559 (c.*168A>G), rs5030700 (c.*931C>T), rs1050960 (c.*1067G>T), rs7623 (c.*1152G>A), rs11841182 (c.*1197T>A), and rs7988691 (c.*1277T>C)); one SNP downstream of the 3′UTR (rs11839674 (c.*1447G>A)).

For the sake of simplicity, when describing the composite genotypes regarding SNPs rs73431557 (c.-410T>C), rs3751385 (c.*84T>C), rs55704559 (c.*168A>G), and rs5030700 (c.*931C>T), the genotype at each position, indicated in order from 5′ to 3′, is designated by A, C, G, or T if homozygous, or by a code letter, according to IUPAC nucleotide ambiguity code, if heterozygous.

The allelic frequencies regarding deletion c.-684_-675del and the 14 SNPs, were determined in the control population and used to test for Hardy-Weinberg equilibrium. The chi-square test was used to compare the allelic frequencies of the patients with those of the normal hearing individuals. Allelic frequencies of the control sample for the 14 SNPs were used to calculate pairwise linkage disequilibrium values. Testing for Hardy-Weinberg equilibrium, calculation of pairwise linkage disequilibrium values, and haplotype estimation (through the expectation maximization algorithm), were performed using SNPAnalyzer 1.2A online software (

2.5. Analysis of mRNA Folding

Mfold ( [23] was used to assess the effect of alleles c.[*168A;*931C], c.[*168G;*931T], c.[*168A;*931T], and c.[*168G;*931C] on the folding of GJB2 mRNA (template sequence: ENST00000382848, retrieved from Ensembl). For each sequence the lowest free-energy structure was considered.

3. Results and Discussion

In the current study, 89 Portuguese HL patients, previously screened for mutations in the GJB2 coding region and acceptor splice site (80 patients presenting no mutation, plus eight heterozygous for coding mutations and one heterozygous for the noncoding variant c.-22-12C>T), and 91 hearing individuals were analyzed as regards the noncoding region immediately upstream of the exon 1 (which includes the basal promoter), the exon 1, and the whole 3′UTR of GJB2 gene. All individuals were also genotyped for c.-684_-675del and 14 SNPs localized therein.

3.1. DNA Sequence Variants

No additional GJB2 variant was found in any of the eight patients previously found to be heterozygous for a coding GJB2 mutation or in the patient heterozygous for the c.-22-12C>T noncoding variant.

Among the remaining 80 patients, six of them presented noncoding variants, which had already been reported (Table 1).

VariantLocationPatients ( )Controls ( )

c.-731C>T5′ of the BP0010
c.-684_-675del5′ of the BP2161
c.-45C>AEx 11010
c.-26G>TEx 10010
c.670A>C (p.Lys224Gln)Ex 2 (CR)0010

One patient, presenting with profound HL was heterozygous for the donor splice site c.-23+1G>A mutation. The patient may just be a carrier, or other GJB2 or GJB6 mutation might remain undetected. One other patient, presenting with moderate to severe HL, harboured in heterozygosity the c.-216T>G variant, located within the basal promoter, between two GT boxes [24, 25]. This variant was previously identified in two HL patients, also in heterozygosity [26]. The c.-45C>A variant in exon 1 was found in heterozygosity in one individual with severe HL. This variant was referred, by Wilch and coworkers [8], as an SNP at position +94 in exon 1. These authors observed expression of the GJB2 allele harbouring the variant but, since a quantitative comparison with wild-type allele was not performed, a possible contribution to HL cannot be excluded. Three affected individuals (two heterozygous and one homozygous) harboured the deletion c.-684_-675del.

No novel putative pathogenic noncoding mutation has been found in the patients, which might be due to the low number of monoallelic individuals and the small sample size. It is also possible that, simply, such mutations are very rare in our population.

Among controls, four novel noncoding variants were identified: c.-731C>T, c.-26G>T, c.*45G>A, and c.*985A>T (rs112400198, rs112875543, rs112399473, and rs111729919, resp.). Each of these variants was identified only once, in heterozygosity, and in different individuals (Table 1). The hearing individual harbouring the novel c.-731C>T variant was also heterozygous for the recessive c.670A>C (p.Lys224Gln) mutation (; phase unknown). One control individual harboured the c.-45C>A exon 1 variant in heterozygosity (Table 1). Interestingly, we found one control subject homozygous for c.-684_-675del (Table 1), which is, to our knowledge, the first case described to date of a normal hearing individual presenting this genotype. This individual did not harbour the c.101T>C mutation. Our finding, together with the previous report of transcription from alleles harbouring c.-684_-675del [16] suggests the nonpathogenicity of the deletion. In addition, six normal hearing heterozygotes for the deletion were also identified (Table 1), with one also heterozygous for c.101T>C.

It should be noted that the pathogenic basal promoter mutation c.-259C>T, identified for the first time in a Portuguese family [18], was not found among the 89 patients and 91 normal hearing individuals here analysed, and neither was it identified in the other studies which analysed the basal promoter [14, 1621]. Therefore, known occurrence of c.-259C>T continues to be restricted to that Portuguese family.

3.2. Genotypic Data and Statistical Analysis

The allelic frequencies and Hardy-Weinberg equilibrium status regarding the deletion c.-684_-675del and the 14 noncoding SNPs were determined (Table 2; see Supplementary Table  1 in Supplementary Material available online at doi: 10.4061/2011/827469).

Variant/SNPAllelesPatients ( alleles)ControlsP value
ObservedExpected( alleles)


rs9550621c.-484 C162165.291690.338941
c.-484 T1612.7113

rs73431557c.-410 C4126.41270.002089
c.-410 T137151.59155

rs9552101 c.-369 A2019.56200.916103
c.-369 G158158.44162

c.*1C>Tc.*1 C177178182ND
c.*1 T100

rs3751385c.*84 C129139.861430.04734
c.*84 T4938.1439

rs7337074c.*104 A174177.02181ND
c.*104 T40.981

rs7329857c.*111 C174177.02181ND
c.*111 T40.981

rs55704559c.*168 A154170.181743.33E-09
c.*168 G247.828

rs5030700c.*931 C155169.201739.18E-07
c.*931 T238.809

rs1050960c.*1067 G1919.56200.893155
c.*1067 T159158.44162

rs7623c.*1152 A165165.291690.93373
c.*1152 G1312.7113

rs11841182c.*1197 A200ND
c.*1197 T176178182

rs7988691c.*1277 C178176.04180ND
c.*1277 T01.962

rs11839674c.*1447 A200ND
c.*1447 G176178182

The allelic frequencies of the deletion c.-684_-675del in patients and controls are not statistically different (Table 2). The allelic frequency observed for this deletion in our control population is close to the one found among the British control population [16], and higher than the one determined in the German control population [22].

The allelic frequencies regarding SNPs c.-410T>C, c.*84T>C, c.*168A>G, and c.*931C>T, were statistically different between patient and control groups (Table 2).

By sorting both patients and controls into groups reflecting the genotypes for these four SNPs altogether, eleven composite genotypes were evidenced (Figure 1). Comparison of the genotypic frequencies in controls and patients promptly revealed an increased frequency in patients of the genotypes YYRY and CTRY, both heterozygous for SNPs c.*168A>G and c.*931C>T. Also, the genotype YCAC was identified in four patients but not found in controls. On the contrary, a decrease was observed in the frequency of the three genotypes that are most represented in controls—TCAC, TYAC, and YYAC. Each of the remaining genotypes was scarcely represented in both controls and patients (0%–2%), and their frequency did not vary more than 2% between the two groups; only 3% of controls and 4% of patients belong to one of these genotypes.

We also observed that, regarding SNPs c.*168A>G and c.*931C>T, nearly all individuals analysed (178/180) were either c.[=;=]+[=;=] or c.[*168A>G(+)*931C>T], which results from LD between these two SNPs (Supplementary Table  2, SNP pair 8 : 9). Interestingly, the overrepresentation of c.[*168A>G(+)*931C>T] genotype among patients, when comparing to hearing controls, is statistically very significant ( ; E-06), thus accounting for the statistically significant differences in the allelic frequencies of these two SNPs between patients and hearing controls.

The statistically significant differences also observed in the allelic frequencies of SNPs c.-410T>C and c.*84T>C seems to be due to the differential association of their variants with the estimated predominant alleles c.[*168A+*931C] and c.[*168G+*931T] (Tables 3(a) and 3(b)). This fact is in accordance with the observed LD between the two SNPs and SNPs c.*168A>G and c.*931C>T (Supplementary Table  2, SNP pairs 2 : 8, 2 :  9, 5 : 8, and 5 : 9).





Allele (c.*168; c.*931)c.-410 T>Cc.*84T>C

AC89.6% T82.1% C
GT100% C100% T

It should be noticed that the presence of genotype YCAC among patients lends some contribute to the difference in allelic frequencies between patients and controls regarding SNP c.-410T>C.

The fact that YCAC genotype is not represented in 91 control individuals while it occurs in 4/89 patients is noteworthy. The presence of genotype YCAC implies the presence of haplotype CCAC, which frequency is of at least 2,2% among patients, and estimated to be null in the control population (as inferred from Table 3(a)). In order to validate a possible association of haplotype CCAC with HL analysis of larger samples of patients and normal hearing individuals is necessary. Interestingly, one of the four patients with the referred composite genotype is a c.457G>A heterozygote (phase unknown).

3.3. 3′UTR Variants and mRNA Folding

Our findings suggest that the c.[*168G;*931T] allele might have a deleterious effect, contributing to HL. We have used Mfold [23] to predict the effect of alleles c.[*168A;*931C], c.[*168G;*931T], c.[*168A;*931T], and c.[*168G;*931C] on mRNA folding. The change c.*168A>G, regardless of genotype at position c.*931, was predicted to alter mRNA folding. On the contrary, the change c.*931C>T, regardless of genotype at position c.*168, is not predicted to alter mRNA folding (Figure 2).

The c.*168A was predicted to be located in an internal loop of a stem-loop structure (Figure 2). Regulatory motifs in mRNA 3′UTR seem to function in the context of specific secondary structure [27]. Stem-loop structures occurring in the 3′UTR have been implicated in gene expression, with roles at the level of mRNA stability (e.g., the SLDE of G-CSF gene [28], the CDE of TNF-alpha gene [27, 29], the complex structure integrating three C-rich elements of alpha-globin gene, the histone mRNA 3′ terminal stem-loops, and the IRE of TFRC gene [27]) or translation (e.g., the common 30–37 nucleotide long element present in the target mRNAs of TIA-1, a translational repressor [30], and the SECIS element [27]). The disruption of the predicted stem-loop structure and/or other adjacent stem-loop structures (Figure 2), induced by the c.*168A>G change, might lead to deregulation of the GJB2 gene expression, thus being a contributor to the hearing loss phenotype. It should be stressed that mRNA folding predictions are fallible. This fact notwithstanding, the simple change of sequence, without affecting the secondary structure, could conceivably disrupt a binding site for a trans-acting factor, also leading to gene expression deregulation. Regarding the c.*931C>T variant, despite the predictions that c.*931C occurs in a helix and that the change from C to T does not have structural implications, the in vivo situation might be different. Functional studies involving constructs containing a reporter gene’s coding sequence fused with GJB2 3′UTR could help elucidating the functional significance of these two sequence variants.

In this study, of a total of 15 patients presenting either a GJB2 coding mutation or a noncoding variant, 14 do not harbour either the c.*168A>G or the c.*931C>T changes, whereas one patient, heterozygous for the controversial c.380G>A mutation, is a compound heterozygote regarding SNPs c.*168A>G and c.*931C>T (phase unknown). Therefore, our data do not allow withdrawal of conclusions concerning a putative role of the two 3′UTR variants in the HL of some monoallelic patients. In this regard, the investigation of the genotypes regarding c.*168A>G and c.931C>T variants in larger samples of monoallelic patients would be interesting. Finally, the finding of one c.*168G homozygote (a c.*931C>T heterozygote, and carrying no GJB2 sequence variant) in our patient cohort, might further support a possible role of c.*168G in HL.

4. Conclusion

This study suggests the association of the noncoding SNPs c.*168A>G and c.*931C>T with HL. The c.*168A>G change is predicted to alter mRNA folding, suggesting a putative role of this SNP in the pathology. Our data also point to a possible association with HL of the haplotype CCAC, comprising SNPs c.-410T>C, c.*84T>C, c.*168A>G, and c.*931C>T, respectively. However, this observation requires validation through analysis of a larger number of subjects. The technique of targeted sequence capture and massively parallel sequencing makes it very easy and cost-effective to screen large numbers of genes, and might cover noncoding sequences of some of them, such as GJB2. This approach could prove to be very useful for genetic diagnosis in cases of NSHL [31], with predictable benefits for genetic counselling of the affected families.


The authors would like to thank the subjects for their contribution to the investigation. This paper was supported by Fundação para a Ciência e a Tecnologia grants SFRH/BD/19988/2004 (T. D. Matos) and SFRH/BD/24575/2005 (H. Simões-Teixeira).

Supplementary Materials

Results of the analysis of Hardy-Weinberg equilibrium and pair-wise linkage disequilibrium regarding the c.-684_-675del and the 14 SNPs studied in this work.

  1. Supplementary Material


  1. P. D. Stenson, M. Mort, E. V. Ball et al., “The human gene mutation database: 2008 update,” Genome Medicine, vol. 1, no. 1, article gm13, 2009. View at: Publisher Site | Google Scholar
  2. I. Lerer, M. Sagi, Z. Ben-Neriah, T. Wang, H. Levi, and D. Abeliovich, “A deletion mutation in GJB6 cooperating with a GJB2 mutation in trans in non-syndromic deafness: a novel founder mutation in Ashkenazi Jews,” Human Mutation, vol. 18, no. 5, article 460, 2001. View at: Google Scholar
  3. I. del Castillo, M. Villamar, M. A. Moreno-Pelayo et al., “A deletion involving the connexin 30 gene in nonsyndromic hearing impairment,” The New England Journal of Medicine, vol. 346, no. 4, pp. 243–249, 2002. View at: Publisher Site | Google Scholar
  4. N. Pallares-Ruiz, P. Blanchet, M. Mondain, M. Claustres, and A. F. Roux, “A large deletion including most of GJB6 in recessive non syndromic deafness: a digenic effect?” European Journal of Human Genetics, vol. 10, no. 1, pp. 72–76, 2002. View at: Publisher Site | Google Scholar
  5. F. J. del Castillo, M. Rodríguez-Ballesteros, A. Álvarez 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,” Journal of Medical Genetics, vol. 42, no. 7, pp. 588–594, 2005. View at: Publisher Site | Google Scholar
  6. J. E. A. Common, M. Bitner-Glindzicz, E. A. O'Toole et al., “Specific loss of connexin 26 expression in ductal sweat gland epithelium associated with the deletion mutation del(GJB6-D13S1830),” Clinical and Experimental Dermatology, vol. 30, no. 6, pp. 688–693, 2005. View at: Publisher Site | Google Scholar
  7. J. Rodriguez-Paris and I. Schrijver, “The digenic hypothesis unraveled: the GJB6 del(GJB6-D13S1830) mutation causes allele-specific loss of GJB2 expression in cis,” Biochemical and Biophysical Research Communications, vol. 389, no. 2, pp. 354–359, 2009. View at: Publisher Site | Google Scholar
  8. E. Wilch, M. Zhu, K. B. Burkhart et al., “Expression of GJB2 and GJB6 is reduced in a novel DFNB1 allele,” American Journal of Human Genetics, vol. 79, no. 1, pp. 174–179, 2006. View at: Publisher Site | Google Scholar
  9. E. Wilch, H. Azaiez, R. A. Fisher et al., “A novel DFNB1 deletion allele supports the existence of a distant cis-regulatory region that controls GJB2 and GJB6 expression,” Clinical Genetics, vol. 78, no. 3, pp. 267–274, 2010. View at: Publisher Site | Google Scholar
  10. R. S. Mani, A. Ganapathy, R. Jalvi et al., “Functional consequences of novel connexin 26 mutations associated with hereditary hearing loss,” European Journal of Human Genetics, vol. 17, no. 4, pp. 502–509, 2009. View at: Publisher Site | Google Scholar
  11. F. Denoyelle, S. Mariin, D. Weil, L. Moatti, and P. Chauvin, “Clinical features of the prevalent form of childhood deafness, DFNB1, due to a connexin-26 gene defect: implications for genetic counselling,” The Lancet, vol. 353, no. 9161, pp. 1298–1303, 1999. View at: Publisher Site | Google Scholar
  12. H. Shahin, T. Walsh, T. Sobe et al., “Genetics of congenital deafness in the Palestinian population: multiple connexin 26 alleles with shared origins in the Middle East,” Human Genetics, vol. 110, no. 3, pp. 284–289, 2002. View at: Publisher Site | Google Scholar
  13. P. Seeman and I. Sakmaryová, “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,” Clinical Genetics, vol. 69, no. 5, pp. 410–413, 2006. View at: Publisher Site | Google Scholar
  14. A. Sirmaci, D. Akcayoz-Duman, and M. Tekin, “The c.IVS1+1G>A mutation inthe GJB2 gene is prevalent and large deletions involving the GJB6 gene are not present in the Turkish population,” Journal of Genetics, vol. 85, no. 3, pp. 213–216, 2006. View at: Publisher Site | Google Scholar
  15. T. Tóth, S. Kupka, B. Haack et al., “Coincidence of mutations in different connexin genes in Hungarian patients,” International Journal of Molecular Medicine, vol. 20, no. 3, pp. 315–321, 2007. View at: Google Scholar
  16. M. J. Houseman, L. A. Ellis, A. Pagnamenta et al., “Genetic analysis of the connexin-26 M34T variant: identification of genotype M34T/M34T segregating with mild-moderate non-syndromic sensorineural hearing loss,” Journal of Medical Genetics, vol. 38, no. 1, pp. 20–25, 2001. View at: Google Scholar
  17. M. H. Chaleshtori, D. D. Farhud, R. Taylor et al., “Deafness—associated connexin 26 gene (GJB2) mutations in Iranian population,” Iranian Journal of Public Health, vol. 31, no. 3, pp. 75–79, 2002. View at: Google Scholar
  18. T. D. Matos, H. Caria, H. Simôes-Teixeira et al., “A novel hearing loss-related mutation occurring in the GJB2 basal promoter,” Journal of Medical Genetics, vol. 44, no. 11, pp. 721–725, 2007. View at: Publisher Site | Google Scholar
  19. A. Pollak, M. Mueller-Malesińska, A. Skórka et al., “GJB2 and hearing impairment: promoter defects do not explain the excess of monoallelic mutations,” Journal of Medical Genetics, vol. 45, no. 9, pp. 607–608, 2008. View at: Publisher Site | Google Scholar
  20. Y. Yuan, Y. You, D. Huang et al., “Comprehensive molecular etiology analysis of nonsyndromic hearing impairment from typical areas in China.,” Journal of Translational Medicine, vol. 7, article 79, 2009. View at: Publisher Site | Google Scholar
  21. Y. Yuan, F. Yu, G. Wang 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,” Journal of Translational Medicine, vol. 8, article 127, 2010. View at: Publisher Site | Google Scholar
  22. B. Zoll, L. Petersen, K. Lange et al., “Evaluation of Cx26/GJB2 in German hearing impaired persons: mutation spectrum and detection of disequilibrium between M34T (c.101T>C) and -493del10,” Human Mutation, vol. 21, no. 1, article 98, 2003. View at: Google Scholar
  23. M. Zuker, “Mfold web server for nucleic acid folding and hybridization prediction,” Nucleic Acids Research, vol. 31, no. 13, pp. 3406–3415, 2003. View at: Publisher Site | Google Scholar
  24. D. T. Kiang, N. Jin, Z. J. Tu, and H. H. Lin, “Upstream genomic sequence of the human connexin 26 gene,” Gene, vol. 199, no. 1-2, pp. 165–171, 1997. View at: Publisher Site | Google Scholar
  25. Z. J. Tu and D. T. Kiang, “Mapping and characterization of the basal promoter of the human connexin26 gene,” Biochimica et Biophysica Acta, vol. 1443, no. 1-2, pp. 169–181, 1998. View at: Publisher Site | Google Scholar
  26. H. Y. Tang, P. Fang, P. A. Ward et al., “DNA sequence analysis of GJB2, encoding connexin 26: observations from a population of hearing impaired cases and variable carrier rates, complex genotypes, and ethnic stratification of alleles among controls,” American Journal of Medical Genetics, Part A, vol. 140, no. 22, pp. 2401–2415, 2006. View at: Publisher Site | Google Scholar
  27. J. M. Chen, C. Férec, and D. N. Cooper, “A systematic analysis of disease-associated variants in the 3′ regulatory regions of human protein-coding genes II: the importance of mRNA secondary structure in assessing the functionality of 3′ UTR variants,” Human Genetics, vol. 120, no. 3, pp. 301–333, 2006. View at: Publisher Site | Google Scholar
  28. C. Y. Brown, C. A. Lagnado, and G. J. Goodall, “A cytokine mRNA-destabilizing element that is structurally and functionally distinct from A+U-rich elements,” Proceedings of the National Academy of Sciences of the United States of America, vol. 93, no. 24, pp. 13721–13725, 1996. View at: Publisher Site | Google Scholar
  29. G. Stoecklin, M. Lu, B. Rattenbacher, and C. Moroni, “A constitutive decay element promotes tumor necrosis factor alpha mRNA degradation via an AU-rich element-independent pathway,” Molecular and Cellular Biology, vol. 23, no. 10, pp. 3506–3515, 2003. View at: Publisher Site | Google Scholar
  30. I. López De Silanes, S. Galbán, J. L. Martindale et al., “Identification and functional outcome of mRNAs associated with RNA-binding protein TIA-1,” Molecular and Cellular Biology, vol. 25, no. 21, pp. 9520–9531, 2005. View at: Publisher Site | Google Scholar
  31. A. E. Shearer, A. P. DeLuca, M. S. Hildebrand et al., “Comprehensive genetic testing for hereditary hearing loss using massively parallel sequencing,” Proceedings of the National Academy of Sciences of the United States of America, vol. 107, no. 49, pp. 21104–21109, 2010. View at: Publisher Site | Google Scholar

Copyright © 2011 T. D. Matos et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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