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Contribution of the GJB2 gene to nonsyndromic sensorineural hearing loss in the portuguese population

Detalhes bibliográficos
Resumo:	Mutations in the GJB2 gene are responsible for a considerable proportion of nonsyndromic sensorineural hearing loss (NSSHL), in several populations. This gene is a member of a gene family coding for connexins, the subunits of the hemichannels (connexons) which form the intercellular channels of the gap junctions existing in the vertebrates and tunicates. Connexin-26 (Cx26), encoded by the GJB2 gene, is expressed in several tissues, including specific epithelial and conjunctive tissues of the cochlea, the auditory organ which is localised to the inner ear. In the cochlea, Cx26 is co-expressed with connexin-30 (Cx30), encoded by the GJB6 gene, in several cell types. Two large GJB6 deletions are involved in several hearing loss (HL) cases, as well, being most of these cases due to compound heterozygosity with a GJB2 mutation. Given the relevance of both GJB2 and GJB6 genes to the HL etiology in several populations, the molecular diagnosis of NSSHL cases with probable genetic cause is usually initiated by the analysis of the GJB2 coding region (in which most pathogenic mutations have been found), followed by the investigation of the presence of the GJB6 deletions in the cases where none or only one GJB2 mutation is found. In some cases, attributing the cause of the HL to the GJB2/GJB6 genotype is complicated because some GJB2 mutations are of unclear or controversial pathogenicity, due to either lack of genetic evidence (evident segregation of the mutation with the HL) or contradictory genetic data (same genotype occurring in both normalhearing and hearing-impaired individuals). In those cases, functional studies constitute an adequate approach for the investigation of the putative pathogenicity of such mutations, several of which have already been studied in this way. One of the aims of this work consisted in the study of the coding region and acceptor splice site of the GJB2 gene in Portuguese individuals presenting with NSSHL. We have further extended the analysis of the gene to noncoding regions (basal promoter and about 700 bp immediately upstream, exon 1, donor splice site and the whole 3’UTR). These noncoding regions have rarely been studied. Nonetheless, two pathogenic noncoding mutations had previously been identified, both in the donor splice site. Initially, we analysed the basal promoter, exon 1 and donor splice site in some patients who only harboured one coding mutation. We then found a novel mutation (c.-259C>T) in the basal promoter, in one of the binding sites for Sp1/Sp3 transcription factors. This mutation was found in a profoundly hearing-impaired patient, in trans with p.Val84Met, that we had identified as a novel mutation, in a previous work. Later, we analised all the fore mentioned noncoding regions in a larger sample, including monoallelic patients and also those harbouring no mutation in the GJB2 coding region (n=89) as well as 91 controls who reported to have normal hearing. The obtained data revealed that the c.[168A>G(+)931C>T] double heterozygous genotype, regarding the rs55704559 and rs5030700 SNPs (both localised to the 3’UTR), respectively, is overrepresented in the patients. In silico analysis predicts that the c.168A>G variant (regardless of the fact that in position c.931 is a cytosine or a thymine) causes an alteration of mRNA folding. Thus, our data suggest that the c.168G allele might be associated with HL. Other aim of this work was to investigate the effects of the mutations c.-259C>T and p.Val84Met, mentioned above, and the mutation p.Met163Leu, also identified for the first time in a previous work, in other Portuguese family. The available genetic data regarding these mutations were insufficient to prove their pathogenicity, and thus we have performed functional studies on the three mutations. The obtained results suggest that the three mutations are indeed pathogenic, exerting their effect in distinct ways. The c.-259C>T mutation decreases very significantly the basal promoter activity. As regards the coding mutations, p.Val84Met alters the ionic and molecular permeability properties of the intercellular channel, which may compromise their function in vivo, while p.Met163Leu leads to cell death, possibly by a mechanism other than the malfunctioning of undocked hemichannels (already described in the literature), and has a partial dominant-negative effect on wild-type Cx26 (wtCx26) and Cx30 (wtCx30).Mutations in the GJB2 gene are responsible for a considerable proportion of nonsyndromic sensorineural hearing loss (NSSHL), in several populations. This gene is a member of a gene family coding for connexins, the subunits of the hemichannels (connexons) which form the intercellular channels of the gap junctions existing in the vertebrates and tunicates. Connexin-26 (Cx26), encoded by the GJB2 gene, is expressed in several tissues, including specific epithelial and conjunctive tissues of the cochlea, the auditory organ which is localised to the inner ear. In the cochlea, Cx26 is co-expressed with connexin-30 (Cx30), encoded by the GJB6 gene, in several cell types. Two large GJB6 deletions are involved in several hearing loss (HL) cases, as well, being most of these cases due to compound heterozygosity with a GJB2 mutation. Given the relevance of both GJB2 and GJB6 genes to the HL etiology in several populations, the molecular diagnosis of NSSHL cases with probable genetic cause is usually initiated by the analysis of the GJB2 coding region (in which most pathogenic mutations have been found), followed by the investigation of the presence of the GJB6 deletions in the cases where none or only one GJB2 mutation is found. In some cases, attributing the cause of the HL to the GJB2/GJB6 genotype is complicated because some GJB2 mutations are of unclear or controversial pathogenicity, due to either lack of genetic evidence (evident segregation of the mutation with the HL) or contradictory genetic data (same genotype occurring in both normalhearing and hearing-impaired individuals). In those cases, functional studies constitute an adequate approach for the investigation of the putative pathogenicity of such mutations, several of which have already been studied in this way. One of the aims of this work consisted in the study of the coding region and acceptor splice site of the GJB2 gene in Portuguese individuals presenting with NSSHL. We have further extended the analysis of the gene to noncoding regions (basal promoter and about 700 bp immediately upstream, exon 1, donor splice site and the whole 3’UTR). These noncoding regions have rarely been studied. Nonetheless, two pathogenic noncoding mutations had previously been identified, both in the donor splice site. Initially, we analysed the basal promoter, exon 1 and donor splice site in some patients who only harboured one coding mutation. We then found a novel mutation (c.-259C>T) in the basal promoter, in one of the binding sites for Sp1/Sp3 transcription factors. This mutation was found in a profoundly hearing-impaired patient, in trans with p.Val84Met, that we had identified as a novel mutation, in a previous work. Later, we analised all the fore mentioned noncoding regions in a larger sample, including monoallelic patients and also those harbouring no mutation in the GJB2 coding region (n=89) as well as 91 controls who reported to have normal hearing. The obtained data revealed that the c.[168A>G(+)931C>T] double heterozygous genotype, regarding the rs55704559 and rs5030700 SNPs (both localised to the 3’UTR), respectively, is overrepresented in the patients. In silico analysis predicts that the c.168A>G variant (regardless of the fact that in position c.931 is a cytosine or a thymine) causes an alteration of mRNA folding. Thus, our data suggest that the c.168G allele might be associated with HL. Other aim of this work was to investigate the effects of the mutations c.-259C>T and p.Val84Met, mentioned above, and the mutation p.Met163Leu, also identified for the first time in a previous work, in other Portuguese family. The available genetic data regarding these mutations were insufficient to prove their pathogenicity, and thus we have performed functional studies on the three mutations. The obtained results suggest that the three mutations are indeed pathogenic, exerting their effect in distinct ways. The c.-259C>T mutation decreases very significantly the basal promoter activity. As regards the coding mutations, p.Val84Met alters the ionic and molecular permeability properties of the intercellular channel, which may compromise their function in vivo, while p.Met163Leu leads to cell death, possibly by a mechanism other than the malfunctioning of undocked hemichannels (already described in the literature), and has a partial dominant-negative effect on wild-type Cx26 (wtCx26) and Cx30 (wtCx30). Mutations in the GJB2 gene are responsible for a considerable proportion of nonsyndromic sensorineural hearing loss (NSSHL), in several populations. This gene is a member of a gene family coding for connexins, the subunits of the hemichannels (connexons) which form the intercellular channels of the gap junctions existing in the vertebrates and tunicates. Connexin-26 (Cx26), encoded by the GJB2 gene, is expressed in several tissues, including specific epithelial and conjunctive tissues of the cochlea, the auditory organ which is localised to the inner ear. In the cochlea, Cx26 is co-expressed with connexin-30 (Cx30), encoded by the GJB6 gene, in several cell types. Two large GJB6 deletions are involved in several hearing loss (HL) cases, as well, being most of these cases due to compound heterozygosity with a GJB2 mutation. Given the relevance of both GJB2 and GJB6 genes to the HL etiology in several populations, the molecular diagnosis of NSSHL cases with probable genetic cause is usually initiated by the analysis of the GJB2 coding region (in which most pathogenic mutations have been found), followed by the investigation of the presence of the GJB6 deletions in the cases where none or only one GJB2 mutation is found. In some cases, attributing the cause of the HL to the GJB2/GJB6 genotype is complicated because some GJB2 mutations are of unclear or controversial pathogenicity, due to either lack of genetic evidence (evident segregation of the mutation with the HL) or contradictory genetic data (same genotype occurring in both normalhearing and hearing-impaired individuals). In those cases, functional studies constitute an adequate approach for the investigation of the putative pathogenicity of such mutations, several of which have already been studied in this way. One of the aims of this work consisted in the study of the coding region and acceptor splice site of the GJB2 gene in Portuguese individuals presenting with NSSHL. We have further extended the analysis of the gene to noncoding regions (basal promoter and about 700 bp immediately upstream, exon 1, donor splice site and the whole 3’UTR). These noncoding regions have rarely been studied. Nonetheless, two pathogenic noncoding mutations had previously been identified, both in the donor splice site. Initially, we analysed the basal promoter, exon 1 and donor splice site in some patients who only harboured one coding mutation. We then found a novel mutation (c.-259C>T) in the basal promoter, in one of the binding sites for Sp1/Sp3 transcription factors. This mutation was found in a profoundly hearing-impaired patient, in trans with p.Val84Met, that we had identified as a novel mutation, in a previous work. Later, we analised all the fore mentioned noncoding regions in a larger sample, including monoallelic patients and also those harbouring no mutation in the GJB2 coding region (n=89) as well as 91 controls who reported to have normal hearing. The obtained data revealed that the c.[168A>G(+)931C>T] double heterozygous genotype, regarding the rs55704559 and rs5030700 SNPs (both localised to the 3’UTR), respectively, is overrepresented in the patients. In silico analysis predicts that the c.168A>G variant (regardless of the fact that in position c.931 is a cytosine or a thymine) causes an alteration of mRNA folding. Thus, our data suggest that the c.168G allele might be associated with HL. Other aim of this work was to investigate the effects of the mutations c.-259C>T and p.Val84Met, mentioned above, and the mutation p.Met163Leu, also identified for the first time in a previous work, in other Portuguese family. The available genetic data regarding these mutations were insufficient to prove their pathogenicity, and thus we have performed functional studies on the three mutations. The obtained results suggest that the three mutations are indeed pathogenic, exerting their effect in distinct ways. The c.-259C>T mutation decreases very significantly the basal promoter activity. As regards the coding mutations, p.Val84Met alters the ionic and molecular permeability properties of the intercellular channel, which may compromise their function in vivo, while p.Met163Leu leads to cell death, possibly by a mechanism other than the malfunctioning of undocked hemichannels (already described in the literature), and has a partial dominant-negative effect on wild-type Cx26 (wtCx26) and Cx30 (wtCx30).Mutations in the GJB2 gene are responsible for a considerable proportion of nonsyndromic sensorineural hearing loss (NSSHL), in several populations. This gene is a member of a gene family coding for connexins, the subunits of the hemichannels (connexons) which form the intercellular channels of the gap junctions existing in the vertebrates and tunicates. Connexin-26 (Cx26), encoded by the GJB2 gene, is expressed in several tissues, including specific epithelial and conjunctive tissues of the cochlea, the auditory organ which is localised to the inner ear. In the cochlea, Cx26 is co-expressed with connexin-30 (Cx30), encoded by the GJB6 gene, in several cell types. Two large GJB6 deletions are involved in several hearing loss (HL) cases, as well, being most of these cases due to compound heterozygosity with a GJB2 mutation. Given the relevance of both GJB2 and GJB6 genes to the HL etiology in several populations, the molecular diagnosis of NSSHL cases with probable genetic cause is usually initiated by the analysis of the GJB2 coding region (in which most pathogenic mutations have been found), followed by the investigation of the presence of the GJB6 deletions in the cases where none or only one GJB2 mutation is found. In some cases, attributing the cause of the HL to the GJB2/GJB6 genotype is complicated because some GJB2 mutations are of unclear or controversial pathogenicity, due to either lack of genetic evidence (evident segregation of the mutation with the HL) or contradictory genetic data (same genotype occurring in both normalhearing and hearing-impaired individuals). In those cases, functional studies constitute an adequate approach for the investigation of the putative pathogenicity of such mutations, several of which have already been studied in this way. One of the aims of this work consisted in the study of the coding region and acceptor splice site of the GJB2 gene in Portuguese individuals presenting with NSSHL. We have further extended the analysis of the gene to noncoding regions (basal promoter and about 700 bp immediately upstream, exon 1, donor splice site and the whole 3’UTR). These noncoding regions have rarely been studied. Nonetheless, two pathogenic noncoding mutations had previously been identified, both in the donor splice site. Initially, we analysed the basal promoter, exon 1 and donor splice site in some patients who only harboured one coding mutation. We then found a novel mutation (c.-259C>T) in the basal promoter, in one of the binding sites for Sp1/Sp3 transcription factors. This mutation was found in a profoundly hearing-impaired patient, in trans with p.Val84Met, that we had identified as a novel mutation, in a previous work. Later, we analised all the fore mentioned noncoding regions in a larger sample, including monoallelic patients and also those harbouring no mutation in the GJB2 coding region (n=89) as well as 91 controls who reported to have normal hearing. The obtained data revealed that the c.[168A>G(+)931C>T] double heterozygous genotype, regarding the rs55704559 and rs5030700 SNPs (both localised to the 3’UTR), respectively, is overrepresented in the patients. In silico analysis predicts that the c.168A>G variant (regardless of the fact that in position c.931 is a cytosine or a thymine) causes an alteration of mRNA folding. Thus, our data suggest that the c.168G allele might be associated with HL. Other aim of this work was to investigate the effects of the mutations c.-259C>T and p.Val84Met, mentioned above, and the mutation p.Met163Leu, also identified for the first time in a previous work, in other Portuguese family. The available genetic data regarding these mutations were insufficient to prove their pathogenicity, and thus we have performed functional studies on the three mutations. The obtained results suggest that the three mutations are indeed pathogenic, exerting their effect in distinct ways. The c.-259C>T mutation decreases very significantly the basal promoter activity. As regards the coding mutations, p.Val84Met alters the ionic and molecular permeability properties of the intercellular channel, which may compromise their function in vivo, while p.Met163Leu leads to cell death, possibly by a mechanism other than the malfunctioning of undocked hemichannels (already described in the literature), and has a partial dominant-negative effect on wild-type Cx26 (wtCx26) and Cx30 (wtCx30).Mutations in the GJB2 gene are responsible for a considerable proportion of nonsyndromic sensorineural hearing loss (NSSHL), in several populations. This gene is a member of a gene family coding for connexins, the subunits of the hemichannels (connexons) which form the intercellular channels of the gap junctions existing in the vertebrates and tunicates. Connexin-26 (Cx26), encoded by the GJB2 gene, is expressed in several tissues, including specific epithelial and conjunctive tissues of the cochlea, the auditory organ which is localised to the inner ear. In the cochlea, Cx26 is co-expressed with connexin-30 (Cx30), encoded by the GJB6 gene, in several cell types. Two large GJB6 deletions are involved in several hearing loss (HL) cases, as well, being most of these cases due to compound heterozygosity with a GJB2 mutation. Given the relevance of both GJB2 and GJB6 genes to the HL etiology in several populations, the molecular diagnosis of NSSHL cases with probable genetic cause is usually initiated by the analysis of the GJB2 coding region (in which most pathogenic mutations have been found), followed by the investigation of the presence of the GJB6 deletions in the cases where none or only one GJB2 mutation is found. In some cases, attributing the cause of the HL to the GJB2/GJB6 genotype is complicated because some GJB2 mutations are of unclear or controversial pathogenicity, due to either lack of genetic evidence (evident segregation of the mutation with the HL) or contradictory genetic data (same genotype occurring in both normalhearing and hearing-impaired individuals). In those cases, functional studies constitute an adequate approach for the investigation of the putative pathogenicity of such mutations, several of which have already been studied in this way. One of the aims of this work consisted in the study of the coding region and acceptor splice site of the GJB2 gene in Portuguese individuals presenting with NSSHL. We have further extended the analysis of the gene to noncoding regions (basal promoter and about 700 bp immediately upstream, exon 1, donor splice site and the whole 3’UTR). These noncoding regions have rarely been studied. Nonetheless, two pathogenic noncoding mutations had previously been identified, both in the donor splice site. Initially, we analysed the basal promoter, exon 1 and donor splice site in some patients who only harboured one coding mutation. We then found a novel mutation (c.-259C>T) in the basal promoter, in one of the binding sites for Sp1/Sp3 transcription factors. This mutation was found in a profoundly hearing-impaired patient, in trans with p.Val84Met, that we had identified as a novel mutation, in a previous work. Later, we analised all the fore mentioned noncoding regions in a larger sample, including monoallelic patients and also those harbouring no mutation in the GJB2 coding region (n=89) as well as 91 controls who reported to have normal hearing. The obtained data revealed that the c.[168A>G(+)931C>T] double heterozygous genotype, regarding the rs55704559 and rs5030700 SNPs (both localised to the 3’UTR), respectively, is overrepresented in the patients. In silico analysis predicts that the c.168A>G variant (regardless of the fact that in position c.931 is a cytosine or a thymine) causes an alteration of mRNA folding. Thus, our data suggest that the c.*168G allele might be associated with HL. Other aim of this work was to investigate the effects of the mutations c.-259C>T and p.Val84Met, mentioned above, and the mutation p.Met163Leu, also identified for the first time in a previous work, in other Portuguese family. The available genetic data regarding these mutations were insufficient to prove their pathogenicity, and thus we have performed functional studies on the three mutations. The obtained results suggest that the three mutations are indeed pathogenic, exerting their effect in distinct ways. The c.-259C>T mutation decreases very significantly the basal promoter activity. As regards the coding mutations, p.Val84Met alters the ionic and molecular permeability properties of the intercellular channel, which may compromise their function in vivo, while p.Met163Leu leads to cell death, possibly by a mechanism other than the malfunctioning of undocked hemichannels (already described in the literature), and has a partial dominant-negative effect on wild-type Cx26 (wtCx26) and Cx30 (wtCx30).
Autores principais:	Matos, Tiago Morim De
Assunto:	Surdez Genética humana Teses de doutoramento - 2012
Ano:	2012
País:	Portugal
Tipo de documento:	tese de doutoramento
Tipo de acesso:	acesso aberto
Instituição associada:	Universidade de Lisboa
Idioma:	inglês
Origem:	Repositório da Universidade de Lisboa

Descrição
Resumo:	Mutations in the GJB2 gene are responsible for a considerable proportion of nonsyndromic sensorineural hearing loss (NSSHL), in several populations. This gene is a member of a gene family coding for connexins, the subunits of the hemichannels (connexons) which form the intercellular channels of the gap junctions existing in the vertebrates and tunicates. Connexin-26 (Cx26), encoded by the GJB2 gene, is expressed in several tissues, including specific epithelial and conjunctive tissues of the cochlea, the auditory organ which is localised to the inner ear. In the cochlea, Cx26 is co-expressed with connexin-30 (Cx30), encoded by the GJB6 gene, in several cell types. Two large GJB6 deletions are involved in several hearing loss (HL) cases, as well, being most of these cases due to compound heterozygosity with a GJB2 mutation. Given the relevance of both GJB2 and GJB6 genes to the HL etiology in several populations, the molecular diagnosis of NSSHL cases with probable genetic cause is usually initiated by the analysis of the GJB2 coding region (in which most pathogenic mutations have been found), followed by the investigation of the presence of the GJB6 deletions in the cases where none or only one GJB2 mutation is found. In some cases, attributing the cause of the HL to the GJB2/GJB6 genotype is complicated because some GJB2 mutations are of unclear or controversial pathogenicity, due to either lack of genetic evidence (evident segregation of the mutation with the HL) or contradictory genetic data (same genotype occurring in both normalhearing and hearing-impaired individuals). In those cases, functional studies constitute an adequate approach for the investigation of the putative pathogenicity of such mutations, several of which have already been studied in this way. One of the aims of this work consisted in the study of the coding region and acceptor splice site of the GJB2 gene in Portuguese individuals presenting with NSSHL. We have further extended the analysis of the gene to noncoding regions (basal promoter and about 700 bp immediately upstream, exon 1, donor splice site and the whole 3’UTR). These noncoding regions have rarely been studied. Nonetheless, two pathogenic noncoding mutations had previously been identified, both in the donor splice site. Initially, we analysed the basal promoter, exon 1 and donor splice site in some patients who only harboured one coding mutation. We then found a novel mutation (c.-259C>T) in the basal promoter, in one of the binding sites for Sp1/Sp3 transcription factors. This mutation was found in a profoundly hearing-impaired patient, in trans with p.Val84Met, that we had identified as a novel mutation, in a previous work. Later, we analised all the fore mentioned noncoding regions in a larger sample, including monoallelic patients and also those harbouring no mutation in the GJB2 coding region (n=89) as well as 91 controls who reported to have normal hearing. The obtained data revealed that the c.[168A>G(+)931C>T] double heterozygous genotype, regarding the rs55704559 and rs5030700 SNPs (both localised to the 3’UTR), respectively, is overrepresented in the patients. In silico analysis predicts that the c.168A>G variant (regardless of the fact that in position c.931 is a cytosine or a thymine) causes an alteration of mRNA folding. Thus, our data suggest that the c.168G allele might be associated with HL. Other aim of this work was to investigate the effects of the mutations c.-259C>T and p.Val84Met, mentioned above, and the mutation p.Met163Leu, also identified for the first time in a previous work, in other Portuguese family. The available genetic data regarding these mutations were insufficient to prove their pathogenicity, and thus we have performed functional studies on the three mutations. The obtained results suggest that the three mutations are indeed pathogenic, exerting their effect in distinct ways. The c.-259C>T mutation decreases very significantly the basal promoter activity. As regards the coding mutations, p.Val84Met alters the ionic and molecular permeability properties of the intercellular channel, which may compromise their function in vivo, while p.Met163Leu leads to cell death, possibly by a mechanism other than the malfunctioning of undocked hemichannels (already described in the literature), and has a partial dominant-negative effect on wild-type Cx26 (wtCx26) and Cx30 (wtCx30).Mutations in the GJB2 gene are responsible for a considerable proportion of nonsyndromic sensorineural hearing loss (NSSHL), in several populations. This gene is a member of a gene family coding for connexins, the subunits of the hemichannels (connexons) which form the intercellular channels of the gap junctions existing in the vertebrates and tunicates. Connexin-26 (Cx26), encoded by the GJB2 gene, is expressed in several tissues, including specific epithelial and conjunctive tissues of the cochlea, the auditory organ which is localised to the inner ear. In the cochlea, Cx26 is co-expressed with connexin-30 (Cx30), encoded by the GJB6 gene, in several cell types. Two large GJB6 deletions are involved in several hearing loss (HL) cases, as well, being most of these cases due to compound heterozygosity with a GJB2 mutation. Given the relevance of both GJB2 and GJB6 genes to the HL etiology in several populations, the molecular diagnosis of NSSHL cases with probable genetic cause is usually initiated by the analysis of the GJB2 coding region (in which most pathogenic mutations have been found), followed by the investigation of the presence of the GJB6 deletions in the cases where none or only one GJB2 mutation is found. In some cases, attributing the cause of the HL to the GJB2/GJB6 genotype is complicated because some GJB2 mutations are of unclear or controversial pathogenicity, due to either lack of genetic evidence (evident segregation of the mutation with the HL) or contradictory genetic data (same genotype occurring in both normalhearing and hearing-impaired individuals). In those cases, functional studies constitute an adequate approach for the investigation of the putative pathogenicity of such mutations, several of which have already been studied in this way. One of the aims of this work consisted in the study of the coding region and acceptor splice site of the GJB2 gene in Portuguese individuals presenting with NSSHL. We have further extended the analysis of the gene to noncoding regions (basal promoter and about 700 bp immediately upstream, exon 1, donor splice site and the whole 3’UTR). These noncoding regions have rarely been studied. Nonetheless, two pathogenic noncoding mutations had previously been identified, both in the donor splice site. Initially, we analysed the basal promoter, exon 1 and donor splice site in some patients who only harboured one coding mutation. We then found a novel mutation (c.-259C>T) in the basal promoter, in one of the binding sites for Sp1/Sp3 transcription factors. This mutation was found in a profoundly hearing-impaired patient, in trans with p.Val84Met, that we had identified as a novel mutation, in a previous work. Later, we analised all the fore mentioned noncoding regions in a larger sample, including monoallelic patients and also those harbouring no mutation in the GJB2 coding region (n=89) as well as 91 controls who reported to have normal hearing. The obtained data revealed that the c.[168A>G(+)931C>T] double heterozygous genotype, regarding the rs55704559 and rs5030700 SNPs (both localised to the 3’UTR), respectively, is overrepresented in the patients. In silico analysis predicts that the c.168A>G variant (regardless of the fact that in position c.931 is a cytosine or a thymine) causes an alteration of mRNA folding. Thus, our data suggest that the c.168G allele might be associated with HL. Other aim of this work was to investigate the effects of the mutations c.-259C>T and p.Val84Met, mentioned above, and the mutation p.Met163Leu, also identified for the first time in a previous work, in other Portuguese family. The available genetic data regarding these mutations were insufficient to prove their pathogenicity, and thus we have performed functional studies on the three mutations. The obtained results suggest that the three mutations are indeed pathogenic, exerting their effect in distinct ways. The c.-259C>T mutation decreases very significantly the basal promoter activity. As regards the coding mutations, p.Val84Met alters the ionic and molecular permeability properties of the intercellular channel, which may compromise their function in vivo, while p.Met163Leu leads to cell death, possibly by a mechanism other than the malfunctioning of undocked hemichannels (already described in the literature), and has a partial dominant-negative effect on wild-type Cx26 (wtCx26) and Cx30 (wtCx30). Mutations in the GJB2 gene are responsible for a considerable proportion of nonsyndromic sensorineural hearing loss (NSSHL), in several populations. This gene is a member of a gene family coding for connexins, the subunits of the hemichannels (connexons) which form the intercellular channels of the gap junctions existing in the vertebrates and tunicates. Connexin-26 (Cx26), encoded by the GJB2 gene, is expressed in several tissues, including specific epithelial and conjunctive tissues of the cochlea, the auditory organ which is localised to the inner ear. In the cochlea, Cx26 is co-expressed with connexin-30 (Cx30), encoded by the GJB6 gene, in several cell types. Two large GJB6 deletions are involved in several hearing loss (HL) cases, as well, being most of these cases due to compound heterozygosity with a GJB2 mutation. Given the relevance of both GJB2 and GJB6 genes to the HL etiology in several populations, the molecular diagnosis of NSSHL cases with probable genetic cause is usually initiated by the analysis of the GJB2 coding region (in which most pathogenic mutations have been found), followed by the investigation of the presence of the GJB6 deletions in the cases where none or only one GJB2 mutation is found. In some cases, attributing the cause of the HL to the GJB2/GJB6 genotype is complicated because some GJB2 mutations are of unclear or controversial pathogenicity, due to either lack of genetic evidence (evident segregation of the mutation with the HL) or contradictory genetic data (same genotype occurring in both normalhearing and hearing-impaired individuals). In those cases, functional studies constitute an adequate approach for the investigation of the putative pathogenicity of such mutations, several of which have already been studied in this way. One of the aims of this work consisted in the study of the coding region and acceptor splice site of the GJB2 gene in Portuguese individuals presenting with NSSHL. We have further extended the analysis of the gene to noncoding regions (basal promoter and about 700 bp immediately upstream, exon 1, donor splice site and the whole 3’UTR). These noncoding regions have rarely been studied. Nonetheless, two pathogenic noncoding mutations had previously been identified, both in the donor splice site. Initially, we analysed the basal promoter, exon 1 and donor splice site in some patients who only harboured one coding mutation. We then found a novel mutation (c.-259C>T) in the basal promoter, in one of the binding sites for Sp1/Sp3 transcription factors. This mutation was found in a profoundly hearing-impaired patient, in trans with p.Val84Met, that we had identified as a novel mutation, in a previous work. Later, we analised all the fore mentioned noncoding regions in a larger sample, including monoallelic patients and also those harbouring no mutation in the GJB2 coding region (n=89) as well as 91 controls who reported to have normal hearing. The obtained data revealed that the c.[168A>G(+)931C>T] double heterozygous genotype, regarding the rs55704559 and rs5030700 SNPs (both localised to the 3’UTR), respectively, is overrepresented in the patients. In silico analysis predicts that the c.168A>G variant (regardless of the fact that in position c.931 is a cytosine or a thymine) causes an alteration of mRNA folding. Thus, our data suggest that the c.168G allele might be associated with HL. Other aim of this work was to investigate the effects of the mutations c.-259C>T and p.Val84Met, mentioned above, and the mutation p.Met163Leu, also identified for the first time in a previous work, in other Portuguese family. The available genetic data regarding these mutations were insufficient to prove their pathogenicity, and thus we have performed functional studies on the three mutations. The obtained results suggest that the three mutations are indeed pathogenic, exerting their effect in distinct ways. The c.-259C>T mutation decreases very significantly the basal promoter activity. As regards the coding mutations, p.Val84Met alters the ionic and molecular permeability properties of the intercellular channel, which may compromise their function in vivo, while p.Met163Leu leads to cell death, possibly by a mechanism other than the malfunctioning of undocked hemichannels (already described in the literature), and has a partial dominant-negative effect on wild-type Cx26 (wtCx26) and Cx30 (wtCx30).Mutations in the GJB2 gene are responsible for a considerable proportion of nonsyndromic sensorineural hearing loss (NSSHL), in several populations. This gene is a member of a gene family coding for connexins, the subunits of the hemichannels (connexons) which form the intercellular channels of the gap junctions existing in the vertebrates and tunicates. Connexin-26 (Cx26), encoded by the GJB2 gene, is expressed in several tissues, including specific epithelial and conjunctive tissues of the cochlea, the auditory organ which is localised to the inner ear. In the cochlea, Cx26 is co-expressed with connexin-30 (Cx30), encoded by the GJB6 gene, in several cell types. Two large GJB6 deletions are involved in several hearing loss (HL) cases, as well, being most of these cases due to compound heterozygosity with a GJB2 mutation. Given the relevance of both GJB2 and GJB6 genes to the HL etiology in several populations, the molecular diagnosis of NSSHL cases with probable genetic cause is usually initiated by the analysis of the GJB2 coding region (in which most pathogenic mutations have been found), followed by the investigation of the presence of the GJB6 deletions in the cases where none or only one GJB2 mutation is found. In some cases, attributing the cause of the HL to the GJB2/GJB6 genotype is complicated because some GJB2 mutations are of unclear or controversial pathogenicity, due to either lack of genetic evidence (evident segregation of the mutation with the HL) or contradictory genetic data (same genotype occurring in both normalhearing and hearing-impaired individuals). In those cases, functional studies constitute an adequate approach for the investigation of the putative pathogenicity of such mutations, several of which have already been studied in this way. One of the aims of this work consisted in the study of the coding region and acceptor splice site of the GJB2 gene in Portuguese individuals presenting with NSSHL. We have further extended the analysis of the gene to noncoding regions (basal promoter and about 700 bp immediately upstream, exon 1, donor splice site and the whole 3’UTR). These noncoding regions have rarely been studied. Nonetheless, two pathogenic noncoding mutations had previously been identified, both in the donor splice site. Initially, we analysed the basal promoter, exon 1 and donor splice site in some patients who only harboured one coding mutation. We then found a novel mutation (c.-259C>T) in the basal promoter, in one of the binding sites for Sp1/Sp3 transcription factors. This mutation was found in a profoundly hearing-impaired patient, in trans with p.Val84Met, that we had identified as a novel mutation, in a previous work. Later, we analised all the fore mentioned noncoding regions in a larger sample, including monoallelic patients and also those harbouring no mutation in the GJB2 coding region (n=89) as well as 91 controls who reported to have normal hearing. The obtained data revealed that the c.[168A>G(+)931C>T] double heterozygous genotype, regarding the rs55704559 and rs5030700 SNPs (both localised to the 3’UTR), respectively, is overrepresented in the patients. In silico analysis predicts that the c.168A>G variant (regardless of the fact that in position c.931 is a cytosine or a thymine) causes an alteration of mRNA folding. Thus, our data suggest that the c.168G allele might be associated with HL. Other aim of this work was to investigate the effects of the mutations c.-259C>T and p.Val84Met, mentioned above, and the mutation p.Met163Leu, also identified for the first time in a previous work, in other Portuguese family. The available genetic data regarding these mutations were insufficient to prove their pathogenicity, and thus we have performed functional studies on the three mutations. The obtained results suggest that the three mutations are indeed pathogenic, exerting their effect in distinct ways. The c.-259C>T mutation decreases very significantly the basal promoter activity. As regards the coding mutations, p.Val84Met alters the ionic and molecular permeability properties of the intercellular channel, which may compromise their function in vivo, while p.Met163Leu leads to cell death, possibly by a mechanism other than the malfunctioning of undocked hemichannels (already described in the literature), and has a partial dominant-negative effect on wild-type Cx26 (wtCx26) and Cx30 (wtCx30).Mutations in the GJB2 gene are responsible for a considerable proportion of nonsyndromic sensorineural hearing loss (NSSHL), in several populations. This gene is a member of a gene family coding for connexins, the subunits of the hemichannels (connexons) which form the intercellular channels of the gap junctions existing in the vertebrates and tunicates. Connexin-26 (Cx26), encoded by the GJB2 gene, is expressed in several tissues, including specific epithelial and conjunctive tissues of the cochlea, the auditory organ which is localised to the inner ear. In the cochlea, Cx26 is co-expressed with connexin-30 (Cx30), encoded by the GJB6 gene, in several cell types. Two large GJB6 deletions are involved in several hearing loss (HL) cases, as well, being most of these cases due to compound heterozygosity with a GJB2 mutation. Given the relevance of both GJB2 and GJB6 genes to the HL etiology in several populations, the molecular diagnosis of NSSHL cases with probable genetic cause is usually initiated by the analysis of the GJB2 coding region (in which most pathogenic mutations have been found), followed by the investigation of the presence of the GJB6 deletions in the cases where none or only one GJB2 mutation is found. In some cases, attributing the cause of the HL to the GJB2/GJB6 genotype is complicated because some GJB2 mutations are of unclear or controversial pathogenicity, due to either lack of genetic evidence (evident segregation of the mutation with the HL) or contradictory genetic data (same genotype occurring in both normalhearing and hearing-impaired individuals). In those cases, functional studies constitute an adequate approach for the investigation of the putative pathogenicity of such mutations, several of which have already been studied in this way. One of the aims of this work consisted in the study of the coding region and acceptor splice site of the GJB2 gene in Portuguese individuals presenting with NSSHL. We have further extended the analysis of the gene to noncoding regions (basal promoter and about 700 bp immediately upstream, exon 1, donor splice site and the whole 3’UTR). These noncoding regions have rarely been studied. Nonetheless, two pathogenic noncoding mutations had previously been identified, both in the donor splice site. Initially, we analysed the basal promoter, exon 1 and donor splice site in some patients who only harboured one coding mutation. We then found a novel mutation (c.-259C>T) in the basal promoter, in one of the binding sites for Sp1/Sp3 transcription factors. This mutation was found in a profoundly hearing-impaired patient, in trans with p.Val84Met, that we had identified as a novel mutation, in a previous work. Later, we analised all the fore mentioned noncoding regions in a larger sample, including monoallelic patients and also those harbouring no mutation in the GJB2 coding region (n=89) as well as 91 controls who reported to have normal hearing. The obtained data revealed that the c.[168A>G(+)931C>T] double heterozygous genotype, regarding the rs55704559 and rs5030700 SNPs (both localised to the 3’UTR), respectively, is overrepresented in the patients. In silico analysis predicts that the c.168A>G variant (regardless of the fact that in position c.931 is a cytosine or a thymine) causes an alteration of mRNA folding. Thus, our data suggest that the c.*168G allele might be associated with HL. Other aim of this work was to investigate the effects of the mutations c.-259C>T and p.Val84Met, mentioned above, and the mutation p.Met163Leu, also identified for the first time in a previous work, in other Portuguese family. The available genetic data regarding these mutations were insufficient to prove their pathogenicity, and thus we have performed functional studies on the three mutations. The obtained results suggest that the three mutations are indeed pathogenic, exerting their effect in distinct ways. The c.-259C>T mutation decreases very significantly the basal promoter activity. As regards the coding mutations, p.Val84Met alters the ionic and molecular permeability properties of the intercellular channel, which may compromise their function in vivo, while p.Met163Leu leads to cell death, possibly by a mechanism other than the malfunctioning of undocked hemichannels (already described in the literature), and has a partial dominant-negative effect on wild-type Cx26 (wtCx26) and Cx30 (wtCx30).