Tiago D. Matos

A novel hearing loss-related mutation occurring in the GJB2 basal promoter

Mutations in the GJB2 gene are a major cause of non-syndromic recessive hearing loss in many countries. In a significant fraction of patients, only monoallelic GJB2 mutations known to be either recessive or of unclear pathogenicity are identified. This paper reports a novel GJB2 mutation, −3438C→T, found in the basal promoter of the gene, in trans with V84M, in a patient with profound hearing impairment. This novel mutation can abolish the basal promoter activity of GJB2. These results highlight the importance of extending the mutational screening to regions outside the coding region of GJB2.

A novel M163L mutation in connexin 26 causing cell death and associated with autosomal dominant hearing loss

Mutations in GJB2 gene (encoding connexin 26) are the most common cause of hereditary non-syndromic sensorineural hearing loss (NSSHL) in different populations. The majority of GJB2 mutations are recessive, but a few dominant mutations have been associated with hearing loss either isolated or associated with skin disease. We describe a novel dominant pathogenic GJB2 mutation, identified in a Portuguese family affected with bilateral mild/moderate high-frequency NSSHL. In vitro functional studies demonstrate that the mutant protein (p.M163L) has defective trafficking to the plasma membrane and is associated with increased cell death.

DFNB1-associated deafness in Portuguese cochlear implant users: prevalence and impact on oral outcome.

OBJECTIVES Hearing loss is a condition that interferes with the development of the child at a cognitive and language level. Therefore, early diagnosis of deafness is important for (re)habilitation, namely through the use of cochlear implant (CI). The present study aimed at screening CI Portuguese individuals for the presence of mutations in the genes GJB2 and GJB6 (DFNB1 locus), and searching a possible correlation between the genotype and the oral habilitation outcome following implantation. METHODS Our sample included 117 CI individuals implanted longer than 5 years. Sequencing of GJB2 entire coding region was first performed. The presence of deletions del(GJB6-D13S1830) and del(GJB6-D13S1854) was subsequently tested by multiplex PCR. To assess the oral outcome of these individuals, a global score is calculated through a formula that integrates the results of a battery of speech and audiological tests routinely used in ORL services. This global oral performance score was used to test whether individuals with DFNB1-associated deafness perform significantly better than individuals without DFNB1-associated deafness. RESULTS In 35% of the cases, deafness was clearly associated to DFNB1. The most common mutated allele was c.35delG (85%). Other variants have also been found, namely p.Gly130Ala, p.Asn206Ser, p.Val37Ile, p.Glu47X, p.Arg184Trp, p.Trp24X and the two common GJB6 deletions, del(GJB6-D13S1854) and del(GJB6-D13S1830), the last one identified for the first time in our population. Regarding the oral outcome, after testing the homogeneity of the two groups it could be observed that, in mean, the individuals with DFNB1-associated deafness perform significantly better (p=0.012) than the individuals without DFNB1-associated deafness. DISCUSSION AND CONCLUSION This first screening of DFNB1 genes in the Portuguese CI population provides clear evidence of the high proportion of DFNB1-associated deafness amongst the Portuguese implanted individuals. DFNB1 status is significantly associated to higher oral performance scores, with DFNB1 individuals performing, on average, 6% better than the individuals without DFNB1-associated deafness.

Spectrum and frequency of GJB2 mutations in a cohort of 264 Portuguese nonsyndromic sensorineural hearing loss patients

Abstract Objective: To assess the spectrum and prevalence of mutations in the GJB2 gene in Portuguese nonsyndromic sensorineural hearing loss (NSSHL) patients. Design: Sequencing of the coding region, basal promoter, exon 1, and donor splice site of the GJB2 gene; screening for the presence of the two common GJB6 deletions. Study sample: A cohort of 264 Portuguese NSSHL patients. Results: At least one out of 21 different GJB2 variants was identified in 80 (30.2%) of the 264 patients analysed. Two mutant alleles were found in 53 (20%) of these probands, of which 83% (44/53) harboured at least one c.35delG allele. Twenty-seven (10.2%) of the probands harboured only one mutant allele. Subsequent analysis revealed that the GJB6 deletion del(GJB6-D13S1854) was present in at least 7.4% (2/27) of the patients carrying only one mutant GJB2 allele. Overall, one in five (55/264) of the patients were diagnosed as having DFNB1-related NSSHL, of which the vast majority (53/55) harboured only GJB2 mutations. Conclusions: This study provides clear demonstration that mutations in the GJB2 gene are an important cause of NSSHL in Portugal, thus representing a valuable indicator as regards therapeutical and rehabilitation options, as well as genetic counseling of these patients and their families.

The Controversial p.Arg127His Mutation in GJB2: Report on Three Portuguese Hearing Loss Family Cases

Mutations in the GJB2 gene account for up to 50% of hereditary nonsyndromic hearing loss in several populations. Over 200 mutations are already described in this gene, and three of them, c.35delG, c.167delT, and c.235delC, are the most frequent in Caucasians, Ashkenazi Jews, and Asians, respectively. Most of GJB2 hearing loss-related mutations are recessive, but a few dominant alleles have also been described. Apart from the clearly pathogenic mutations, there are some other variants whose pathogenicity is still controversial, such as p.Met34Thr, p.Val37Ile, p.Arg127His, and p.Val153Ile. The p.Arg127His allele has been found in some mono- and biallelic hearing-impaired patients from several countries. In this article we report on some Portuguese patients harboring this mutation. Taking into consideration the analysis of these Portuguese cases as well as the genetic and functional data regarding p.Arg127His available in the literature, we conclude that this variant may be a cause of hearing loss depending on environmental factors and/or genetic background.

Novel splice-site mutation c.1615-2A>G (IVS14-2A>G) in the SLC26A4 gene causing Pendred syndrome in a consanguineous Portuguese family.

Novel Splice-Site Mutation c.1615-2A>G (IVS14-2A>G) in the SLC26A4 Gene Causing Pendred Syndrome in a Consanguineous Portuguese Family Helena Sim~oes-Teixeira, Tiago D. Matos, Marta Canas Marques, Óscar Dias, M ario Andrea, Eduardo Barreiros, Lu ıs Barreiros, Felipe Moreno, Graça Fialho, Helena Caria, and Ignacio del Castillo* Faculty of Science, Centre for Biodiversity, Functional and Integrative Genomics (BioFIG), University of Lisbon, Lisbon, Portugal Unidad de Gen etica Molecular, Hospital Universitario Ram on y Cajal, IRYCIS, Madrid, Spain ORL Service, Hospital de Santa Maria, Centro Hospitalar Lisboa Norte, EPE, Lisbon, Portugal Endocrinology Department, Hospital de Santa Maria, Centro Hospitalar Lisboa Norte, EPE, Lisbon, Portugal Centro de Investigaci on Biom edica en Red de Enfermedades Raras (CIBERER), Madrid, Spain Higher School of Health, Polytechnic Institute of Set ubal, Set ubal, Portugal

A novel p.Leu213X mutation in GJB2 gene in a Portuguese family

INTRODUCTION Hearing loss is the most common sensory disability and is present in about 1.9 per 1000 infants at birth. The DFNB1 locus (13q11-q12) includes the genes GJB2, coding for connexin 26, and GJB6, encoding connexin 30. More than 100 mutations have been identified associated with autosomal dominant and recessive hearing loss in the GJB2 gene. OBJECTIVES The aim of the present study was to identify the genetic aetiology of deafness in two Portuguese individuals, presenting nonsyndromic sensorineural moderate and severe hearing loss, respectively. PATIENTS AND METHODS The individuals were evaluated in both ears by pure tone audiometry and blood samples were collected after written informed consent was signed. DNA extraction and PCR amplification of GJB2 coding region followed standard methodologies. PCR products were automatically sequenced in both directions. RESULTS We identified a novel mutation, c.638T>A (p.Leu213X), in GJB2 gene. This nonsense mutation was found in both siblings, and was inherited from their hearing father. Molecular analysis showed that the two siblings were also heterozygous for c.333-334delAA, a previously described GJB2 deletion. This novel mutation was not found in a random control sample of 480 individuals that were screened for coding region of GJB2 gene. p.Leu213X mutation identified in this study for the first time changes the codon 213, coding for a highly conserved and slowly evolving residue of connexin 26, localised to the C-terminus domain of the protein, to a STOP codon, leading to the deletion of the last 14 amino acids of the protein. CONCLUSION We can conclude that the aetiology of deafness in these individuals is due to the GJB2 genotype involving the c.333-334delAA deletion and the novel p.Leu213X mutation in compound heterozygosity.

The controversial p.Met34Thr variant in GJB2 gene: Two siblings, one genotype, two phenotypes

INTRODUCTION Recent advances in molecular genetics have increased the identification of genes and mutations responsible for inherited forms of hearing loss (HL), enabling early detection of these cases. Approximately, 60% of early-onset HL cases are due to genetic causes, of which 70% are non-syndromic. Of these, 75-80% are inherited in an autosomal recessive pattern (DFNB). Mutations in GJB2 gene, coding for connexin 26 (Cx26), are the major cause of autosomal recessive hereditary HL, but some GJB2 mutations are yet of unclear or controversial significance. OBJECTIVES The aim of the present study was to identify the etiology of hearing loss, and correlate genotype-phenotype, in two Portuguese siblings with profound and moderate non-syndromic sensorineural bilateral HL. MATERIAL AND METHODS The affected subjects and their parents underwent audiological and genetic study. Molecular analysis of GJB2 gene was performed, searching for mutations in the coding region and receptor splicing site by automated sequencing. RESULTS The onset and the degree of HL were different in the two affected subjects. However, the same GJB2 genotype [p.Met34Thr]+[p.Arg184Pro] was identified in both siblings. The c.551G>C (p.Arg184Pro) and c.101T>C (p.Met34Thr) missense variants were inherited from the father and mother, respectively, both heterozygous carriers of these variants. CONCLUSION The clinical and genetic data here presented suggest that the non-syndromic sensorineural HL of these two Portuguese siblings might be due to the presence of p.Met34Thr and p.Arg184Pro variants in compound heterozygosity. If so, p.Met34Thr variant could have function as a hypomorphic allele that may cause HL depending on the opposing GJB2 allele. The observed phenotypic variability may not, however, be solely explained by variable expression of this genotype. A putative modifier gene or mutations in another HL-associated gene could probably be contributing to the severe HL in one of the siblings.

Report on a Novel Mutation, p.Leu213X, in Connexin-26 Protein

Objectives: Nonsyndromic sensorineural hearing loss (HL) is predominantly inherited in an autosomal recessive pattern. The DFNB1 locus, at chromosome 13q11-q12, includes the GJB2 and GJB6 genes, which encode connexin-26 (Cx26) and connexin-30 (Cx30), respectively. The objective of this study was to assess the genetic etiology of HL in a Portuguese family, presenting two siblings with nonsyndromic sensorineural moderate and severe HL, respectively. Methods: The individuals from one Portuguese family were evaluated by pure tone audiometry and blood samples were collected for DNA extraction. Polymerase chain reaction (PCR) amplification of GJB2 coding region followed standard methodologies. PCR products were automatically sequenced in both directions. A random control sample of 480 Portuguese individuals was also screened for the GJB2 coding region. Results: A novel mutation, c.638T>A (p.Leu213X), was identified in the GJB2 gene. This nonsense mutation was found in both siblings, and was inherited from their hearing father. Molecular analysis showed that the two siblings were also heterozygous for c.333-334delAA, a previously described GJB2 deletion, inherited from their hearing mother. The novel mutation wasn’t found in the control sample. The mutation p.Leu213X is located to the C-terminus domain of the connexin-26 and changes the codon 213, that codes for a highly conserved and slowly evolving residue of this protein, to a STOP codon, leading to the deletion of the last 14 amino acids of the protein. Conclusions: The HL of the two Portuguese siblings is most probably due to the presence of the c.333-334delAA deletion and the p.Leu213X novel mutation, in compound heterozygosity.