Connexin 26 mutations in autosomal recessive deafness disorders: A ...

Original Article International Journal of Audiology 2007; 46:75 81

Stacey A. Apps* Wayne A. Rankin$ Andrew P. Kurmis$ *School of Speech Pathology and Audiology, Faculty of Health Sciences, Flinders University, Adelaide, Australia $ School of Medicine, Faculty of Health Sciences, Flinders University, Adelaide, Australia

Key Words Connexin 26 Gene mutations Autosomal hearing loss

Connexin 26 mutations in autosomal recessive deafness disorders: A review Mutaciones de Conexina 26 en condiciones de sordera autoso´mica recesiva: Una revisioń Abstract

Sumario

This review explores the association between GJB2 gene mutations, encoding connexin 26 (Cx26), and nonsyndromic hearing loss. Connexins are proteins that form intracellular membrane channels and regulate ion movement between contiguous fluid spaces. A family of autosomal gene mutations has been identified that lead to abnormal connexin expression within the inner ear that are associated with hearing loss. The exact mechanism by which this link is elicited remains unclear. We aim to highlight the clinically underestimated prevalence of GJB2 gene mutations, to explore the influential role of ethnic diversity in mutation frequency, and to provide a framework for hearing specialists in considering the differential diagnosis of nonsyndromic hearing loss. By linking an observed phenotype associated with abnormal Cx26 expression to the current understanding of the biological and genetic basis underlying it will allow a more accurate clinical description of associated hearing loss, and therefore enable more effective patient management and genetic counselling.

Esta revisioń explora la asociacioń entre las mutaciones del gen GJB2, la codificacioń de conexina 26 (Cx26) y los trastornos auditivos no sindro´micas. Las conexinas son proteıńas que forman canales membranosos intracelulares y regulan el movimiento de los iones entre espacios lı´quidos contiguos. Se ha identificado una familia de mutaciones en genes autoso´micos que conducen a una expresioń anormal de la conexina, que afectan el oı´do interno y se asocian a hipoacusia. El mecanismo exacto por el que se produce este efecto auń no es claro. Nuestro propo´sito es destacar la prevalencia clıńicamente subestimada de las mutaciones del gen GJB2, explorar el papel que la diversidad e´tnica tiene sobre la frecuencia de tales mutaciones, y aportar un marco con el que los especialistas en audicioń puedan establecer el diagno´stico diferencial de la hipoacusia no sindro´mica. Relacionando un fenotipo observado que se asocie a la expresioń anormal de la Cx26, de acuerdo a las bases biolo´gicas y gene´ticas actualmente conocidas, permitira´ una descripcioń clıńica ma´s exacta del trastorno auditivo asociado, y por lo tanto, facilitara´ un ma´s efectivo manejo del paciente y un mejor consejo gene´tico.

Hearing impairment is the most common sensory disorder worldwide (Bitner-Glindzicz, 2002) and genetic inheritance represents a major source of auditory system dysfunction resulting in hearing loss. A number of gene mutations, some of which are yet to be definitively identified, have been associated with recessive hearing impairment by causing mechanical defects in the auditory system (Hardisty et al, 1999; Scott et al, 1998a). Mutations of the GJB2 gene (gap junction protein b2), encoding connexin 26 (Cx26) (Erbe et al, 2004), are the most common cause of hereditary, prelingual, nonsyndromic hearing loss (Oguchi et al, 2005; Seeman et al, 2005). While several mutations of the GJB2 gene have also been linked to syndromic forms of deafness such as Keratitis-Ichthyosis-Deafness (KID) syndrome (Richard et al, 2002; Yotsumoto et al, 2003), Vohwinkel’s syndrome (Common et al, 2005; Richard et al, 2004), and Bart-Pumphrey syndrome (Alexandrino et al, 2005; Richard et al, 2004) their details fall beyond the scope of the present work, and will not be considered further in this review. The link between GJB2 mutations and hearing loss is well established, although the precise strength of the correlations between genotype and phenotypical expression are not clear (Rabionet et al, 2002). Similarly, while many GJB2 mutations are widely understood to follow autosomal recessive inheritance

ISSN 1499-2027 print/ISSN 1708-8186 online DOI: 10.1080/14992020600582190 # 2007 British Society of Audiology, International Society of Audiology, and Nordic Audiological Society

Accepted: January 11, 2006

pathways, symptomatic expression in heterozygote individuals has been reported previously (Denoyelle et al, 1998; Morle et al, 2000), although recent advances in mutation analysis may have provided an explanation for such an occurrence (del Castillo et al, 2002; Lerer et al, 2001). Investigations of the epidemiology of abnormal Cx26 expression, their contribution to hearing loss, and cultural variations are discussed to determine the variability and validity of reported research findings and to provide insight for the practising clinician.

Connexins, connexons and gap junctions Twenty-one genes have been identified in the human genome coding for connexin protein expression (Sohl & Willecke, 2003). Hexamers of connexins join within the cellular plasma membrane to form a single connexon (del Castillo et al, 2003; Chang et al, 2003). Connexons from adjacent cells then unite, forming gap junctions, which serve as intercellular communication channels (Rabionet et al, 2002; Sabag et al, 2005; Segretain & Falk, 2004). Connexons themselves may be homomeric (composed of identical subunits) or heteromeric (of divergent subunits). Similarly, gap junctions may be homotypic (composed of two identical connexons) or hetero-

Dr Andrew P. Kurmis, School of Medicine, L5 Flinders Medical Centre, Flinders Drive, Bedford Park, South Australia, 5042, Australia. E-mail: [email protected]

Figure 1. A schematic cross-sectional representation of the inner ear, demonstrating the regions of connexin 26 expression (dashed arrows) and their role in fluid ion movement; adapted from Steel (1999). typic (composed of connexons with differing connexin isotypes) (Wei et al, 2004). Connexins and gap junctions are essential for normal auditory function (Martin et al, 1999) and are found abundantly in the epithelial and connective tissue cells of the cochlea (KammenJolly et al, 2001; Sabag et al, 2005) (Figure 1). Gap junctions provide intercellular bridges between adjacent cochlear cells (Kkuchi et al, 1995; Martin et al, 1999), and provide the structural basis for the recirculation of potassium ions between the fluids of the inner ear by controlling cellular ion exchange (del Castillo et al, 2003). The scala vestibuli, scala tympani, and scala media (in the cochlear duct), contain ion-rich fluids essential for auditory function. Under normal conditions, the endolymph of the scala media has a high concentration of potassium and a low concentration of both sodium and calcium, whereas the perilymph of the scala vestibuli and tympani have the reverse. Following transduction, electrolytes (in particular potassium ions that flow into the perilymph at the base of the hair cells) are recycled via the gap junctions to the endolymph via the stria vascularis and spiral ligament. In addition, connexons are also found as single units on the scala interface surface of epithelial cells, where they provide a direct potassium ion exchange pathway between cells and cochlear fluids, thus completing the communication pathway for ion recycling (Lefebvre & van de Water 2000). These processes maintain the balance and distribution of electrolytes necessary for normal auditory function within the cochlea.

Connexin 26 Cx26 is the most prevalent of the connexins cochlea (Martin et al, 1999). It has been the nonsensory epithelial cells around the including the supporting cells of the organ of

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expressed in the identified along outer hair cells Corti, inner and

outer sulcus cells, and the interdental cells of the spiral limbus. Connexin 26 is also expressed in the connective tissue of the inner ear (particularly in the strial basal and intermediate cells), the mesenchymal cells lining the scala vestibuli, and fibrocytes in the spiral limbus and ligament (Kikuchi et al, 1995). Clinical studies have suggested that mutations in the gene encoding Cx26 expression (GJB2 ) may disrupt the recycling of potassium ions to the endolymph, resulting in progressive intoxication of the organ of Corti (Lefebvre & van de Water 2000), leading to cellular dysfunction and ultimately cell death. The complex details of the pathophysiological basis underlying deafness in connexin-associated GJB2 mutations falls beyond the intended clinical scope of this review. Readers interested in an in-depth account of this topic are directed to a series of dedicated recent works, including: Chen et al, 2005; Piazza et al, 2005; Sun et al, 2005; and Zhao et al, 2005.

Mutations of GJB2 Approximately 90 GJB2 mutations leading to abnormal Cx26 expression have been reported and linked to hearing impairment, although the strength of their association is variable (Azaiez et al, 2004; Cryns et al, 2004). These mutations may be characterised as truncating or nontruncating (Azaiez et al, 2004). Truncating mutations lead to the loss of Cx26 expression in homozygotes, with heterozygotes often displaying reduced, although sometimes normal, expression. Truncating mutations involve nonsense (stop) mutations, splice-site, and frameshift mutations (caused by insertions or deletions). Nontruncating mutations include insertions and deletions which do not otherwise alter the open reading frame, and missense mutations (abnormal amino acid substitution) that may interfere with post-translational processing and intra-

International Journal of Audiology, Volume 46 Number 2

cellular trafficking of the resultant proteins (Martin et al, 1999). If successfully assembled into gap junctions, these abnormal proteins may result in nonfunctional channels, or channels with altered gating or trafficking of ions and signalling molecules (Beltramello et al, 2005; Martin et al, 1999; Mese et al, 2004). Up to 85% of abnormal Cx26 expression may be attributed to the 35delG mutation (Denoyelle et al, 1997; Estivill et al, 1998), a truncating deficit that introduces a premature stop codon (Martin et al, 1999; Reardon, 1998; Smith & van Camp, 1999). This mutation occurs in a series of six consecutive guanines in the Cx26 gene sequence, and as it is therefore not possible to determine exactly which base is deleted, this mutation is sometimes also referred to as 30delG (Denoyelle et al, 1997; Morell et al, 1998). Individuals homozygous for this mutation manifest a spectrum of clinical phenotypes ranging from a moderate to a profound hearing loss (Denoyelle et al, 1997; Green et al, 1999), suggesting a role for both genetic modifiers (Gerido & White 2004) and environmental factors. Among the nontruncating mutations, the mutation M34T (101T0/C), and its role in nonsyndromic hearing impairment, has been extensively investigated. This mutation was originally suggested to be an autosomal dominant mutation leading to hearing loss (Kelsell et al, 1997). However, doubt was cast upon this assertion by the identification of individuals heterozygous for this mutation but with normal hearing, and some considered this mutation to be a polymorphism only (Feldmann et al, 2004; Kelley et al, 1998; Scott et al, 1998b). This argument was countered by in vitro studies that have shown that M34T results in the inefficient formation of gap junctions, and that the resultant gap junctions are indeed functionally defective (Manthey et al, 2001; Thonnissen et al, 2002). Moreover, the identification of M34T in compound heterozygote individuals who are hearing impaired suggests that it is a recessive allele, and that its effects may be dependent upon the nature of the mutation in the second allele (Cucci et al, 2000). Additionally, the dominant effects of this mutation may be related to a deletion in the 5? UTR of the Cx26 gene (Houseman et al, 2001; Zoll et al, 2003). For several years, clinicians and researchers struggled to provide a valid explanation for the seemingly rare phenotypic expression of Cx26-related deafness, in otherwise heterozygote individuals (del Castillo et al, 2003; Santos et al, 2005). Based largely on family lineage studies, many concluded simply that while the majority of GJB2 mutations were observed to follow recessive inheritance, certain variants could exist in forms inducing dominant patterns of deafness. At the turn of the new century, a truncating deletion mutation in the GJB6 gene, Delta (GJB6 -D13S1830), which codes for connexin 30 (Cx30) and lies upstream from GJB2 on the long arm of chromosome 13, was identified (del Castillo et al, 2002; del Castillo et al, 2003; Lerer et al, 2001) which further complicated the contemporary state of understanding. Although closely related, this deletion does not directly affect the coding sequence of the GJB2 gene (Common et al, 2005). Both Cx26 and Cx30 proteins are expressed in the inner ear under normal conditions (Kelley et al, 1999; Lautermann et al, 1998; del Castillo et al, 2002). Compound heterozygosity, involving mutations in both GJB2 and GJB6 , results in hearing impairment with affected

Connexin 26 mutations in autosomal recessive deafness disorders: A review

individuals demonstrating a phenotype similar to GJB2 mutation homozygosity (Stinckens et al, 2004). It has since been suggested that the Delta (GJB6 -D13S1830) mutation has a relatively high carrier frequency, albeit variable between populations (del Castillo et al, 2003; Frei et al, 2004b; Pandya et al, 2003; Seeman et al, 2005), and may be a significant co-contributor to nonsyndromic deafness. Thus, it is now recognized that three distinct patterns of inheritance exist which may contribute to connexinrelated, nonsyndromic deafness: . homozygote autosomal recessive; . heterozygote (or rare homozygote) autosomal dominant; and . compound autosomal heterozygosity (i.e., concurrent defects in both Cx26 and Cx30 expression). The large number of mutations leading to abnormal Cx26 expression and function, and the examples discussed above, highlight the complexity of, and potential and variable effects of, these mutations and the resulting degree and configuration of associated hearing loss.

Prevalence of mutations in the Cx26 gene Studies to identify the prevalence and carrier frequency of mutations in the Cx26 gene (GJB2 ) associated with hearing loss have been performed worldwide. Cohn et al (1999) estimated that 1 in 1000 children have nonsyndromic recessive hearing loss, proposing that the most common cause is GJB2 gene mutations, accounting for 50% of these cases (i.e., 1 in 2000 children). These findings were supported by the 1999 study of Denoyelle et al, who carried out mutation analysis on 140 children affected by sensorineural hearing loss, and the recent similar work of Frei et al (2004a) who found that almost 50% of such subjects had abnormally expressed Cx26 proteins. A separate multicontinent analysis of families with severe and profound prelingual hearing loss from France, the United Kingdom, and New Zealand revealed that 50% of the families studied exhibited mutations in the gene encoding for Cx26. The 35delG mutation accounted for 70% of the GJB2 mutations, with a carrier rate as high as 4% in these populations. This would suggest that 35delG has prevalence equal to that of the DF508 mutation of the CFTR gene, which leads to cystic fibrosis, the most prevalent disease-causing genetic mutation yet identified in the human population (Marlin et al, 2001). In a broad epidemiological study, Orzan et al (1999) provided further evidence of this high prevalence, adding weight to the suggestion that mutations in the GJB2 gene are likely to be one of the most frequent hereditary defects in humans. In an Australian population study by Dahl et al (2001), 243 children attending hearing loss clinics were screened to determine the prevalence of GJB2 mutations and the frequency of relevant subtype classifications. Mutation analysis was performed on DNA extracted from the patients’ blood and revealed a heterozygous carrier rate of 1 in 54 for all GJB2 mutations and 1 in 100 for the specific 35delG. It was concluded from the high carrier frequency and prevalence of this mutation, that mutations in the GJB2 gene were likely to represent the most common cause of prelingual hearing loss in Australia. Statistical analysis of epidemiological survey data of patients obtained by Bitner-Glindzicz et al (2002) found that 50% of

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Clinical characterisation of the audiological phenotype that correlates with the Cx26 genotype remains unresolved (Rabionet et al, 2002), with variable audiological findings reported throughout the literature. To determine the genetic description associated with GJB2 mutations, Denoyelle et al (1999) examined 140 children affected by sensorineural hearing loss with an autosomal recessive mode of inheritance. Analysis of the inner ear defects of prelingually deaf children identified with GJB2 mutations (affected cases) was studied in comparison with prelingually deaf children without GJB2 mutations (nonaffected cases). Audiometric configuration testing demonstrated that the hearing loss was flat in those affected, whereas U-shaped curves and flat sloping losses were observed in the nonaffected individuals. Vestibular function in those affected appeared normal in this study. However, those unaffected by the Cx26 abnormalities presented evidence of vestibular abnormalities. Additional examination of those affected was conducted to determine the clinical characterisation of GJB2 mutations. Results indicated the hearing loss to be prelingual, bilaterally symmetrical, ranging from mild to profound, affecting all frequencies, variable between siblings, and generally not progressive. However, the authors speculated that if two audiometric tests were done at least 10 years apart, the progression of the hearing loss resulting from mutations in the GJB2 gene could be followed and that a large number of children would show progressive hearing loss. The work of Cohn et al (1999) examined 46 individuals of European decent with GJB2 mutations to determine the degree, stability, and symmetry associated with this genotype. This research, and that of others, also revealed the hearing loss to be prelingual, symmetrical, effecting all frequencies equally, and of variable severity ranging from mild to profound (Cohn et al, 1999; Orzan et al, 1999) indicating that no consistent audiological phenotype correlated with this genotype (Cohn et al, 1999; Rabionet et al, 1999). However, in contrast to previous studies,

the results of this investigation indicated that the hearing loss was progressive in a number of cases, with a loss of at least 1 decibel of pure-tone average per year. The progression observed in this study was apparent in one third of cases and was not related to gender or to the type of mutation present. However, the sample size was limited for a comparison between genotypic groups. The findings of Orzan et al (1999) were at odds with the suggestion of progressive hearing loss in the investigation of 94 nonsyndromal hearing-impaired children and young adults. Followed up after a mean period of ten years, the authors found no evidence of progressive hearing loss. This observation was supported by the subsequent work of Bitner-Glindzicz et al (2002) who extensively studied the clinical phenotype of connexin disorders, concluding that in the majority of cases the hearing loss was stable, but conceded that progression had been demonstrated in previous studies. The work of Orzan et al (1999) did, however, find agreement with the reported outcomes of Cohn et al (1999), in being unable to demonstrate a clinical phenotype with the given gene mutation. In addition, substantial variability in the degree of hearing impairment was noted amongst individuals in both interfamilial and intrafamilial cases. This diversity in clinical presentation provides further evidence to support the suggestion that both genetic modifiers and environmental factors play influential roles in determining the degree and configuration of consequential hearing loss (Marlin et al, 2005), confounding both acute diagnosis and projected rehabilitation outcomes. Clinical characterisation of the degree and nature of the hearing loss was largely variable between studies. One hypothesis to explain these findings is that yet-to-be-described co-existing mutations may affect the potential of the gene and influence the severity of hearing impairment associated with GJB2 mutations (Cohn et al, 1999; Orzan et al, 1999; Rabionet et al, 2000). On this basis, results that indicate progressive hearing loss should be validated with periodical reassessment to confirm their accuracy and rule out confounding factors that may contribute to a progressive deficit of hearing. Data that conclusively establishes the progressive nature of Cx26 associated hearing disorders has high clinical significance in instances where an individual with a mild or moderate degree of hearing loss is seen to progress to a severe or even profound loss. Clearly, such understanding greatly impacts upon patient management and the nature of interventional strategies employed, ultimately influencing outcome efficacy. While there has been marked progress in the identification of GJB2 gene mutations directly associated with hearing impairment, the multifactorial influence of concurrent mutagenic anomalies resulting in hearing loss are yet to be fully described (Hardisty et al, 1999). This is inherently problematic for those researchers using small sample sizes and thus obtaining limited data cohorts. Given that only a small number of auditory links to genetic mutations have been definitively confirmed to date, the likelihood of identifying previously undescribed mutations is low. In order to allow more precise genotype-phenotype correlation, improvement in genetic counselling, provision of new data for evaluating different forms of hearing loss and accurate diagnosis and effective treatment (Orzan et al, 2002), a more complete picture must be established by further advances in the identification of gene mutations associated with hearing loss (van Hauwe et al, 1999).

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recessive nonsyndromic hearing loss in the Caucasian population could be attributed to mutations in the gene encoding Cx26. This research demonstrated that most of these recessive forms of hearing impairment cause a phenotypically identical severe-toprofound prelingual hearing loss. The significance of broadening this data to the wider community suggests both a high prevalence rate and carrier frequency of GJB2 mutations within the population. The clinical significance of extrapolating such data holds immediate relevance for hearing specialists in instances where the cause of hearing loss is seen to be sporadic (Feldmann et al, 2004; Estivill et al, 1998) or has otherwise unknown aetiology. With increasing societal awareness of genetic disorders, hearing-impaired individuals and their families are increasingly eager to learn the cause of their auditory deficit and to confirm or exclude differential diagnoses of genetic origin. Thorough investigation of the aetiology of hearing loss and identification of heterozygote and homozygote individuals by genetic testing is a necessary precursor for accurate and effective diagnosis, instigation of appropriate clinical management, patient-targeted genetic counselling, and monitoring of transmission and penetrance (Estivill et al, 1998; Oguchi et al, 2005).

Phenotypical expression of mutations in the Cx26 gene

Ethnic diversity The prevalence of specific mutations expressed within different cultural and racial groups suggests that certain mutations may be influenced by ethnic background and are, therefore, likely to vary between populations (Andrea et al, 2002; Reardon 1998). Rabionet et al (2000) examined mutations in Cx26-expressing gap junction genes and determined that certain subtype mutations are particularly common among specific populations, describing a positive correlation with background ethnicity. The 35delG mutation showed a high prevalence among Caucasian populations (Marlin et al, 2005) while the 235delC mutation was found predominantly within individuals of Asian decent. In addition, the carrier frequencies in these populations varied greatly (between 1 in 30 and 1 in 75 respectively). Furthermore, this disparity has been supported by subsequent epidemiological studies. Liu et al (2002) studied the prevalence of GJB2 mutations in the Chinese population, concluding that 235delC was the most frequent cause of recessive hearing impairment among this racial group, not 35delG, which otherwise accounts for up to 70% of cases in Caucasian populations (Marlin et al, 2005). Similar to these findings, Wang et al (2002) who studied a cohort of 169 Asian children with prelingual deafness and Abe et al (2000) who studied 35 Japanese families with bilateral sensorineural hearing loss, also found the 235delC defect to be most prevalent mutation affecting Cx26 expression amongst this population, with no evidence of the 35delG mutation. The subsequent investigation by Andrea et al (2002), in a similar population demographic, also failed to identify the presence of the 35delG mutation, despite the apparent familial heriditary communication of an autosomal hearing loss pattern. Similar investigations have suggested ethnic association to other specific GJB2 mutations, including high prevalence of: . the W24X mutation among Spanish/Slovak Romany (gypsy) (Alvarez et al, 2005; Minarik et al, 2003) and Indian (Maheshwari et al, 2003) populations; . R143W mutation among isolated African subgroups (Brobby et al, 1998; Hamelmann et al, 2001); and . 167delT among Ashkenazi Jews (Bors et al, 2004; Lerer et al, 2000; Morell et al, 1998). These mutations have been estimated to account for 79%, 91%, and 70%, of recessive hearing impairment amongst each of the respective ethnic groups. Beyond simply identifying specific gene mutations prevalent amongst different populations, Bitner-Glindzicz et al (2002) also examined the link to ethnicity and racial background, determining that the demographic spread of specific connexin mutations could be broadly defined based on continental region. Their findings indicated that the 35delG is the most prevalent mutation in Caucasian populations of Europe, America, and the Mediterranean, whereas the 235delC deletion was more prevalent in the East Asian populations. Based on these studies, it has been suggested that this difference may be attributable to a historical founder effect (Andrea et al, 2002; Bitner-Glindzicz 2002; Tekin et al, 2001). The high frequency of GJB2 mutations causing hearing loss in both Caucasian and Eastern Asian populations is noteworthy and hearing specialists should be aware of its likelihood as a


potential differential diagnosis in the setting whereby the general aetiology of prelingual hearing loss is otherwise unknown.

Conclusions It has been demonstrated that GJB2 gene mutations play a vital role in the pathophysiology of Cx26-associated, nonsyndromic, hearing impairment. Recognising the prevalence of GJB2 mutations and early identification of individuals exhibiting this genotype allows the clinician to consider the role of this defect in prelingual hearing loss of unknown aetiology. Previous research indicates inconsistent findings in genotypephenotype correlation between GJB2 gene mutations and associated hearing loss. In addition, a definitive clinical description of the type, degree, and nature of the hearing loss associated with abnormal Cx26 expression remains undefined. It has been theorized that the variable clinical presentations may be attributed in part to factors such as ethnic diversity in the expression of Cx26 defects, or to as-yet-unidentified confounding co-existing gene mutations or genetic modifiers. The clinical phenotype is further complicated by the role of environmental factors, which may be influential in varying the degree of hearing impairment. These inconsistencies necessitate the need for future, comprehensive research to allow a definitive description of the pathoclinical development of hearing loss associated with GJB2 mutations. Given the current state of scientific disagreement, further longitudinal research is also warranted to confirm either the presence or absence of progression in hearing impairment associated with GJB2 mutations. Information gained from such investigations would facilitate the instigation of more consistent and appropriate clinical management, including patient-focussed genetic counselling.

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Apps/Rankin/Kurmis

Additional resources For further information on the current advancements in the field of connexin-related hearing research and disorders, interested readers are directed to the following online resources: The Connexin-deafness homepage (http://davinci.crg.es/deafness/); and The Hereditary Hearing Loss Homepage (http://webhost. ua.ac.be/hhh/). Both provide easily accessible, regularly updated, evidence-supported information which may prove beneficial for clinical settings in guiding the management of patients with known, or suspected, connexin-related hearing loss.

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