Loss of function mutations of the GJB2 gene detected in patients with DFNB1-associated hearing impairment

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Abstract

Mutations in GJB2, which encodes the gap junction protein connexin 26 (Cx26), are one of the major causes for inherited and sporadic nonsyndromic hearing impairment. This study aimed to functionally characterize more frequent GJB2 mutations identified in patients showing nonsyndromic hearing impairment. Following injection of wild type and mutated cRNA in Xenopus oocytes, Cx26 hemichannel activity was measured by depolarization activated conductance in noncoupled oocytes. All mutants showed a partially or completely defective phenotype, except V27ICx26, a polymorphism tested as positive control. Coexpression of wild type and mutant Cx26 injected at equimolar levels revealed that p.M34T, p.V37I and p.I82M, but not p.G59V, p.L90P, p.R127H and p.R143W exert a dominant inhibitory effect. When coexpressed with Cx30, a connexin partially colocalized with Cx26 in the cochlea, all mutants had a dominant behavior. This study provides data that might be important for the improvement of genetic diagnosis and counseling for patients with hearing impairment.

Introduction

Nonsyndromic neurosensory hearing impairment is the most common human sensory disorder. Approximately one in thousand children is born with prelingual hearing loss, and in about 50% of the cases, the disease is inherited (Fraser, 1971, Morton, 1991). The pattern of inheritance is autosomal recessive (70%), autosomal dominant (15%), X-linked (1–3%) and mitochondrial (<1%). To date, more than 70 loci involved in nonsyndromic forms of deafness have been identified (http://dnalab-www.uia.ac.be/dnalab/hhh/). A single locus, DFNB1, accounts for a major proportion of nonsyndromic autosomal recessive deafness. The most frequently involved gene in DFNB1 is GJB2 which encodes the protein connexin 26 (Cx26).

Cx26 is a member of a family of transmembrane proteins that permit intercellular communication (Kumar and Gilula, 1996, Lee and Rhee, 1998). Connexins have four transmembrane domains connected by two extracellular and one intracellular loop, with the amino and carboxy termini in the cytoplasm. Six connexins assemble to form a hexameric hemichannel (connexon) that can aggregate with another connexon present in the membrane of neighboring cells to form a functional gap junction (Kumar and Gilula, 1996) allowing the passage of ions, second messengers and metabolites between cells (Nicholson et al., 2000). About 20 connexins have been identified in the human genome. The formation of multiple heteromeric hemichannels and heterotypic gap junctions may provide charge and size selectivity of the intercellular channels.

In the inner ear, Cx26 is expressed in the epithelial and the connective cellular network, and it has been suggested to play a role in endolymph potassium recycling (Kikuchi et al., 2000). Targeted ablation of Cx26 in the epithelial network demonstrated its essential role for cochlear function and cell survival (Cohen-Salmon et al., 2002).

The most common inherited mutation in the GJB2 gene is c.35delG (Guilford et al., 1994). This deletion generates a frameshift leading to a premature stop codon and a nonfunctional protein. While the c.35delG mutation shows highest frequencies in countries adjacent to the Mediterranean Sea (70%) (Cryns et al., 2004), its allele frequencies are lower in other parts of the world. Numerous additional mutations in the Cx26 gene have been described so far, most of which show very low frequencies (<0.1%) among deaf populations (http://www.crg.es/deafness). The functional implications of most mutants and often also the mode of inheritance are still elusive. This fact particularly aggravates genetic diagnosis and counseling for patients with hearing impairment which are offered by many clinics and other facilities in the meantime.

In this study, we applied heterologous expression in Xenopus laevis oocytes and subsequent dual electrode voltage clamp to analyze the electrophysiological properties of four mutations of questionable functionality (p.G59V [c.176C > G], p.I82M [c.246C > G], p.R127H [c.380G > A] and p.R143W [c.427C > T]), three mutations showing higher allele frequencies (p.M34T [c.101T > C], p.V37I [c.G > A], p.L90P [c.269T > C]) and the polymorphism p.V27I [c.79G > A] as a positive control.

Section snippets

Constructs and site-directed mutagenesis

The GJB2 coding region isolated from healthy individuals and patients was subcloned into the HindIII and BamHI sites of pSP64 Poly(A) expression vector (Promega GmbH, Mannheim, Germany). The p.V27I, p.M34T, p.V37I, p.G59V, p.I82M, p.L90P and p.R143W Cx26 mutants were generated by site-directed mutagenesis (GeneEditor™ in vitro Site-Directed Mutagenesis System, Promega GmbH, Mannheim, Germany) with the primers mut_V27I (5′ CTGGCTCACCATCCTCTTCATTTTTC 3′); mut_M34T (5′ CGCATTACGATCCTCG 3′);

Mutations

Among the many mutations identified, eight were selected for functional analysis due to their more frequent occurrence and thus higher clinical relevance. Mutations p.V27I [c.79G > A], p.M34T [c.101T > C], p.V37I [c.G > A], p.I82M [c.246C > G], p.L90P [c.269T > C] and p.R143W [c.427C > T] are present in the transmembrane domains, p.G59V [c.176C > G] is positioned in the first extracellular loop and p.R127H [c.380G > A] in the intracellular loop (Fig. 1). All mutations identified in patients

Discussion

The study describes the functional implications of eight point mutations present in the coding region of the GJB2 gene. While all mutants were translated properly, only the V27ICx26 mutant retained functional activity. Several lines of evidence suggest that this mutation is a polymorphism (Kelley et al., 1998, Abe et al., 2000, Kudo et al., 2000, Park et al., 2000). Therefore, it had been included in this study as a positive control. Western blots of plasma membrane preparations showed that all

Acknowledgments

The authors acknowledge the assistance of P. Heidrych in cloning several constructs and of B. Haack in generating some mutants. They are grateful to B. Noll for expert technical assistance and to T. Loch and L. Subasic for meticulous preparation of the manuscript. The study was supported by the intramural project “fortuene no. 1028-0-0” to S. Kupka and F. Lang.

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