Human Molecular Genetics, 2001, Vol. 10, No. 25 2945-2951
© 2001 Oxford University Press
Mutations in GJA1 (connexin 43) are associated with non-syndromic autosomal recessive deafness
1Department of Human Genetics, Medical College of Virginia of Virginia Commonwealth University, Richmond, VA 23298-0033, USA, 2Department of Otolaryngology, University of Miami, Miami, FL 33101, USA, 3Department of Otolaryngology, Harvard Medical School and Massachusetts Eye and Ear Infirmary and 4Neurology Department, Massachusetts General Hospital and Neurobiology Department, Harvard Medical School, Boston, MA 02114, USA and 5Department of Biology, Gallaudet University, 800 Florida Avenue NE, Washington, DC 20002, USA
Received August 21, 2001; Revised and Accepted September 24, 2001.
DDBJ/EMBL/GenBank accession no. X17027459.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in four members of the connexin gene family have been shown to underlie distinct genetic forms of deafness, including GJB2 [connexin 26 (Cx26)], GJB3 (Cx31), GJB6 (Cx30) and GJB1 (Cx32). We have found that alterations in a fifth member of this family, GJA1 (Cx43), appear to cause a common form of deafness in African Americans. We identified two different GJA1 mutations in four of 26 African American probands. Three were homozygous for a Leu
Phe substitution in the absolutely conserved codon 11, whereas the other was homozygous for a ValAla transversion at the highly conserved codon 24. Neither mutation was detected in DNA from 100 control subjects without deafness. Cx43 is expressed in the cochlea, as is demonstrated by PCR amplification from human fetal cochlear cDNA and by RT–PCR of mouse cochlear tissues. Immunohistochemical staining of mouse cochlear preparations showed immunostaining for Cx43 in non-sensory epithelial cells and in fibrocytes of the spiral ligament and the spiral limbus. To our knowledge this is the first 
connexin gene to be associated with non-syndromic deafness. Cx43 must also play a critical role in the physiology of hearing, presumably by participating in the recycling of potassium to the cochlear endolymph. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Hearing loss is an important cause of human morbidity that eventually affects at least 15% of the population. The incidence of profound prelingual deafness is approximately one per 1000 at birth and includes many known genetic and environmental causes (1). Dramatic progress has been made in the localization of 71 loci that can cause dominant or recessive forms of non-syndromic deafness, and, of these, 22 have been cloned (G.Van Camp and R.J.H.Smith, Hereditary Hearing Loss Homepage. World Wide Web address: http:dnalab-www.uia.ac.be/dnalab/hhh/). Mutations in GJB2 [connexin 26 (Cx26)] can give rise to recessive non-syndromic deafness or, more rarely, dominantly transmitted deafness (2). Mutations involving this locus are the most common cause of deafness in many populations. One particular mutation, 30delG or 35delG, is especially common, accounting for two-thirds of all pathologic Cx26 mutations in most populations. Cx31 (GJB3) is also expressed in the cochlea and is the cause of deafness in several Chinese pedigrees (3,4). Likewise, mutations in GJB6 (Cx30) are the apparent cause of deafness in a few small families (http:dnalab-www.uia.ac.be/dnalab/hhh/) (5), whereas mutations in GJB1 (Cx32) lead to an X-linked form of Charcot-Marie-Tooth disease (CMTX) associated with hearing loss (http:dnalab-www.uia.ac.be/dnalab/hhh/) (6).
Connexins are the proteins which line the intercellular channels or gap junctions that connect adjacent cells and facilitate the exchange of ions, secondary messengers and small molecules (7). The numbers assigned to the various connexins refer to their approximate molecular weight. In addition, the human connexins can be classified into sub groups: a, b, and g, based on similarities at the nucleotide and amino acid level. Six connexin molecules assemble to form a half-channel or connexon, which docks with its counterpart in an adjacent cell to form a complete intercellular channel or gap junction. In the sensory epithelia of the inner ear, it is thought that an important function of these gap junctions is to facilitate the recycling of potassium ions from the hair cells back into the cochlear endolymph during auditory transduction (8). The involvement of several members of the connexin gene family in deafness suggests that others should be considered as candidates for non-syndromic deafness. To pursue this possibility, we initiated a study to determine whether there are mutations in GJA1, encoding Cx43, which lead to deafness and to document the expression of Cx43 in the inner ear.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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In total, 26 deaf African American probands were screened for Cx43 mutations, including five from multiplex and 21 from simplex families. In addition, screening was conducted on several control groups including 40 African American subjects with normal hearing. Four of the deaf probands who were born to non-consanguineous parents in three simplex (NSDF 056, 062 and 205) and one multiplex family (NSDF 158) were found to have pathological mutations in GJA1 (see below). Clinical and audiological evaluation showed that all four probands had a profound bilateral non-syndromic sensorineural loss that appeared to have been congenital with no evidence for progression, which is the most common type of audiograms in non-syndromic recessive deafness (9). Physical examination of probands in families NSDF 056, 062 and 205 showed no dysmorphic features of the face, jaw, palate, or external ears with no history of vertigo or evidence for vestibular dysfunction. No other clinical abnormalities were noted. Likewise, the proband in family NSDF 158 had no reported abnormalities. In family NSDF 158 (Fig. 1A), there is a history of a brother who, by report, is also deaf but not available for testing.
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The DNA samples from probands were screened for Cx43 mutations by a single-strand conformation polymorphism (SSCP)-sequencing approach after initial testing for mutations in Cx26, Cx30 and Cx31 with negative results (data not shown). GJA1 contains a single, uninterrupted ORF of 1148 nt (10). Eight pairs of overlapping primers covering the entire coding region of GJA1 were used for the SSCP analysis (see Materials and Methods). Variant SSCP patterns for the PCR of primer 1FR were observed in four African American patients (data not shown). The four affected subjects were all homozygous for one or the other of two variants, whereas the remaining 22 black probands and 40 black control subjects with normal hearing all had normal SSCP patterns. Sequence analysis of the SSCP variants demonstrated that all three deaf individuals from families NSDF 056, 062 and 205 were homozygous for a 30C
T transversion which leads to LeuPhe substitution at codon 11 (L11F) (Fig. 1B). In family NSDF 205 (Fig. 1A), a hearing sister (II.2) and the mother (I.2) were found to be heterozygous for the L11F mutation. The other mutant L11F allele was apparently inherited from the unaffected father, who was not available for testing. In family NSDF 158, the affected individual (II.1, Fig. 1B) was homozygous for a 71TC change which creates a ValAla substitution at codon 24 (V24A). To exclude the possibility that these mutations are simply polymorphisms, we have studied samples from 100 unrelated control subjects (including 40 African Americans and 60 from other populations) for both mutations. Neither of the two mutations was detected in the control panel. The mutations are believed to be pathological, first, because of their location and conservation (see below) and, secondly, because the changes were not observed in a series of normal controls. To search for the mutations in other racial or population groups, we screened 510 deaf probands from other ethnic backgrounds obtained in part from the National DNA Repository for Genetic Deafness at the Medical College of Virginia (11). None carried the two mutations found in our patients.
Like other connexins, Cx43 consists of four transmembrane domains linked by one cytoplasmic and two extracellular loops, with cytoplasmic C- and N-terminal ends (Fig. 2). GJA1 is located on human chromosome 6q21–q23.2 (10). Currently, none of the known loci for non-syndromic deafness maps to this chromosomal region. The only human disease previously attributed to GJA1 was a recessive form of lateralization defects (heterotaxy) in which the substitutions of phosphoratable Ser or Thr residues in the cytoplamic tail domain were reported (12). However, intensive efforts to confirm these findings in a large number of patients have failed to reveal similar findings (13). To date, mutations in six connexin genes are known to cause a variety of clinical abnormalities (14). In addition to Cx43, four other connexins are associated with hearing impairment (http:dnalab-www.uia.ac.be/dnalab/hhh/).
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The altered Leu residue in families NSDF 056, 062 and 205 lies in the cytoplasmic N-terminal domain (NT, Fig. 2) in a stretch of 14 residues that exhibit a high degree of evolutionary conservation across species in all Cx43 genes studied to date as well as in other members of the connexin family (Fig. 3). This domain plays a role in the voltage-gating of gap junction channels and in the insertion of connexins into membranes (15,16). In GJB2 (Cx26), more than 50 different mutations have been identified in patients with non-syndromic deafness, including missense, nonsense and small deletions or insertions across all protein domains. More than five of these Cx26 and two Cx30 mutations lie within the N-terminal domain, and a number of mutations in this region occur in Cx32 in patients with CMTX as well as in Cx31 in patients with erythrokeratodermia variabilis. Moreover, mutations in residues close to the Leu at codon 11 altered in the families reported here, namely T8 and G12 in Cx26, as well as T5 and G11 in Cx30, have been reported previously in patients with hearing impairment (http:dnalab-www.uia.ac.be/dnalab/hhh/) (5). Mutations in the N-terminal domain of Cx32 or Cx26 have been shown to either reverse the gating polarity or cause defective trafficking of proteins (15,16). Interestingly, a substitution of the Gly residue 12 to Ser (G12S) in Cx32, has been shown to cause defective trafficking with no measurable conductance (16). Therefore, it is highly likely that the Leu11Phe substitution we observed could alter the structure of the N-terminus, in a way that might block the assembly, insertion or cellular processing of the Cx43 subunit. The identification of pathological mutations associated with hearing loss in the homologous region of several other connexins supports the view that this domain is of functional importance for the Cx43 protein.
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The Val24Ala substitution in family NSDF 158 occurs in the first putative transmembrane domain (M1) of Cx43, which is critical for the normal formation of the pore of gap junction channels (13,17). Moreover, this Val is located at the NT/M1 boundary (Fig. 2) and is conserved across connexins from many species, whereas others contain a structurally conservative isoleucine substitution at this position (Fig. 3). Amino acids at this transmembrane border form part of a charged complex that is a component of the connexin voltage sensor (15). Non-conservative connexin mutations affecting this Val and adjacent residues are pathologic (14). Interestingly, two substitutions involving this residue, Val23Ala and Val23Glu in Cx32, have been implicated in CMTD (18). A mutation involving the adjacent Trp24 in Cx26 has also been reported previously in patients with deafness (http:dnalab-www.uia.ac.be/dnalab/hhh/). The mutation in the neighboring S26 residue in Cx32 reduces channel permeability by decreasing the pore size (19). Since the Val24Ala substitution that we observed introduces a less hydrophobic residue at the membrane cytoplasm boundary (Fig. 2), functionally significant changes in the structure of M1 might be expected.
To support the causal relation between Cx43 mutations and deafness in our patients, we characterized the expression of Cx43 by RT–PCR of mouse cochlear tissues and by PCR amplification from human fetal cochlear cDNA (data not shown). We detected GJA1 expression in mouse cochlear tissues, and were able to amplify Cx43 in human fetal cochlear cDNA from the Morton cochlear library.
Using two independent affinity purified antibodies to Cx43, we performed immunohistochemical localization of the Cx43 molecule in sections of mouse cochlea. The pattern of staining for Cx43 was dependent upon fixation conditions of the tissue. In tissue fixed with formalin\glutaraldehyde, staining was strongest and most consistently present in non-sensory epithelial cells (closed arrows, Fig. 4A), in the spiral ligament and in the spiral limbus (open arrows, Fig. 4A). In the ligament, type I fibrocytes were much more darkly stained than adjacent type III fibrocytes. Other positive staining cells included the interdental cells of the spiral limbus and outer sulcus and the root cells beneath the spiral prominence. The pattern of staining was essentially the same with both antibodies, with the Zymed antibody usually providing superior results. A similar distribution of positive staining cells was also found in rat and guinea pig cochlea (data not shown). In tissue fixed in ethanol\acetic acid, staining was limited to the apices of supporting cells of the organ of Corti (Fig. 4B and C). Previous work on the distribution patterns of Cx26, Cx30 and Cx31, including ultrastructural confirmation of the locations of gap junctions within the cochlea, led to the conclusion that gap junctional systems provided the pathways by which potassium ions are recirculated from the organ of Corti to the stria vascularis (8). According to this model, the potassium ions that enter hair cells in response to acoustic activation of the organ of Corti are expelled basolaterally by hair cells and are accumulated by supporting cells. From the organ of Corti the ions moved laterally through gap junctions to the spiral ligament, where they expelled from the terminal cells of the epithelial gap junctional system and are accumulated by type II fibrocytes, from which they are transported to the stria vascularis via the gap junctional system of the connective tissue cells of the spiral ligament. Cx26 and Cx30 are present in both the epithelial and connective tissue gap junctional systems and mutations in Cx26 and Cx30 genes can result in sensorineural hearing loss (2,5). On the other hand, Cx31 is present primarily in type II fibrocytes (20) and mutations in that gene can also result in hearing loss (3,4). Apparently failure of gap junctions that are either distributed throughout the cochlea or are limited to key cells can lead to hearing loss. The present results indicate that Cx43 is either present in a highly restricted group of supporting cells (Fig. 4B and C) or is found throughout non-sensory epithelial cells and in type I fibrocytes. Apparently this issue will have to be resolved by using in situ hybridization. In either case, it appears that the contribution of Cx43 to the function of cochlear gap junctions is critical for normal hearing. These immunohistochemical data are consistent with the idea that the Cx43 mutation is the underlying cause of deafness in our four families.
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To gain further insight into the ontogeny of GJA1 expression, we have characterized the expression profiles of mouse cochlea at three developmental stages by Genechip analysis (21). Cx43 was expressed in the mouse cochlea at P1, P2 and P32, and at significantly higher levels in P32 (>2-fold greater than at P1 or P2). Cx43 was also expressed during early development of the mouse utricle (Z.-Y.Chen, unpublished data). Cx43, Cx30 and Cx26 all increased their expression from P1 to P32, with the highest increase for Cx30 (>5-fold) and the smallest for Cx26 (marginally). In the developing utricle, in contrast to Cx43, both Cx26 and Cx30 are up-regulated postnatally. The observed variation in the expression of the three connexins may indicate that they have distinct but overlapping functional roles in the inner ear.
The present data demonstrate that Cx43 is responsible for a recessive form of non-syndromic deafness. The identification of mutations in four of 26 African American families suggests that Cx43 mutations could be a common cause of deafness in this group, and like Cx26, its prevalence in the United States could be influenced by the mating structure of the deaf population (22). Additional data will be required to determine the frequency of GJA1 mutations as a cause for deafness in other populations. Since the connexins function by forming heteromeric or homomeric connexons, it is interesting to notice that all pairs of the mutations identified in deaf patients to date have involved changes in the same connexin subunit. It seems possible that mutations involving different but interacting connexins may exist in some deaf individuals. Our findings of mutations in Cx43 in four deaf families and information on its localization and the expression profile should aid identification of epistatic interactions of this type.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Clinical evaluations of patients
Probands were ascertained through the Genetics Clinic at Virginia Commonwealth University in Richmond and the Genetics Program at Gallaudet University in Washington, DC. Informed consent was obtained from all participants or from parents of patients younger than 18 years of age. In total, 26 African American probands with recessive or sporadic non-syndromic deafness were included in this study. All probands had profound prelingual deafness. The clinical and family history was obtained on each proband and complete physical examinations were performed on three of the four probands by one of the investigators. The hearing of all affected individuals in the present series was examined using pure tone audiometry. Air conduction thresholds were measured at 250 Hz, 500 Hz, 1 kHz, 2 kHz, 4 kHz, 6 kHz and 8 kHz. DNA samples were obtained from peripheral blood in the study families and controls.
SSCP and sequence analysis of Cx43
GJA1 has a single exon with a coding region of 1148 bp. Eight pairs of overlapping primers were designed from the complete coding and flanking UTR sequence of Cx43 (GenBank accession no. XM027459) using the Primer 3 program (http://www-genome.wi.mit.edu/genome-software/other/primer3.html). The amount of overlap between each of the primer pairs ranged from 34 to 65 bp. Primer sequences for the SSCP screen of Cx43 will be provided by request. The coding region of the Cx43 gene was amplified from genomic DNA by PCR using the above primers. The amplification conditions were: 95°C for 5 min, then 30 cycles of 95°C for 1 min, 60°C for 1 min and 72°C for 1 min, with a final extension for 5 min at 72°C.
For mutation analysis, the PCR products were initially run on a 1 mm thick 8% non-denaturing polyacrylamide gel (acrylamide:N,N'-methylene bisacrylamide, 49:1) at 4°C. SSCPs were detected using silver staining as previously described by Liu et al. (23). Direct sequencing of PCR products from patients with SSCP variants was then performed on both strands using the fluorescent dideoxy terminator method and an ABI 377 DNA sequencer.
RT–PCR
We performed reverse transcription reactions with the Superscript reverse transciptase (Gibco BRL, Boston, MA) following the manufacturer’s protocol. All soft tissues from cochleas of young male CBA/CaJ mice were collected. Total RNA was isolated using TRIzol reagent (Gibco BRL) in accordance with the manufacturer’s directions. PCR conditions were: 93°C for 2 min, followed by 39 cycles of 93°C for 1 min, 56°C for 1 min and 72°C for 1 min. The final primer extension was at 72°C for 7 min. The primers used for RT–PCR were: forward 5'-TGCGGTCTACACCTGCAAGA-3'; reverse 5'-ACCAAGGACACCACCAGCAT-3'.
Immunohistochemical analysis
CBA/CaJ mice were deeply anesthetized and perfused intracardially with phosphate buffered saline (PBS) and then 10% formalin in PBS. The stapes was removed, the round window pierced and formalin gently perfused through the cochlear scalae. In some cases the fixative also included 0.1% glutaraldehyde. In other cases, no aldehyde fixative was used. Instead, following exsanguination with PBS, a solution of 50% ethanol and 5% acetic acid was perfused through the cochlear scalae. In all cases, the head was immersed in fixative for 2 h. Then a block of tissue including the temporal bones was removed from the skull and placed in a large volume of EDTA (pH 7.0) and gently agitated for 10–14 days until the bone was decalcified. The tissue was then dehydrated and embedded in paraffin.
Serial 8 µm sections were mounted on slides. Selected slides were dewaxed in xylene, hydrated and immunostained for Cx43. Primary antibodies included two well characterized affinity-purified rabbit antibodies (Zymed Labs, San Francisco, CA and a gift antiserum Petunia from Dr David Paul). Following 30–60 min in 5% normal horse serum to block non-specific IgG binding, serial dilutions of the primary antibodies were performed and the tissue sections bathed in primary antibody overnight at room temperature. A biotinylated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch Labs, West Grove, PA) was applied for 1 h, followed by an avidin–biotin–HRP complex (Standard ABC kit; Vector Laboratories, Burlingame, CA) for 1 h, biotinylated tyramine/H2O2 for 10 min (24), ABC solution for 30 min and then diaminobenzidine/H2O2 for 2 min. Copious washing in PBS followed each step.
Following the final step, sections were dehydrated and coverslipped in Permount. Inner ears of rat and guinea pig were also immunostained using the same protocol.
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ACKNOWLEDGEMENTS |
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We would like to thank the families for their contribution to this study. We thank Dr C.C.Morton for providing human fetal cochlear cDNA and Wanda Hunt for her technical help. This work was supported by NIH grants DC 05575 and DC 04530 to X.Z.L., DC 02530 and DC 04293 to W.E.N., DC 03929 to J.A. and DC 04546 to Z.Y.C.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Department of Otolaryngology (D-48), University of Miami, 1666 NW 12th Avenue, Miami, FL 33136, USA. Tel: +1 305 243 4923; Fax: +1 305 243 4925; Email: xliu@med.miami.edu
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