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At least 19 recordsLinked to original sources
PubMed2005

Gene structure and promoter analysis of the human GJB6 gene encoding connexin 30.

Connexins (Cx) are the protein subunits of gap junctions, which play an important role in cell-to-cell communication. We characterized the genomic structure of the human GJB6 gene, encoding connexin 30 (C x 30), and showed that it differs from most connexin-encoding genes. GJB6 presents six different exons, some of which can be alternatively spliced. We also mapped a basal promoter sequence active in a human keratinocyte cell line which responds to the activation of the EGF receptor. One of the non-encoding exons of GJB6, which has been described in brain C x 30 cDNA, was not found in cDNA obtained from human keratinocytes, suggesting tissue-specific splicing.

5' Flanking Region↗
PubMed2002

Functional studies of human skin disease- and deafness-associated connexin 30 mutations.

Connexin 30 (Cx30) is a component of the gap junction complex. Dominant and recessive mutations in the GJB6 gene encoding Cx30 are associated with a variety of human inherited diseases primarily affecting the epidermis, hair, nail, and/or the inner ear. The underlying mechanism of disease associated with different GJB6 mutations such as the disruption of gap junction mediated intercellular communication is unknown. Towards understanding these disease mechanisms, transfection studies were performed in a keratinocyte cell line and in HeLa cells using EGFP tagged wildtype Cx30 and mutant Cx30 constructs harbouring dominant disease-associated GJB6 mutations. For all three of the skin disease-associated Cx30 mutations investigated, impaired trafficking of the protein to the plasma membrane was observed thus preventing the formation of functional Cx30 gap junctions. In contrast, the deafness-associated mutation T5M-Cx30/EGFP trafficked to the membrane but defective channel activity was observed following dye transfer studies.

Base Sequence↗
PubMed2003

Use of a multiplex PCR/sequencing strategy to detect both connexin 30 (GJB6) 342 kb deletion and connexin 26 (GJB2) mutations in cases of childhood deafness.

Hearing loss is a common congenital disorder that is frequently associated with mutations in the Cx26 gene (GJB2). Three recent reports that found a large deletion in another DFNB1 gene, Cx30 (GJB6), suggest that this defect may cause nonsyndromic recessive hearing loss through either a homozygous deletion of Cx30, or digenic inheritance of a Cx30 deletion and a Cx26 mutation in trans. We designed a simple diagnostic strategy with multiplex PCR followed by direct sequencing to allow for the simultaneous detection of Cx26 mutations and Cx30 deletions, and evaluated its effectiveness as a clinical genetic test by examining 200 DNA samples. In the 108 samples from deaf subjects, two digenic mutations were identified in Cx26 and Cx30 (E47X/342 kb deletion and 167delT/342 kb deletion); 69 had only Cx26 mutations (29 biallelic, 40 singleton), including two novel frameshift mutations 511-512insAACG and 358-360delAG; and 37 had no detectable mutation in either Cx26 or Cx30. Our deletion mapping suggested that the proximal breakpoint of all reported Cx30 large deletions are between the nucleotide 444 and 627 at the Cx30 coding region within a maximal interval of 78 or 184 bp. This simultaneous examination of Cx26 and Cx30 is a practical and efficient diagnostic approach for patients with nonsyndromic congenital deafness.

Base Sequence↗
PubMed1999

Human connexin 30 (GJB6), a candidate gene for nonsyndromic hearing loss: molecular cloning, tissue-specific expression, and assignment to chromosome 13q12.

Mutations in connexin 26 are responsible for approximately 20% of genetic hearing loss and 10% of all childhood hearing loss. However, only about 75% of the mutations predicted to be in Cx26 are actually observed. While this may be due to mutations in noncoding regulatory regions, an alternative hypothesis is that some cases may be due to mutations in another gene immediately adjacent to Cx26. Another gap junction gene, connexin 30 (HGMW-approved symbol GJB6), is found to lie on the same PAC clone that hybridizes to chromosome 13q12. Human connexin 26 and connexin 30 are expressed in the same cells of the cochlea. Cx26 and Cx30 share 77% identity in amino acid sequence but Cx30 has an additional 37 amino acids at its C-terminus. These considerations led us to hypothesize that mutations in Cx30 might also be responsible for hearing loss. Eight-eight recessive nonsyndromic hearing loss families from both American and Japanese populations were screened for mutations. In addition, 23 dominant hearing loss families and 6 singleton families presumed to be recessive were tested. No significant mutation has been found in the dominant or recessive families.

Amino Acid Sequence↗
PubMed2000

Expression of connexin 30 in Xenopus embryos and its involvement in hatching gland function.

Connexins are a family of proteins that assemble to form gap junction channels. Cell-cell communication through gap junctions mediates many important events in embryogenesis, including limb patterning, lens physiology, neuronal function, left-right asymmetry, and secretion from gland tissue. We studied the expression of connexin 30 (Cx30) in the Xenopus embryo and find that it is expressed in the developing hatching gland and pronephros. To determine whether its expression plays a functional role in the activity of the hatching gland, we exposed pre-hatching embryos to drugs that block gap junctional communication. This resulted in a continuation of normal growth and development but specifically abolished hatching. The treatment did not affect Cx30 or Xenopus hatching enzyme transcription, suggesting a post-transcriptional effect on Cx30 gap junctions. We conclude that junctional communication, possibly mediated by Cx30, is involved in secretion of hatching enzyme in Xenopus.

Administration, Topical↗
PubMed2001

Expression of connexin 30 in the developing mouse cochlea.

Mutations in the GJB6 gene encoding connexin 30 (Cx30) can cause dominant forms of nonsyndromic deafness. By studying immunohistochemical localization of Cx30 in the mouse cochlea at different ages from 0 to 30 days after birth, we found that the expression of Cx30 is nearly the same as that of Cx26. These findings suggest that as well as Cx26, Cx30 may also contribute to the generation and maturation of endocochlear potential.

Age Factors↗
PubMed2004

Connexin 26 and connexin 30 mutations in children with nonsyndromic hearing loss.

OBJECTIVES/HYPOTHESIS: Mutations in the connexin 26 (Cx26) or gap junction beta 2 gene are the leading cause of hereditary nonsyndromic sensorineural hearing loss in Caucasians. The Cx26 coding region of 68 children with nonsyndromic sensorineural hearing loss was sequenced to determine the frequency and type of Cx26 mutations in this population. Screening was also performed for a common connexin 30 (Cx30) or gap junction beta 6 mutation (del [GJB6-D13S1830]). Children also underwent audiological testing to determine whether any correlation exists between Cx26 mutations and severity of hearing loss. STUDY DESIGN: In all, 68 children with nonsyndromic sensorineural hearing loss were screened for Cx26 and Cx30 mutations by polymerase chain reaction and direct sequencing. METHODS: Genomic DNA was amplified by polymerase chain reaction using primers that flank the entire Cx26 coding region. Screening for the 342-kb Cx30 deletion was performed using primers that amplified the breakpoint junction of the deletion. The amplicons were then sequenced in both directions and analyzed for mutations. Audiometric testing, including pure-tone audiometry and auditory evoked brainstem response, was also performed to determine the degree of hearing loss. RESULTS: Twenty-seven of 68 children tested had mutations in Cx26 with 35delG being the most prevalent. Ten additional Cx26 mutations were detected including a novel compound heterozygote. Two children were heterozygous for the Cx30 del (GJB6-D13S1830) mutation. CONCLUSION: Cx26 and Cx30 mutations were present in 41.2% of children tested in the study population. Audiometric data supported previous studies demonstrating a greater degree of hearing loss in subjects who are homozygous for the 35delG mutation.

Adolescent↗
PubMed2002

A novel connexin 30 mutation in Clouston syndrome.

Clouston syndrome (hidrotic ectodermal dysplasia) is an autosomal dominant ectodermal dysplasia characterized by alopecia, palmoplantar hyperkeratosis, and nail dystrophy. Recently, mutations in the GJB6 gene encoding the gap junction protein connexin 30 have been shown to cause this disorder. To date, all mutations have involved two codons: G11R and A88V. Here, we report a novel mutation V37E within the first transmembrane domain of connexin 30 in a spontaneous case of Clouston syndrome. The mutation was detected in genomic DNA, confirmed in reverse transcription polymerase chain reaction products, and was excluded from 100 ethnically matched control individuals by restriction enzyme analysis.

Adult↗
PubMed2004

Longitudinal phenotypic analysis in patients with connexin 26 (GJB2) (DFNB1) and connexin 30 (GJB6) mutations.

In 15 Belgian subjects with prelingual sensorineural hearing impairment, the connexin 26 (GJB2) gene and the connexin 30 (GJB6) gene were analyzed for the presence of the 35delG mutation and the delta(GJB6-D13S1830) deletion first described by del Castillo et al in 2002. Seven patients were found to be homozygous for the 35delG mutation; 7 were combined heterozygotes for the 35delG mutation and the GJB6 deletion. In 11 subjects, phenotype and genotype were correlated. Significant, transient progression, in the range of 1.7 to 2.7 dB/y, was only found in 2 patients in the first part of the second decade of life. Hearing impairment was otherwise stable, with mean thresholds of 75, 90, and 100 dB at 0.125, 0.25, and 0.5 kHz, respectively, and 100 dB or higher at 1 to 4 kHz. There was no significant difference in hearing impairment between the patients with the homozygous 35delG mutation in GJB2 and those who are heterozygous for both the 35delG mutation and the deletion encompassing part of GJB6.

Adolescent↗
PubMed2003

Mutations in the gene for connexin 26 (GJB2) that cause hearing loss have a dominant negative effect on connexin 30.

Mutations in the gene (GJB2) encoding connexin 26 (cx26) have been linked to sensorineural hearing loss either alone or as part of a syndrome. Here we compare the properties of four cx26 mutants derived from point mutations associated with dominantly inherited hearing loss, either non-syndromic (W44S, R75W) or with various skin disorders (G59A, D66H, R75W). Since cx26 and cx30 are co-localized within the inner ear the effect of the dominant cx26 mutations on both of these wild-type proteins was determined. Communication-deficient HeLa cells were transiently transfected with the various cDNA constructs by microinjection. Dye transfer studies using the gap junction permeant tracer Cascade Blue demonstrated a disruption to the intercellular coupling for all four of the mutant proteins. Immunostaining of the transfected cells revealed that for the G59A and D66H mutants this correlated with impaired intracellular trafficking and targeting to the plasma membrane, as both proteins had a perinuclear localization. The impaired trafficking was rescued by oligomerization both with cx26 and with cx30, suggesting that cx26 and cx30 can form heteromeric connexons. Significantly reduced dye transfer rates were observed between cells co-expressing either cx26 or cx30 together with W44S or R75W compared with the wild-type proteins alone. The dominant actions of the G59A and D66H mutants were only on cx30 and cx26, respectively. We suggest that cx26 and cx30 form heteromeric connexons in vivo, within the inner ear, with particular properties essential for hearing. Disruption of these heteromeric channels by certain mutations may underlie the non-syndromic nature of the deafness.

Cell Communication↗
PubMed2002

A deletion involving the connexin 30 gene in nonsyndromic hearing impairment.

BACKGROUND: Inherited hearing impairment affects about 1 in 2000 newborns. Up to 50 percent of all patients with autosomal recessive nonsyndromic prelingual deafness in different populations have mutations in the gene encoding the gap-junction protein connexin 26 (GJB2) at locus DFNB1 on chromosome 13q12. However, a large fraction (10 to 42 percent) of patients with GJB2 mutations have only one mutant allele; the accompanying mutation has not been identified. DFNB1-linked familial cases with no mutation in GJB2 have also been reported. METHODS: We evaluated 33 unrelated probands with nonsyndromic prelingual deafness who had only one GJB2 mutant allele. Nine subjects had evidence of linkage to DFNB1. We used haplotype analysis for markers on 13q12 to search for mutations other than the one involving GJB2. RESULTS: We identified a 342-kb deletion in the gene encoding connexin 30 (GJB6), a protein that is reported to be expressed with connexin 26 in the inner ear. The deletion extended distally to GJB2, which remained intact. The break-point junction of the deletion was isolated and sequenced, and a specific diagnostic test was developed for this common mutation. Twenty-two of the 33 subjects were heterozygous for both the GJB6 and GJB2 mutations, including all 9 with evidence of linkage to DFNB1. Two subjects were homozygous for the GJB6 mutation. CONCLUSIONS: A 342-kb deletion in GJB6 is the second most frequent mutation causing prelingual deafness in the Spanish population. Our data suggest that mutations in the complex locus DFNB1, which contains two genes (GJB2 and GJB6), can result in a monogenic or a digenic pattern of inheritance of prelingual deafness.

Base Sequence↗
PubMed2003

A mutation in the connexin 30 gene in Chinese Han patients with hidrotic ectodermal dysplasia.

BACKGROUND: hidrotic ectodermal dysplasia (HED) or Clouston syndrome is a rare autosomal dominant disorder affecting the skin and its derivatives. It is characterized by the triad of nail dystrophy, alopecia, and palmoplantar hyperkeratosis. To date, all mutations have been involving in three codons: G11R, A88V and V37E in the connexin 30 (Cx30) gene have been shown to cause this disorder. OBJECTIVE: in order to analyze the mutations of the Cx30 gene in Chinese Han patients with HED. METHODS: we collected a large Chinese HED family consisting of a total of 81 individuals including 28 HED patients (14 males and 14 females). The whole coding region of Cx30 was amplified by polymerase chain reaction and products analyzed by direct sequencing, then further confirmed at the mRNA level by RT-PCR. RESULTS: we detected a transition, 31(G-->A), leading to a missense mutation (G11R) in genomic DNAs of 18 patients, and the point mutation was not found in 16 normal individuals in this HED family and in 188 unrelated, population-match control individuals. The transcription of mutated allele was confirmed by RT-PCR of Cx30 mRNA. CONCLUSION: our data suggests that a G11R missense mutation in the Cx30 gene can cause HED in Chinese Han population and emphasizes the importance of screening for this as well as other Cx30 gene mutations in the HED.

Adult↗
PubMed2005

Hearing impairment in Dutch patients with connexin 26 (GJB2) and connexin 30 (GJB6) mutations.

OBJECTIVE: Despite the identification of mutations in the connexin 26 (GJB2) gene as the most common cause of recessive nonsyndromic hearing loss, the pattern of hearing impairment with these mutations remains inconsistent. Recently a deletion encompassing the GJB6 gene was identified and hypothesized to also contribute to hearing loss. We hereby describe the hearing impairment in Dutch patients with biallelic connexin 26 (GJB2) and GJB2+connexin 30 (GJB6) mutations. METHODS: The audiograms of patients who were screened for GJB2 and GJB6 mutations were analysed retrospectively. Standard statistical testing was done for symmetry and shape, while repeated measurement analysis was used to assess the relation between mutation and severity. Progression was also studied via linear regression analysis. RESULTS: Of 222 hearing-impaired individuals, 35 exhibited sequence variations; of these 19 had audiograms for study. Hearing loss in patients with biallelic "radical" (i.e. deletions, nonsense and splice site) mutations was significantly worse than in the wild type and heterozygotes (SAS proc GENMOD, p=0.013). The presence of at least one missense mutation in compound heterozygotes tends to lead to better hearing thresholds compared to biallelic radical mutations (p=0.08). One patient with the [35delG]+[del(GJB6-D13S1830)] genotype was severely impaired. Non-progressive hearing impairment was demonstrated in five 35delG homozygotes in individual longitudinal analyses. However a patient with the [299A>C]+[416G>A] genotype showed significant threshold progression in the lower frequencies. Findings on asymmetry and shape were inconclusive. CONCLUSIONS: Our data support the hypothesis that severity is a function of genotype and its effect on the amino acid sequence. A bigger cohort is required to establish non-progressivity more definitively.

Adolescent↗
PubMed2005

Localization of connexin 30 in the luminal membrane of cells in the distal nephron.

Several isoforms of the gap junction protein connexin (Cx) have been identified in a variety of tissues that communicate intercellular signals between adjacent cells. In the kidney, Cx37, Cx40, and Cx43 are localized in the vasculature, glomerulus, and tubular segments in a punctuate pattern, typical of classic gap junction channels. We performed immunohistochemistry in the mouse, rat, and rabbit kidney to study the localization of Cx30 protein, a new member of the Cx family. The vasculature, glomerulus, and proximal nephron segments were devoid of staining in all three species. Unexpectedly, Cx30 was found throughout the luminal membrane of select cells in the distal nephron. Expression of Cx30 was highest in the rat, which also showed some diffuse cytosolic labeling, continuous from the medullary thick ascending limb to the collecting duct system, and with the highest level in the distal convoluted tubule. Labeling in the mouse and rabbit was much less, limited to intercalated cells in the connecting segment and cortical collecting duct, where the apical signal was particularly strong. A high-salt-containing diet and culture medium upregulated Cx30 expression in the rat inner medulla and in M1 cells, respectively. The distinct, continuous labeling of the luminal plasma membrane and upregulation by high salt suggest that Cx30 may function as a hemichannel involved in the regulation of salt reabsorption in the distal nephron.

Animals↗
PubMedDate not supplied

Frequency and distribution of GJB2 (connexin 26) and GJB6 (connexin 30) mutations in a large North American repository of deaf probands.

PURPOSE: Profound hearing loss occurs with a frequency of 1 in 1000 live births, half of which is genetic in etiology. The past decade has witnessed rapid advances in determining the pathogenesis of both syndromic and nonsyndromic deafness. The most significant clinical finding to date has been the discovery that mutations of GJB2 at the DFNB1 locus are the major cause of profound prelingual deafness in many countries. 1 More recently, GJB2 mutations have been shown to cause deafness when present with a deletion of the GJB6 gene. We report on the prevalence of GJB2 and GJB6 mutations in a large North American Repository of DNA from deaf probands and document the profound effects of familial ethnicity and parental mating types on the frequency of these mutations in the population. METHODS: Deaf probands were ascertained through the Annual Survey of Deaf and Hard of Hearing Children and Youth, conducted at the Research Institute of Gallaudet University. Educational, etiologic, and audiologic information was collected after obtaining informed consent. DNA studies were performed for the GJB2 and GJB6 loci by sequencing and PCR methods. RESULTS: GJB2 mutations accounted for 22.2% of deafness in the overall sample but differed significantly among Asians, African-Americans and Hispanics and for probands from deaf by deaf and deaf by hearing matings, as well as probands from simplex and multiplex sibships of hearing parents. In our sample, the overall incidence of GJB2/GJB6 deafness was 2.57%. CONCLUSION: GJB2 mutations account for a large proportion of deafness in the US, with certain mutations having a high ethnic predilection. Heterozygotes at the GJB2 locus should be screened for the GJB6 deletion as a cause of deafness. Molecular testing for GJB2 and GJB6 should be offered to all patients with nonsyndromic hearing loss.

Alleles↗
PubMed2005

Ethnicity and mutations in GJB2 (connexin 26) and GJB6 (connexin 30) in a multi-cultural Canadian paediatric Cochlear Implant Program.

OBJECTIVE: To determine the relationship between ethnicity and mutations in the GJB2 and GJB6 genes in multi-cultural patients enrolled in a Canadian paediatric Cochlear Implant Program. METHODS: Blood was analyzed from 65 paediatric cochlear implant users by direct sequencing of the coding region and intron/exon boundaries of the GBJ2 gene. Individuals heterozygous for one mutation in GJB2 or in whom mutations in GJB2 were not detected were analyzed for the common 342 kb deletion mutation D13S1830 in the GJB6 gene. Information regarding ethnicity of patients' families was obtained from patient records and/or interview. RESULTS: GJB2 mutations were found in 36.9% of paediatric cochlear implant users tested. Nine different GJB2 mutations were identified among individuals from 14 different countries of origin. Seventy-eight percent of all identified pathogenic GJB2 mutations were 35delG. Biallelic GJB2 mutations were found in 16 cochlear implant users (66.7% of GJB2 mutations). Three novel GJB2 sequence changes were identified: (1) a missense mutation T107C (L36P) in an individual of African decent; (2) a missense mutation G475T (D159Y) in an individual of Caribbean decent; (3) a regulatory region change 1-34C to T in an individual of African decent. GJB6-D13S1830 mutations were not found in any of the patients tested. Individuals of African, Caribbean and East Indian decent had different GJB2 mutations than the remainder of individuals tested. Patients of Asian, Italian, Spanish, Polish and Armenian decent were not found to carry mutations in GJB2 or the common GJB6-D13S1830 mutation. CONCLUSIONS: This study represents the largest number of biallelic GJB2 mutations isolated in a group of paediatric cochlear implant users to date. Numerous and diverse GJB2 mutations were found in this multi-cultural group of children. Even though GJB2 mutations have been widely reported in the literature, this discussion represents the first report of GJB2 mutations in a multi-ethnic population (Canadian), as compared with previous studies that investigated fairly homogeneous populations. The diversity of GJB2 mutations identified reinforces the importance of testing for changes in GJB2 by direct sequencing of the entire coding region rather than testing only for common mutations.

Canada↗
PubMed2002

Functional significance of gap junctional coupling in preimplantation development.

Gap junctional intercellular coupling allows cells to share low molecular weight metabolites and second messengers, thus facilitating homeostatic and developmental processes. Gap junctions make their appearance very early in rodent development, during compaction in the eight-cell stage. Surprisingly, preimplantation mouse embryos lacking the gap junction protein connexin 43 develop normally and establish full-term pregnancies despite severely reduced gap junctional coupling. It was suggested that this might be explained by the presence of at least five additional connexins known to be expressed in blastocysts. In the present study, we set out to clarify the number of connexins present in preimplantation rodent embryos and the role of gap junctional coupling, if any, in blastocyst development. We provide evidence from reverse transcription-polymerase chain reaction analysis that the genes encoding 3 additional connexins (connexin 30 or beta6, connexin 36 or alpha9, and connexin 57 or alpha10) are also transcribed in preimplantation mouse embryos. Furthermore, we show that multiple connexins are expressed in rat preimplantation embryos, indicating that multiplicity of connexin expression may be a common feature of early mammalian embryogenesis. We could detect no up-regulation of any of 3 coexpressed connexins examined in mouse embryos lacking connexin 43. Impaired intercellular coupling caused either by the loss of connexin 43 or by treatment of cultured embryos with the gap junctional coupling blocker 18alpha-glycyrrhetinic acid (AGA) had no discernable effect on either apoptosis or glucose utilization, parameters known to be affected by gap junctional coupling in other contexts. These results, taken together with the reported inability of AGA to perturb blastocyst formation, imply that gap junctional coupling is not essential during this developmental period. We propose that connexin expression and the assembly of multiple types of gap junction channels in preimplantation embryos facilitates the diversification of communication pathways that will appear during postimplantation development. New evidence of this diversification is presented using rat blastocyst outgrowths.

Animals↗