Mechanism of a novel missense mutation, p.V174M, of the human connexin31 (GJB3) in causing nonsyndromic hearing loss

2014 ◽  
Vol 92 (4) ◽  
pp. 251-257 ◽  
Author(s):  
Tung-Cheng Li ◽  
Yu-Hsiang Kuan ◽  
Tzu-Yu Ko ◽  
Chuan Li ◽  
Jiann-Jou Yang
Keyword(s):  
Hearing Loss ◽  
Gap Junction ◽  
Missense Mutation ◽  
Functional Change ◽  
Dominant Negative ◽  
Mutant Protein ◽  
Dominant Negative Effect ◽  
Nonsyndromic Hearing Loss ◽  
Dye Transfer ◽  
Gap Junction Channel

Hearing loss is the most common sensory disorder, worldwide. In a recent study, we have identified a missense mutation, p.V174M, in the connexin 31 encoded by the GJB3 gene, in a patient with nonsyndromic hearing loss. However, the functional change in the CX31V174M mutant remains unknown. This study compared the intracellular distribution and assembly of the mutant CX31V174M with that of the wild-type (WT) CX31 in HeLa cells, and it examined the effect that the mutant protein had on those cells. A fluorescent localization assay of WT CX31 showed the typical punctuate pattern of a gap junction channel between the neighboring expression cells. Conversely, the p.V174M missense mutation resulted in the accumulation of the mutant protein in the lysosomes rather than in the cytoplasmic membrane. Moreover, dye transfer experiments have also demonstrated that the CX31V174M mutant did not form functional gap junction channels, probably due to the incorrect assembly or the altered properties of the CX31 channels. In addition, we found that CX31V174M-transfection can cause cell death by MTT assay. CX31V174M co-expressed with either CX31WT or CX26WT studies, suggested the impairment of the ability of CX26WT proteins to intracellular trafficking and targeting to the plasma membrane, but did not influence the trafficking of CX31WT. Based on these findings, we suggest that the CX31V174M mutant may have an effect on the formation and function of the gap junction, and CX31V174M has a trans-dominant negative effect on the function of wild types CX26. These results provide a novel molecular explanation for the role that GJB3 plays in hearing loss.

scholarly journals The Functional Role of CONNEXIN 26 Mutation in Nonsyndromic Hearing Loss, Demonstrated by Zebrafish Connexin 30.3 Homologue Model

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1291
Author(s):  
Hsuan-An Su ◽  
Ting-Wei Lai ◽  
Shuan-Yow Li ◽  
Tzu-Rong Su ◽  
Jiann-Jou Yang ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Structural Changes ◽  
Dominant Negative ◽  
Gjb2 Gene ◽  
Dominant Negative Effect ◽  
Connexin 26 ◽  
Transgenic Zebrafish ◽  
Nonsyndromic Hearing Loss ◽  
Transgenic Lines

Nonsyndromic hearing loss (NSHL) is of great clinical importance, and mutations in the GJB2 gene and the encoded human CONNEXIN 26 (CX26) protein play important roles in the genetic pathogenesis. The CX26 p.R184Q mutation was shown to be a dominant-negative effect in our previous study. Previously, we also demonstrated that zebrafish Cx30.3 is orthologous to human CX26. In the present study, we established transgenic zebrafish models with mutated Cx30.3 specifically expressed in the supporting cells of zebrafish inner ears driven by the agr2 promoter, to demonstrate and understand the mechanism by which the human CX26 R.184 mutation causes NSHL. Our results indicated that significant structural changes in the inner ears of transgenic lines with mutations were measured and compared to wild-type zebrafish. Simultaneously, significant alterations of transgenic lines with mutations in swimming behavior were analyzed with the zebrafish behavioral assay. This is the first study to investigate the functional results of the CX26 p.R184Q mutation with in vivo disease models. Our work supports and confirms the pathogenic role of the CX26 p.R184Q mutation in NSHL, with a hypothesized mechanism of altered interaction among amino acids in the connexins.

Genetics Landscape of Nonsyndromic Hearing Loss in Indian Populations

2021 ◽  
Author(s):  
Manisha Ray ◽  
Saurav Sarkar ◽  
Mukund Namdev Sable
Keyword(s):  
Hearing Loss ◽  
Gap Junction ◽  
Genetic Heterogeneity ◽  
Target Gene ◽  
Indian Population ◽  
Nonsyndromic Hearing Loss ◽  
Indian Populations ◽  
Chronic Disorder

AbstractCongenital nonsyndromic hearing loss (NSHL) has been considered as one of the most prevalent chronic disorder in children. It affects the physical and mental conditions of a large children population worldwide. Because of the genetic heterogeneity, the identification of target gene is very challenging. However, gap junction β-2 (GJB2) is taken as the key gene for hearing loss, as its involvement has been reported frequently in NSHL cases. This study aimed to identify the association of GJB2 mutants in different Indian populations based on published studies in Indian population. This will provide clear genetic fundamental of NSHL in Indian biogeography, which would be helpful in the diagnosis process.

A Missense Mutation (Tyr88 to Cys) in the Platelet Membrane Glycoprotein Ibβ Gene Affects GPIb/IX Complex Expression

2001 ◽  
Vol 86 (11) ◽  
pp. 1249-1256 ◽  
Author(s):  
Yumi Kurokawa ◽  
Takehiko Kamijo ◽  
Shinji Kunishima ◽  
Dermot Kenny ◽  
Kiyoshi Kitano ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Platelet Glycoprotein ◽  
Homozygous Mutation ◽  
Sequencing Analysis ◽  
Giant Platelets ◽  
Giant Platelet ◽  
Negative Effect ◽  
Dna Sequencing Analysis

SummaryThis study examined the molecular basis of a missense mutation of the platelet glycoprotein (GP) Ibβ gene in two families. In the propositus with a novel form of Bernard-Soulier syndrome (BSS) from Family I, only GPIbα was detectable in reduced amounts on platelet surfaces by flow cytometry. There were no GPIX or GPIbβ found by immunoblotting. DNA sequencing analysis showed a homozygous mutation in the GPIbβ gene which changed Tyr (TAC) to Cys (TGC) at residue 88. Her parents were heterozygous for Tyr88Cys in the GPIbβ gene. In transient transfection studies on 293T cells, both Tyr88Cys and Tyr88Ala mutations suppressed the expression of GPIb/IX complexes. In addition, Tyr88Cys GPIbβ mutation was found to exert a dominant negative effect on the GPIb expression.Five individuals from Family II, four of whom reported elsewhere as having giant platelet disorders with normal aggregation (BLOOD, 1997; 89: 2404) and one newly analyzed in this study, were heterozygous for Tyr88Cys in the GPIb gene. Microsatellite analysis of chromosome 22 showed a common haplotype in 8 of the individuals with Tyr88Cys mutations in Families I and II. Tyr88 in the GPIbβ gene plays a significant role in the GPIb/IX expression; the defect causes BSS in a homozygous form and possibly giant platelets in a heterozygous form.

scholarly journals Mutation of a Conserved Threonine in the Third Transmembrane Helix of α- and β-Connexins Creates a Dominant-negative Closed Gap Junction Channel

2005 ◽  
Vol 281 (12) ◽  
pp. 7994-8009 ◽  
Author(s):  
Derek L. Beahm ◽  
Atsunori Oshima ◽  
Guido M. Gaietta ◽  
Galen M. Hand ◽  
Amy E. Smock ◽  
...  
Keyword(s):  
Gap Junction ◽  
Transmembrane Helix ◽  
Dominant Negative ◽  
The Third ◽  
Gap Junction Channel

Phenotypic behavior of caveolin-3 R26Q, a mutant associated with hyperCKemia, distal myopathy, and rippling muscle disease

2003 ◽  
Vol 285 (5) ◽  
pp. C1150-C1160 ◽  
Author(s):  
Federica Sotgia ◽  
Scott E. Woodman ◽  
Gloria Bonuccelli ◽  
Franco Capozza ◽  
Carlo Minetti ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Cell Function ◽  
Cultured Cells ◽  
Dominant Negative ◽  
Muscle Disease ◽  
Mutant Protein ◽  
Dominant Negative Effect ◽  
Distal Myopathy ◽  
Wild Type ◽  
Disease Phenotypes

Four different phenotypes have been associated with CAV3 mutations: limb girdle muscular dystrophy-1C (LGMD-1C), rippling muscle disease (RMD), and distal myopathy (DM), as well as idiopathic and familial hyperCKemia (HCK). Detailed molecular characterization of two caveolin-3 mutations (P104L and ΔTFT), associated with LGMD-1C, shows them to impart a dominant-negative effect on wild-type caveolin-3, rendering it dysfunctional through sequestration in the Golgi complex. Interestingly, substitution of glutamine for arginine at amino acid position 26 (R26Q) of caveolin-3 is associated not only with RMD but also with DM and HCK. However, the phenotypic behavior of the caveolin-3 R26Q mutation has never been evaluated in cultured cells. Thus we characterized the cellular and molecular properties of the R26Q mutant protein to better understand how this mutation can manifest as such distinct disease phenotypes. Here, we show that the caveolin-3 R26Q mutant is mostly retained at the level of the Golgi complex. The caveolin-3 R26Q mutant formed oligomers of a much larger size than wild-type caveolin-3 and was excluded from caveolae-enriched membranes. However, caveolin-3 R26Q did not behave in a dominant-negative fashion when coexpressed with wild-type caveolin-3. Thus the R26Q mutation behaves differently from other caveolin-3 mutations (P104L and ΔTFT) that have been previously characterized. These data provide a possible explanation for the scope of the various disease phenotypes associated with the caveolin-3 R26Q mutation. We propose a haploinsufficiency model in which reduced levels of wild-type caveolin-3, although not rendered dysfunctional due to the caveolin-3 R26Q mutant protein, are insufficient for normal muscle cell function.

scholarly journals Novel Loss-of-Function Mutations in COCH Cause Autosomal Recessive Nonsyndromic Deafness

2020 ◽  
Author(s):  
Kevin T Booth ◽  
Amama Ghaffar ◽  
Muhammad Rashid ◽  
Luke T Hovey ◽  
Mureed Hussain ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Rna Splicing ◽  
Autosomal Recessive ◽  
Dominant Negative ◽  
Nonsyndromic Hearing Loss ◽  
Loss Of Function ◽  
Functional Studies ◽  
Pathogenic Variants ◽  
Exon Splicing ◽  
Coding Variants

AbstractCOCH is the most abundantly expressed gene in the cochlea. Unsurprisingly, mutations in COCH underly deafness in mice and humans. Two forms of deafness are linked to mutations in COCH, the well-established autosomal dominant nonsyndromic hearing loss, with or without vestibular dysfunction (DFNA9) via a gain-of-function/dominant-negative mechanism, and more recently autosomal recessive nonsyndromic hearing loss (DFNB110) via nonsense variants. Using a combination of targeted gene panels, exome sequencing and functional studies, we identified four novel pathogenic variants (two nonsense variants, one missense and one inframe deletion) in COCH as the cause of autosomal recessive hearing loss in a multi-ethnic cohort. To investigate whether the non-truncating variants exert their effect via a loss-of-function mechanism, we used mini-gene splicing assays. Our data showed both the missense and inframe deletion variants altered RNA-splicing by creating an exon splicing silencer and abolishing an exon splicing enhancer, respectively. Both variants create frameshifts and are predicted to result in a null allele. This study confirms the involvement of loss-of-function mutations in COCH in autosomal recessive nonsyndromic hearing loss, expands the mutational landscape of DFNB110 to include coding variants that alter RNA-splicing, and highlights the need to investigate the effect of coding variants on RNA-splicing.

Expression of a dominant negative inhibitor of intercellular communication in the early Xenopus embryo causes delamination and extrusion of cells

Development ◽  
1995 ◽  
Vol 121 (2) ◽  
pp. 371-381
Author(s):  
D.L. Paul ◽  
K. Yu ◽  
R. Bruzzone ◽  
R.L. Gimlich ◽  
D.A. Goodenough
Keyword(s):  
Gap Junction ◽  
Intercellular Communication ◽  
Lucifer Yellow ◽  
Single Cells ◽  
Positional Information ◽  
Embryonic Cell ◽  
Dominant Negative ◽  
Dye Transfer ◽  
Cell Stage ◽  
The Right

A chimeric construct, termed 3243H7, composed of fused portions of the rat gap junction proteins connexin32 (Cx32) and connexin43 (Cx43) has been shown to have selective dominant inhibitory activity when tested in the Xenopus oocyte pair system. Co-injection of mRNA coding for 3243H7 together with mRNAs coding for Cx32 or Cx43 completely blocked the development of channel conductances, while the construct was ineffective at blocking intercellular channel assembly when coinjected with rat connexin37 (Cx37). Injection of 3243H7 into the right anterodorsal blastomere of 8-cell-stage Xenopus embryos resulted in disadhesion and delamination of the resultant clone of cells evident by embryonic stage 8; a substantial number, although not all, of the progeny of the injected cell were eliminated from the embryo by stage 12. A second construct, 3243H8, differing from 3243H7 in the relative position of the middle splice, had no dominant negative activity in the oocyte pair assay, nor any detectable effects on Xenopus development, even when injected at four-fold higher concentrations. The 3243H7-induced embryonic defects could be rescued by coinjection of Cx37 with 3243H7. A blastomere reaggregation assay was used to demonstrate that a depression of dye-transfer could be detected in 3243H7-injected cells as early as stage 7; Lucifer yellow injections into single cells also demonstrated that injection of 3243H7 resulted in a block of intercellular communication. These experiments indicate that maintenance of embryonic cell adhesion with concomitant positional information requires gap junction-mediated intercellular communication.

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