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.

scholarly journals Spectrum and Frequency of the GJB2 Gene Mutations Among Latvian Patients with Prelingual Nonsyndromic Hearing Loss

2009 ◽  
Vol 63 (4-5) ◽  
pp. 198-203
Author(s):  
Olga Šterna ◽  
Natālija Proņina ◽  
Ieva Grīnfelde ◽  
Sandra Kušķe ◽  
Astrīda Krūmiņa ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Gene Mutations ◽  
Gjb2 Gene ◽  
Connexin 26 ◽  
Compound Heterozygous ◽  
Nonsyndromic Hearing Loss ◽  
Profound Hearing Loss ◽  
Common Cause ◽  
Gjb2 Mutation ◽  
35Delg Mutation

Spectrum and Frequency of the GJB2 Gene Mutations Among Latvian Patients with Prelingual Nonsyndromic Hearing Loss Mutations in the GJB2 gene (connexin 26) are the most common cause of congenital nonsyndromic severe-to-profound hearing loss. Sixty-five hearing impaired probands from Latvia were tested for mutations in the GJB2 gene to determine the percentage of hearing loss attributed to connexin 26 and the types of mutations in this population. A total of 62% of patients tested had GJB2 mutations. Four different mutations in the GJB2 gene were identified in Latvian patients with nonsyndromic sensorineural hearing loss: 35delG, 311-324del14, 235delC and M34T. The most prevalent mutation is 35delG (47% of all probands were homozygous and 8% compound heterozygous). Our findings support the conclusion that the 35delG mutation is the most prevalent GJB2 mutation and that it is the common cause of hereditary nonsyndromic hearing loss in populations of European descent.

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 Mutation R184Q of connexin 26 in hearing loss patients has a dominant-negative effect on connexin 26 and connexin 30

2010 ◽  
Vol 18 (9) ◽  
pp. 1061-1064 ◽  
Author(s):  
Ching-Chyuan Su ◽  
Shuan-Yow Li ◽  
Mao-Chang Su ◽  
Wei-Chi Chen ◽  
Jiann-Jou Yang
Keyword(s):  
Hearing Loss ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Connexin 26 ◽  
Negative Effect ◽  
Connexin 30

scholarly journals Functional Evaluation of a Rare Variant c.516G>C (p.Trp172Cys) in the GJB2 (Connexin 26) Gene Associated with Nonsyndromic Hearing Loss

Biomolecules ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 61
Author(s):  
Ekaterina A. Maslova ◽  
Konstantin E. Orishchenko ◽  
Olga L. Posukh
Keyword(s):  
Hearing Loss ◽  
Transmembrane Protein ◽  
Amino Acid Position ◽  
Indigenous Populations ◽  
Gjb2 Gene ◽  
Connexin 26 ◽  
Functional Evaluation ◽  
Nonsyndromic Hearing Loss ◽  
Extracellular Loop ◽  
Gene Encoding

Mutations in the GJB2 gene encoding transmembrane protein connexin 26 (Cx26) are the most common cause for hearing loss worldwide. Cx26 plays a crucial role in the ionic and metabolic homeostasis in the inner ear, indispensable for normal hearing process. Different pathogenic mutations in the GJB2 gene can affect all stages of the Cx26 life cycle and result in nonsyndromic autosomal recessive (DFNB1) or dominant (DFNA3) deafness and syndromes associating hearing loss with skin disorders. This study aims to elucidate the functional consequences of a rare GJB2 variant c.516G>C (p.Trp172Cys) found with high frequency in deaf patients from indigenous populations of Southern Siberia (Russia). The substitution c.516G>C leads to the replacement of tryptophan at a conserved amino acid position 172 with cysteine (p.Trp172Cys) in the second extracellular loop of Cx26 protein. We analyzed the subcellular localization of mutant Cx26-p.Trp172Cys protein by immunocytochemistry and the hemichannels permeability by dye loading assay. The GJB2 knockout HeLa cell line has been generated using CRISPR/Cas9 genome editing tool. Subsequently, the HeLa transgenic cell lines stably expressing different GJB2 variants (wild type and mutations associated with hearing loss) were established based on knockout cells and used for comparative functional analysis. The impaired trafficking of mutant Cx26-p.Trp172Cys protein to the plasma membrane and reduced hemichannels permeability support the pathogenic effect of the c.516G>C (p.Trp172Cys) variant and its association with nonsyndromic hearing loss. Our data contribute to a better understanding of the role of mutations in the second extracellular loop of Cx26 protein in pathogenesis of deafness.

The prevalence and expression of inherited connexin 26 mutations associated with nonsyndromic hearing loss in the Israeli population

2000 ◽  
Vol 106 (1) ◽  
pp. 50-57 ◽  
Author(s):  
T. Sobe ◽  
S. Vreugde ◽  
H. Shahin ◽  
M. Berlin ◽  
N. Davis ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Connexin 26 ◽  
Nonsyndromic Hearing Loss ◽  
Israeli Population

scholarly journals Impaired cytoplasmic domain interactions cause co-assembly defect and loss of function in the p.Glu293Lys KNCJ2 variant isolated from an Andersen–Tawil syndrome patient

2020 ◽  
Author(s):  
Szilvia Déri ◽  
János Borbás ◽  
Teodóra Hartai ◽  
Lidia Hategan ◽  
Beáta Csányi ◽  
...  
Keyword(s):  
Salt Bridge ◽  
Cytoplasmic Domain ◽  
Inward Rectifier ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Causative Role ◽  
Loss Of Function ◽  
Subunit Interactions ◽  
Negative Effect

Abstract Aims Subunit interactions at the cytoplasmic domain interface (CD-I) have recently been shown to control gating in inward rectifier potassium channels. Here we report the novel KCNJ2 variant p.Glu293Lys that has been found in a patient with Andersen–Tawil syndrome type 1 (ATS1), causing amino acid substitution at the CD-I of the inward rectifier potassium channel subunit Kir2.1. Neither has the role of Glu293 in gating control been investigated nor has a pathogenic variant been described at this position. This study aimed to assess the involvement of Glu293 in CD-I subunit interactions and to establish the pathogenic role of the p.Glu293Lys variant in ATS1. Methods and results The p.Glu293Lys variant produced no current in homomeric form and showed dominant-negative effect over wild-type (WT) subunits. Immunocytochemical labelling showed the p.Glu293Lys subunits to distribute in the subsarcolemmal space. Salt bridge prediction indicated the presence of an intersubunit salt bridge network at the CD-I of Kir2.1, with the involvement of Glu293. Subunit interactions were studied by the NanoLuc® Binary Technology (NanoBiT) split reporter assay. Reporter constructs carrying NanoBiT tags on the intracellular termini produced no bioluminescent signal above background with the p.Glu293Lys variant in homomeric configuration and significantly reduced signals in cells co-expressing WT and p.Glu293Lys subunits simultaneously. Extracellularly presented reporter tags, however, generated comparable bioluminescent signals with heteromeric WT and p.Glu293Lys subunits and with homomeric WT channels. Conclusions Loss of function and dominant-negative effect confirm the causative role of p.Glu293Lys in ATS1. Co-assembly of Kir2.1 subunits is impaired in homomeric channels consisting of p.Glu293Lys subunits and is partially rescued in heteromeric complexes of WT and p.Glu293Lys Kir2.1 variants. These data point to an important role of Glu293 in mediating subunit assembly, as well as in gating of Kir2.1 channels.

Update of the spectrum of GJB2 gene mutations in Tunisian families with autosomal recessive nonsyndromic hearing loss

Gene ◽  
2013 ◽  
Vol 525 (1) ◽  
pp. 1-4 ◽  
Author(s):  
Zied Riahi ◽  
Hassen Hammami ◽  
Houyem Ouragini ◽  
Habib Messai ◽  
Rim Zainine ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Autosomal Recessive ◽  
Gene Mutations ◽  
Gjb2 Gene ◽  
Nonsyndromic Hearing Loss

scholarly journals Entamoeba histolyticaExpressing a Dominant Negative N-Truncated Light Subunit of Its Gal-Lectin Are Less Virulent

2002 ◽  
Vol 13 (12) ◽  
pp. 4256-4265 ◽  
Author(s):  
Uriel Katz ◽  
Serge Ankri ◽  
Tamara Stolarsky ◽  
Yael Nuchamowitz ◽  
David Mirelman
Keyword(s):  
Antisense Rna ◽  
Mammalian Cells ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Impaired Ability ◽  
Reducing Conditions ◽  
Negative Effect ◽  
Terminal Amino ◽  
Heterodimeric Complex

The 260-kDa heterodimeric Gal/GalNAc-specific Lectin (Gal-lectin) of Entamoeba histolytica dissociates under reducing conditions into a heavy (hgl, 170 kDa) and a light subunit (lgl, 35 kDa). We have previously shown that inhibition of expression of the 35-kDa subunit by antisense RNA causes a decrease in virulence. To further understand the role of the light subunit of the Gal-lectin in pathogenesis, amoebae were transfected with plasmids encoding intact, mutated, and truncated forms of the light subunit lgl1 gene. A transfectant in which the 55 N-terminal amino acids of the lgl were removed, overproduced an N-truncated lgl protein (32 kDa), which replaced most of the native 35-kDa lgl in the formation of the Gal-lectin heterodimeric complex and exerted a dominant negative effect. Amoebae transfected with this construct showed a significant decrease in their ability to adhere to and kill mammalian cells as well as in their capacity to form rosettes with and to phagocytose erythrocytes. In addition, immunofluorescence confocal microscopy of this transfectant with anti–Gal-lectin antibodies showed an impaired ability to cap. These results indicate that the light subunit has a role in enabling the clustering of Gal-lectin complexes and that its N-truncation affects this function, which is required for virulence.

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