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Functional study of GJB2 in hereditary hearing loss.

Authors
Choung, YH; Moon, SK; Park, HJ
Citation
The Laryngoscope, 112(9):1667-1671, 2002
Journal Title
The Laryngoscope
ISSN
0023-852X1531-4995
Abstract
OBJECTIVES/HYPOTHESIS: The gene of the gap junction protein connexin 26 (Cx26) was found to be the main causative gene of autosomal recessive nonsyndromic hearing loss (DFNB1). Although 35delG has been known as the major mutation in Western countries, 235delC was reported to be a specific form of mutation in Asian populations. The objective of the study was to identify how 235delC and E114G changes found in the Korean population affected the function of using molecular biological techniques.



METHODS: Genes containing 235delC and E114G were cloned into the pcDNA3 vector, and HeLa cells were transfected with the recombinant DNA samples by the liposome complex method. The expression and subcellular localization of Cx26 were determined, using antibodies against amino acid sequences in the intracellular loop (IL) and N-terminal (NT) portions of Cx26. To analyze functions of the as a gap junction channel, we examined Lucifer yellow dye transfer between cells with a scrape-loaded technique. Wild-type (WT) with normal hearing was used as a positive control, and mock transfected cells were used as a negative control.



RESULTS: Immunocytochemical analysis showed that cells transfected with E114G and WT gave characteristic punctate patterns of reaction in the cell membrane with both antibodies. However, 235delC cells were not stained with anti-IL antibody but stained slightly just around the nucleus only with anti-NT antibody. In a functional study of, transfer of Lucifer yellow into contiguous cells was detected in both WT and E114G, but no transfer activity was observed in 235delC.



CONCLUSIONS: The 235delC mutation showed a loss of targeting activity to the cell membrane and severe deterioration of gap junction activity. For the E114G, we did not find any difference from WT transfected cells.
MeSH terms
Asian Continental Ancestry Group/geneticsCodonConnexins/*genetics*Frameshift MutationGap Junctions/*geneticsGenes, RecessiveHearing Disorders/congenital/ethnology/*geneticsHela CellsHumansKorea/epidemiologyMutation, MissensePolymerase Chain ReactionTransfection
DOI
10.1097/00005537-200209000-00026
PMID
12352684
Appears in Collections:
Journal Papers > School of Medicine / Graduate School of Medicine > Otolaryngology
AJOU Authors
정, 연훈문, 성균
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