KISEP Otology Korean J Otolaryngol 2001;44: 유전성난청과동반된 GJB2 유전자변이의세포간극에대한기능적연구 정연훈 유상준 이준호 박홍준 Functional Study of Gap Junction in GJB2 Mutations

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1 KISEP Otology Korean J Otolaryngol 2001;44: 유전성난청과동반된 GJB2 유전자변이의세포간극에대한기능적연구 정연훈 유상준 이준호 박홍준 Functional Study of Gap Junction in GJB2 Mutations Associated with Hereditary Hearing Loss Yun Hoon Choung, MD, DDS, Sang Jun Ryu, MD, Joon Ho Lee, MD and Hong-Joon Park, MD Department of Otolaryngology, Ajou University School of Medicine, Suwon, Korea ABSTRACT Background and ObjectivesGJB2 Connexin 26, the gene of the gap-junction proteins, was found to be the main causative gene of autosomal recessive nonsyndromic hearing loss DFNB1. Whereas 35delG was known as the major type mutation in the western countries, 235delC was reported as the specific form of mutation in Asian population. The objective of this study is to identify how two mutations 235delC, E114G found in the Korean population affect the function of GJB2 using the molecular biology techniques. Materials and Methods235delC and E114G types of mutations were cloned in the pcdna3 vector. HeLa cells were transfected with these cloned vectors by the liposome complex method. 1 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. 2 To analyze functions of the GJB2, we examined the lucifer yellow dye transfer between cells with scrape-loaded technique. We used the wild-type WT Cx26 of normal hearing as a positive control, and mock cells as a negative control. ResultsThe immunocytochemical analysis showed that cells transfected with E114G and WT gave characteristic punctuated patterns of reaction in the cell membrane with both antibodies. However, 235delC cells were not stained with the anti-il antibody but only with the anti-nt antibody slightly around the nucleus regions. In the functional study of GJB2, transfer of lucifer yellow dye into contiguous cells was detected in E114G but not in 235delC. ConclusionThe 235delC type of mutation showed loss of their targeting activity on the cell membrane. As a result, the function of gap junction channels were severely deteriorated. With the E114G type mutation, we didn t find any difference when compared with the WT transfected cells. Above data indicate that types of GJB2 mutation are closely related to the status of hearing loss due to altered function of gap junction protein. Korean J Otolaryngol 2001;44: KEY WORDSHereditary hearing loss Gap junction protein Connexin 26 Functional study. 239

2 240 효소중합반응 Transformation Fig. 1. Schema of this experiment. This study was carried out by 3 steps of transformation, transfection, subcellular localization of Cx26, and functional study of gap junction using 235delC, E114G, wild-type Cx26, and mock cells. The expression and subcellular localization of Cx26 was determined using antibodies against amino acid sequences in the intracellular loop and N-terminal of Cx26. To analyze functions of the gap junction, we examined Lucifer yellow dye transfer between cells with scrape-loaded technique. Korean J Otolaryngol 2001;44:239-45

3 Plasmid DNA preparation Cx26 발현및세포내위치확인 241

4 간극결합단백질의기능적연구 242 Fig. 2. The molecular structure of connexin delc and E114G are located in M2 and IL, respectively. Epitopes which are used in the immunocytochemistry A N- terminal, B int-racellular loop. M 1-4transmembrane domains, ILintra-cellular loop. Korean J Otolaryngol 2001;44:239-45

against intracellular loop portion of Cx26.

immunofl-uorescent reactions in the cell membranes.

respectively Fluorescent microscopic view.

detected in WT and E114G but not in 235delC transfected

DLight microscopic view of HeLa cells with scrape-loaded

5 A B A B C D C D Fig. 3. Immunocytochemistry of HeLa cells using primary antibody against intracellular loop portion of Cx26. Awildtype WT BE114G C235delC Dmock transfected cells, respectively. Both WT and E114G transfected HeLa cells showed positive immunofl-uorescent reactions in the cell membranes. However, 235delC transfected cells showed no reaction like as mock transfected cells. Fig. 5. Functional study of GJB2 using scrape-loaded dye Lucifer yellow technique. Awild-type WT BE114G C 235delC trans-fected cells, respectively Fluorescent microscopic view. Transfer of Lucifer yellow dye into contiguous cells were detected in WT and E114G but not in 235delC transfected HeLa cells. DLight microscopic view of HeLa cells with scrape-loaded dye technique. A C Fig. 4. Immunocytochemistry of HeLa cells using primary antibody against N-terminal portion of Cx26. Awild-type WT BE114G C235delC Dmock transfected cells, respectively. Both WT and E114G transfected HeLa cells showed positive immunofluorescent reactions in the cell membranes. However, 235delC transfected cells showed weak reactions only around the nucleus regions. Mock transfected cells showed no reaction. B D 243

6 244 Korean J Otolaryngol 2001;44:239-45

7 REFERENCES 1) Park HJ, Park KH, Oho JH, Lee JS. TEOAE as a newborn hearing screening. Korean J tolaryngol 1999;42: ) Morton NE. Genetic epidermiology of hearing impairment. Ann NY Acd Sci 1991;630: ) Zelante L, Gasparini P, Estivill X. Connexin 26 mutations associated with the most common form of non-syndromic neurosensory autosomal recessive deafness (DFNB1) in mediterraneans. Hum Mol Genet 1997;6: ) Estivill X, Fortina P, Surrey S, Rabionet R, Melchionda S, D Agruma L. Connexin-26 mutation in sporadic and inherited sensorineural deafness. Lancet 1998;35: ) Cohn ES, Kelly PM, Fowler TW, Gorga MP, Lefkowitz DM, Kuehn HJ, et al. Clinical Studies of families with hearing loss attributable to mutations in the connexin 26 gene (GJB2/DFNB1). Pediatrics 1999;103: ) Kumar NM, Gilula NB. The gap junction communication channel. Cell 1996;84: ) Forge A, Becker D, Evans WH. Gap junction connexin isoforms in the inner ear of gerbil and guinea pigs. Br J Audio 1997;31: ) Forge A, Becker D, Casalotti S, Edwards J, Evans WH, Lench N, Souter M. Gap junctions and connexin expression in the inner ear. In: Cardew G, editor. Gap Junction-Mediated Intercellular Signaling in Health and Disease. Novartis Foundation Symposium; Wiley Chichester UK.;1999. p ) Denoyelle F, Weil D, Maw MA. Prelingual deafness: High prevalence of a 30delG mutation in the connexin 26 gene. Hum Mol Gen 1997;6: ) Morell RJ, Kim HJ, Hood LJ. Mutation in the connexin 26 gene (GJB2) among Ashkenazi Jews with nonsyndromic recessive deafness. N Engl J Med 1998;339: ) Martin PEM, Coleman SL, Casalotti SO, Forge A, Evans WH. Properties of connexin 26 gap junctional proteins derived from mutations associated with non-syndromal hereditary deafness. Hum Mol Genet 1999;8: ) Park HJ, Park KH, Song JW, Choung YH, Choi HS. Molecular genetic analysis of Connexin 26 in Korean congenital hearing loss. Korean J Otolaryngol 2000;43: ) Park HJ, Hahn SH, Chun YM, Park KH, Kim HN. Connexin 26 mutations associated with nonsyndromic hearing loss. Laryngoscope 2000;110: ) El-Fouly MH, Trosko JE, Chang CC. Scrapeloading and dye transfer: A rapid and simple technique to study gap junctional intercellular communication. Exp Cell Res 1987;168: ) Lowenstein WR. Junctional intecellular communication and the control of growth. Biochem Biophys Acta 1979;560: ) Holt JR, Corey DP. Ion channel defects in hereditary hearing loss. Neuron 1999;22: ) Kikuchi T, Kimura RS, Paul D, Adams JC. Gap junctions in the rat cochlea: Immunohistochemical and ultrastructural analysis. Anat Embryol 1995;191: ) Bruzzone R, White TW, Paul DL. Connections with connexinsthe molecular-basis of direct intercellular signalling. Eur J Biochem 1996;238: ) George CH, Martin PEM, Evans WH. Rapid determination of gap junction formation using HeLa cells microinjected with cdnas encoding wild-type and chimeric connexins. Biochem Biophys Res Commun 1998;284:

Connexin 26 의유전자변이 서론 재료및방법 대상 방법 Korean J Audiol Volume 5 December, 2001

Connexin 26 의유전자변이 서론 재료및방법 대상 방법 Korean J Audiol Volume 5 December, 2001 KISEP Original Articles Korean J Audiol 52:105-110, 2001 선천성고도난청에서 Connexin 26 의유전자변이의형태분석 이상흔 1 이정래 1 이지은 1 김태정 2 강봉석 2 Molecular Genetic Analysis of Connexin 26 in Congenital Profound Hearing Loss Sang