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Digenic inheritance of non-syndromic deafness caused by mutations at the gap junction proteins Cx26 and Cx31

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Abstract

Mutations in the genes coding for connexin 26 (Cx26) and connexin 31 (Cx31) cause non-syndromic deafness. Here, we provide evidence that mutations at these two connexin genes can interact to cause hearing loss in digenic heterozygotes in humans. We have screened 108 GJB2 heterozygous Chinese patients for mutations in GJB3 by sequencing. We have excluded the possibility that mutations in exon 1 of GJB2 and the deletion of GJB6 are the second mutant allele in these Chinese heterozygous probands. Two different GJB3 mutations (N166S and A194T) occurring in compound heterozygosity with the 235delC and 299delAT of GJB2 were identified in three unrelated families (235delC/N166S, 235delC/A194T and 299delAT/A194T). Neither of these mutations in Cx31 was detected in DNA from 200 unrelated Chinese controls. Direct physical interaction of Cx26 with Cx31 is supported by data showing that Cx26 and Cx31 have overlapping expression patterns in the cochlea. In addition, by coimmunoprecipitation of mouse cochlear membrane proteins, we identified the presence of heteromeric Cx26/Cx31 connexons. Furthermore, by cotransfection of mCherry-tagged Cx26 and GFP-tagged Cx31 in human embryonic kidney (HEK)-293 cells, we demonstrated that the two connexins were able to co-assemble in vitro in the same junction plaque. Together, our data indicate that a genetic interaction between these two connexin genes can lead to hearing loss.

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References

  • Abrams CK, Freidin MM, Verselis VK, Bargiello TA, Kelsell DP, Richard G, Bennett MVL, Bukauskas FF (2006) Properties of human connexin 31, which is implicated in hereditary dermatological disease and deafness. Proc Natl Acad Sci 103:5213–5218

    Article  PubMed  CAS  Google Scholar 

  • Ahmad S, Chen S, Sun J, Lin X (2003) Connexins 26 and 30 are co-assembled to form gap junctions in the cochlea of mice. Biochem Biophys Res Commun 307:362–368

    Article  PubMed  CAS  Google Scholar 

  • Alexandrino F, Oliveira CA, Reis FC, Maciel-Guerra AT, Sartorato EL (2004) Screening for mutations in the GJB3 gene in Brazilian patients with nonsyndromic deafness. J Appl Genet 45:249–254

    PubMed  Google Scholar 

  • Beblo DA, Veenstra RD (1997) Monovalent cation permeation through the connexin40 gap junction channel. Cs, Rb, K, Na, Li, TEA, TMA, TBA, and effects of anions Br, Cl, F, acetate, aspartate, glutamate, and NO3. J Gen Physiol 109:509–522

    Article  PubMed  CAS  Google Scholar 

  • Bonifacino JS, Cosson P, Shah N, Klausner RD (1991) Role of potentially charged transmembrane residues in targeting proteins for retention and degradation within the endoplasmic reticulum. EMBO J 10:2783–2793

    PubMed  CAS  Google Scholar 

  • Bruzzone R, Veronesi V, Gomes D, Bicego M, Duval N, Marlin S, Petit C, D’Andrea P, White TW (2003) Loss-of-function and residual channel activity of connexin26 mutations associated with non-syndromic deafness. FEBS Lett 533:79–88

    Article  PubMed  CAS  Google Scholar 

  • Cao F, Eckert R, Elfgang C, Nitsche JM, Snyder SA, H-ulser DF, Willecke K, Nicholson BJ (1998) A quantitative analysis of connexin-specific permeability differences of gap junctions expressed in HeLa transfectants and Xenopus oocytes. J Cell Sci 111:31–43

    PubMed  CAS  Google Scholar 

  • Coucke P, Van Camp G, Djoyodiharjo B, Smith SD, Frants RR, Padberg GW, Darby JK, Huizing EH, Cremers C, Kimberling WJ, Oostra BA, Van de Heyning PH, Willems PJ (1994) Linkage of autosomal dominant hearing loss to the short arm of chromosome 1 in two families. N Engl J Med 331:425–431

    Article  PubMed  CAS  Google Scholar 

  • Dai P, Yu F, Han B, Yuan Y, Li Q, Wang G, Liu X, He J, Huang D, Kang D, Zhang X, Yuan H, Schmitt E, Han D, Wong LJ (2007) The prevalence of the 235delC GJB2 mutation in a Chinese deaf population. Genet Med 9:283–289

    Article  PubMed  CAS  Google Scholar 

  • Del Castillo I, Moreno-Pelayo MA, Del Castillo FJ, Brownstein Z, Marlin S, Adina Q, Cockburn DJ, Pandya A, Siemering KR, Chamberlin GP, Ballana E, Wuyts W, Maciel-Guerra AT, Alvarez A, Villamar M, Shohat M, Abeliovich D, Dahl HH, Estivill X, Gasparini P, Hutchin T, Nance WE, Sartorato EL, Smith RJ, Van Camp G, Avraham KB, Petit C, Moreno F (2003) Prevalence and evolutionary origins of the del(GJB6-D13S1830) mutation in the DFNB1 locus in hearing-impaired subjects: a multicenter study. Am J Hum Genet 73:1452–1458

    Article  PubMed  CAS  Google Scholar 

  • del Castillo FJ, Rodríguez-Ballesteros M, Alvarez A, Hutchin T, Leonardi E, de Oliveira CA, Azaiez H, Brownstein Z, Avenarius MR, Marlin S, Pandya A, Shahin H, Siemering KR, Weil D, Wuyts W, Aguirre LA, Martín Y, Moreno-Pelayo MA, Villamar M, Avraham KB, Dahl HH, Kanaan M, Nance WE, Petit C, Smith RJ, Van Camp G, Sartorato EL, Murgia A, Moreno F, del Castillo I (2005) A novel deletion involving the connexin-30 gene, del(GJB6-d13s1854), found in trans with mutations in the GJB2 gene (connexin-26) in subjects with DFNB1 non-syndromic hearing impairment. J Med Genet 42:588–594

    Article  PubMed  CAS  Google Scholar 

  • Diez JA, Ahmad S, Evans WH (1999) Assembly of heteromeric connexons in guinea-pig liver en route to the Golgi apparatus, plasma membrane and gap junctions. Eur J Biochem 262:142–148

    Article  PubMed  CAS  Google Scholar 

  • Elfgang C, Eckert R, Lichtenberg-Fraté H, Butterweck A, Traub O, Klein RA, Hülser DF, Willecke K (1995) Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells. J Cell Biol 129:805–817

    Article  PubMed  CAS  Google Scholar 

  • Feng Y, He C, Xiao J, Fang J, Zhao S, Tian X, Mei L, Xia K, Tang X (2002) An analysis of a large hereditary postlingually deaf families and detecting mutation of the deafness genes. Chin J Otorhinolaryngol 37:348–351

    Google Scholar 

  • Frei K, Ramsebner R, Hamader G, Lucas T, Schoefer C, Baumgartner WD, Wachtler FJ, Kirschhofer K (2004) Lack of association between Connexin 31 (GJB3) alterations and sensorineural deafness in Austria. Hear Res 194:81–86

    Article  PubMed  CAS  Google Scholar 

  • Grifa A, Wagner CA, D’Ambrosio L, Melchionda S, Bernardi F, Lopez-Bigas N, Rabionet R, Arbones M, Monica MD, Estivill X, Zelante L, Lang F, Gasparini P (1999) Mutations in GJB6 cause nonsyndromic autosomal dominant deafness at DFNA3 locus. Nat Genet 23:16–18

    PubMed  CAS  Google Scholar 

  • Gualandi E, Ravani A, Berto A et al (2004) Occurrence of del(GIB6–D13S1830) mutation in Italian non-syndromic hearing loss patients carrying a single GJB2 mutated allele. Acta Otolaryngol Suppl 552:29–34

    Article  PubMed  CAS  Google Scholar 

  • Gunther B, Steiner A, Nekahm-Heis, Albegger K, Zorowka P, Utermann G, Janecke A (2003) The 342-kb deletion in GJB6 is not present in patients with non-syndromic hearing loss from Austria. Hum Mutat 22:180–184

  • Hamelmann C, Amedofu GK, Albrecht K, Muntau B, Gelhaus A, Brobby GW, Horstmann RD (2001) Pattern of connexin 26 (GJB2) mutations causing sensorineural hearing impairment in Ghana. Hum Mutat 18:84–85

    Article  PubMed  CAS  Google Scholar 

  • Janssen EAM, Kemp S, Hensels GW, Sie OG, de Die-Smulders CEM, Hoogendijk JE, de Visser M, Bolhuis PA (1997) Connexin32 gene mutations in X-linked dominant Charcot-Marie-Tooth disease. Hum Genet 99:501–505

    Article  PubMed  CAS  Google Scholar 

  • Kelsell DP, Dunlop J, Stevens HP, Lench NJ, Liang JN, Parry G, Mueller RF, Leigh IM (1997) Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature 387:80–83

    Article  PubMed  CAS  Google Scholar 

  • Kumar NM, Friend DS, Gilula NB (1995) Synthesis and assembly of human β1 gap junctions in BHK cells by DNA transfection with the human β1 cDNA. J Cell Sci 108:3725–3734

    PubMed  CAS  Google Scholar 

  • Lopez-Bigas N, Rabionet R, Martinez E, Banchs I, Volpini V, Vance JM, Arbones ML, Estivill X (2000) Identification of seven novel SNPS (five nucleotide and two amino acid substitutions) in the Connexin 31 (GJB3) gene. Hum Mutat 15:481–482

    Article  PubMed  CAS  Google Scholar 

  • Lopez-Bigas N, Olive M, Rabionet R, Ben-David O, Martinez-Matos JA, Bravo O, Banchs I, Volpini V, Gasparini P, Avraham KB, Ferrer I, Arbones ML, Estivill X (2001) Connexin 31 (GJB3) is expressed in the peripheral and auditory nerves and causes neuropathy and hearing impairment. Hum Mol Genet 10:947–952

    Article  PubMed  CAS  Google Scholar 

  • Liu XZ, Xia XJ, Xu LR, Pandya A, Liang CY, Blanton SH, Brown SD, Steel KP, Nance WE (2000) Mutations in connexin31 underlie recessive as well as dominant nonsyndromic hearing loss. Hum Mol Genet 9:63–67

    Article  PubMed  CAS  Google Scholar 

  • Liu Y, Ke X, Qi Y, Zhu P (2002a) Connexin 26 gene (GJB2): prevalence of mutations in Chinese population. J Hum Genet 47:688–690

    Article  PubMed  CAS  Google Scholar 

  • Liu XZ, Xia XJ, Ke XM, Ouyang XM, Du LL, Liu YH, Angeli S, Telischi FF, Nance WE, Balkany T, Xu LR (2002b) The prevalence of connexin 26 (GJB2) mutations in the Chinese population. Hum Genet 111:394–397

    Article  PubMed  CAS  Google Scholar 

  • Liu XZ, Ouyang XM, Yan D (2006) The genetic deafness in Chinese population (in English). J Otol 1:1–10

    CAS  Google Scholar 

  • Machamer CE, Grim MG, Esquela A, Chung SW, Rolls M, Ryan K, Swift AM (1993) Retention of a cis Golgi protein requires polar residues on one face of a predicted α-helix in the transmembrane domain. Mol Biol Cell 4:695–704

    PubMed  CAS  Google Scholar 

  • Marlin S, Feldmann D, Blons H, Loundon N, Rouillon I, Albert S, Chauvin P, Garabedian EN, Couderc R, Odent S, Joannard A, Schmerber S, Delobel B, Leman J, Journel H, Catros H, Lemarechal C, Dollfus H, Eliot MM, Delaunoy JL, David A, Calais C, Drouin-Garraud V, Obstoy MF, Goizet C, Duriez F, Fellmann F, Helias J, Vigneron J, Montaut B, Matin-Coignard D, Faivre L, Baumann C, Lewin P, Petit C, Denoyelle F (2005) GJB2 and GJB6 mutations: genotypic and phenotypic correlations in a large cohort of hearing-impaired patients. Arch Otolaryngol Head Neck Surg 131:481–487

    Article  PubMed  Google Scholar 

  • Martin PE, Coleman SL, Casalotti SO, Forge A, Evans WH (1999) Properties of connexin26 gap junctional proteins derived from mutations associated with non-syndromal heriditary deafness. Hum Mol Genet 8:2369–2376

    Article  PubMed  CAS  Google Scholar 

  • Marziano NK, Casalotti SO, Portelli AE, Becker DL, Forge A (2003) Mutations in the gene for connexin 26 (GJB2) that cause hearing loss have a dominant negative effect on connexin 30. Hum Mol Genet 12:805–812

    Article  PubMed  CAS  Google Scholar 

  • Matos T, Caria H, Galhardo I, Dias O, Andrea M, Correia C, Fialho G (2003) A novel M163L mutation in GJB2 gene associated with autosomal dominant isolated hearing loss. Eur J Hum Genet 11(Suppl 1):211

    Google Scholar 

  • Mese G, Londin E, Mui R, Brink PR, White TW (2004) Altered gating properties of functional Cx26 mutants associated with recessive non-syndromic hearing loss. Hum Genet 115:191–199

    Article  PubMed  CAS  Google Scholar 

  • Mhatre AN, Weld E, Lalwani AK (2003) Mutation analysis of Connexin 31 (GJB3) in sporadic non-syndromic hearing impairment. Clin Genet 63:154–159

    Article  PubMed  CAS  Google Scholar 

  • Musil LS, Goodenough DA (1993) Multisubunit assembly of an integral plasma-membrane channel protein, gap junction connexin43, occurs after exit from the ER. Cell 74:1065–1077

    Article  PubMed  CAS  Google Scholar 

  • Neocleous V, Aspris A, Shahpenterian V, Nicolaou V, Panagi C, Ioannis Ioannou I, Kyamides Y, Anastasiadou V, Leonidas AP (2006) High frequency of 35delG mutation and absence of del(GJB6–D13S1830) in Greek Cypriot patients with nonsyndromic hearing loss. Genet Test 10:285–289

    Article  PubMed  CAS  Google Scholar 

  • Plantard L, Huber M, Macari F, Meda P, Hohl D (2003) Molecular interaction of connexin 30.3 and connexin 31 suggests a dominant-negative mechanism associated with erythrokeratodermia variabilis. Hum Mol Genet 12:3287–3294

    Article  PubMed  CAS  Google Scholar 

  • Richard G, Lin JP, Smith L, Whyte YM, Itin P, Wollina U, Epstein E Jr, Hohl D, Giroux JM, Charnas L, Bale SJ, DiGiovanna JJ (1997) Linkage studies in erythrokeratodermias: fine mapping, genetic heterogeneity and analysis of candidate genes. J Invest Derm 109:666–671

    Article  PubMed  CAS  Google Scholar 

  • Richard G, Smith LE, Bailey RA, Itin P, Hohl D, Epstein EH Jr, DiGiovanna JJ, Compton JG, Bale SJ (1998) Mutations in the human connexin gene GJB3 cause erythrokeratodermia variabilis. Nat Genet 20:366–369

    Article  PubMed  CAS  Google Scholar 

  • Richard G, Brown N, Smith LE, Terrinoni A, Melino G, MacKie RM, Bale SJ, Uitto J (2000) The spectrum of mutations in erythrokeratodermias—novel and de novo mutations in GJB3. Hum Genet 106:321–329

    Article  PubMed  CAS  Google Scholar 

  • Rickard S, Kelsell DP, Sirimana T, Rajput K, MacArdle B, Bitner-Glindzicz M (2001) Recurrent mutations in the deafness gene GJB2 (connexin 26) in British Asian families. J Med Genet 38:530–533

    Article  PubMed  CAS  Google Scholar 

  • Simon AM, Goodenough DA (1998) Diverse functions of vertebrate gap junctions. Trends Cell Biol 8:477–483

    Article  PubMed  CAS  Google Scholar 

  • Sun J, Ahmad S, Chen S, Tang W, Zhang Y, Chen P, Lin X (2005) Cochlear gap junctions coassembled from Cx26 and 30 show faster intercellular Ca2+ signaling than homomeric counterparts. Am J Physiol Cell Physiol 288:C613–C623

    Article  PubMed  CAS  Google Scholar 

  • Trexler EB, Bukauskas FF, Kronengold J, Bargiello TA, Verselis VK (2000) The first extracellular loop domain is a major determinant of charge selectivity in connexin46 channels. Biophys J 79:3036–3051

    Article  PubMed  CAS  Google Scholar 

  • Uyguner O, Emiroglu M, Uzumcu A, Hafiz G, Ghanbari A, Baserer N, Yuksel-Apak M, Wollnik B (2003) Frequencies of gap- and tight junction mutations in Turkish families with autosomal-recessive nonsyndromic hearing loss. Clin Genet 64:65–69

    Article  PubMed  CAS  Google Scholar 

  • Van Camp G, Coucke PJ, Kunst H, Schatteman I, Van Velzen D, Marres H, van Ewijk M, Declau F, Van Hauwe P, Meyers J, Kenyon J, Smith SD, Smith RJ, Djelantik B, Cremers CW, Van de Heyning PH, Willems PJ (1997) Linkage analysis of progressive hearing loss in five extended families maps the DFNA2 gene to a 1.25-Mb region on chromosome 1p. Genomics 41:70–74

    Article  PubMed  Google Scholar 

  • Van Hauwe P, Coucke PJ, Declau F, Kunst H, Ensink RJ, Marres HA, Cremers CW, Djelantik B, Smith SD, Kelley P, Van de Heyning PH, Van Camp G (1999) Deafness linked to DFNA2: one locus but how many genes? Nat Genet 21(3):263

    Article  PubMed  Google Scholar 

  • Veenstra RD (1996) Size and selectivity of gap junction channels formed from different connexins. J Bioenerg Biomemb 28:327–337

    Article  CAS  Google Scholar 

  • Verselis VK, Ginter CS, Bargiello TA (1994) Opposite voltage gating polarities of two closely related connexins. Nature 368:348–351

    Article  PubMed  CAS  Google Scholar 

  • White TW, Bruzzone R, Wolfram S, Paul DL, Goodenough DA (1994) Selective interactions among the multiple connexin proteins expressed in the vertebrate lens: the second extracellular domain is a determinant of compatibility between connexins. J Cell Biol 125:879–892

    Article  PubMed  CAS  Google Scholar 

  • White TW, Paul DL (1999) Genetic diseases and gene knockouts reveal diverse connexin functions. Annu Rev Physiol 61:283–310

    Article  PubMed  CAS  Google Scholar 

  • Xia JH, Liu CY, Tang BS, Pan Q, Huang L, Dai HP, Zhang BR, Xie W, Hu DX, Zheng D, Shi XL, Wang DA, Xia K, Yu KP, Liao XD, Feng Y, Yang YF, Xiao JY, Xie DH, Huang JZ (1998) Mutations in the gene encoding gap junction protein beta-3 associated with autosomal dominant hearing impairment. Nat Genet 20:370–373

    Article  PubMed  CAS  Google Scholar 

  • Yan D, Park HJ, Ouyang XM, Pandya A, Doi K, Erdenetungalag R, Du LL, Matsushiro N, Nance WE, Griffith AJ, Liu XZ (2003) Evidence of a founder effect for the 235delC mutation of GJB2 (connexin 26) in East Asians. Hum Genet 114:44–50

    Article  PubMed  CAS  Google Scholar 

  • Yum SW, Zhang J, Valiunas V, Kanaporis G, Brink PR, White TW, Scherer SS (2007) Human connexin26 and connexin30 form functional heteromeric and heterotypic channels. Am J Physiol Cell Physiol 293:C1032–C1048

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We thank the families for their kind participation in this study. This work was supported by NIH grants DCR01 05575 and NSFC 30528025 and 30728030 (China).

Conflict of interest statement

We have no conflict of interest to report.

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Authors and Affiliations

  1. Department of Otolaryngology (D-48), University of Miami, 1666 NW 12th Avenue, Miami, FL, 33136, USA

    Xue-Zhong Liu, Denise Yan, Xiao Mei Ouyang & Li Lin Du

  2. Department of Otorhinolaryngology Head and Neck Surgery, Institute of Otolaryngology, Chinese PLA General Hospital, Beijing, 100853, People’s Republic of China

    Yongyi Yuan, Yu Fei, Huijun Yuan, Xin Zhang, Guojian Wang, Dong Yang Kang & Pu Dai

  3. Department of Otolaryngology, Emory University School of Medicine, Atlanta, GA, 30322, USA

    Emilie Hong Ding, Wenxue Tang, Qing Chang, Shoeb Ahmad & Xi Lin

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  1. Xue-Zhong Liu
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  2. Yongyi Yuan
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  3. Denise Yan
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  4. Emilie Hong Ding
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  5. Xiao Mei Ouyang
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  6. Yu Fei
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  7. Wenxue Tang
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  8. Huijun Yuan
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  9. Qing Chang
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  10. Li Lin Du
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  11. Xin Zhang
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  12. Guojian Wang
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  13. Shoeb Ahmad
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  14. Dong Yang Kang
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  15. Xi Lin
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  16. Pu Dai
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Correspondence to Xue-Zhong Liu or Pu Dai.

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Liu, XZ., Yuan, Y., Yan, D. et al. Digenic inheritance of non-syndromic deafness caused by mutations at the gap junction proteins Cx26 and Cx31. Hum Genet 125, 53–62 (2009). https://doi.org/10.1007/s00439-008-0602-9

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