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Connexin 26: required for normal auditory function
Brain Research Reviews 32 Ž2000. 184–188 www.elsevier.comrlocaterbres
Short review
Connexin 26: required for normal auditory function Philip M. Kelley ) , Edward Cohn, William J. Kimberling Center for Hereditary Communication Disorders, Boys Town National Research Hospital, Omaha, NE 68131, USA
Abstract A single base deletion mutation, 35delG, in the gene ŽGJB2rDFNB1.ŽOMIM 121011r220290. encoding the gap junction protein, connexin 26 is the most important single cause of genetic hearing loss in European and American populations. It is the cause of one of the most common human genetic disorders with a frequency similar to cystic fibrosis. Mutations in this connexin are associated with skin disorders. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Connexin 26; Hearing loss; Deafness; Mutation
Contents 1. Introduction .
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2. Discovery and prevalence of Cx26 mutations associated with hearing loss .
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5. Mutational hot spot
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6. Clinical phenotype
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3. Spectrum of Cx26 recessive mutations . 4. Cx26 dominant mutations .
Acknowledgements . References
1. Introduction Connexin 26 ŽCx26., connexin 31 ŽCx31., and connexin 32 ŽCx32. are associated with hearing loss w2,14,33x. Connexin 30 ŽCx30., also a beta class connexin, is expressed in mammalian cochlea in many of the same cell types as Cx26 but hearing loss has not been associated with mutations in this gene w17,18x. Two of these connexins are associated with human skin disorders w25,26x. A single
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author.
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base deletion mutation in Cx26rGJB2rDFNB1, 35delG, is responsible for 10% of all childhood hearing loss and for 20% of all childhood hereditary hearing loss w12x. It is the cause of one of the most common human genetic diseases with a frequency similar to cystic fibrosis w12x. Cx26 was discovered by subtractive hybridization, selecting for mRNAs expressed in normal human mammary epithelial cells but not in mammary tumor cell lines w19x. Kikuchi et al. w15x studied the expression of Cx26 by immunostaining the rat cochlea. He found staining in non-sensory epithelial cells, including the interdental cells of the spiral limbus, inner sulcus cells, organ of Corti supporting cells, outer sulcus cells, and cells within the
0165-0173r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 1 7 3 Ž 9 9 . 0 0 0 8 0 - 6
P.M. Kelley et al.r Brain Research ReÕiews 32 (2000) 184–188
root processes of the spiral ligament w15x. Kikuchi et al. w15x also detected staining in connective tissue cells of the cochlea including various fibrocyte types of the spiral limbus and spiral ligament, basal and intermediate cells of the stria vascularis, and mesenchymal cells which line the scala vestibuli. This evidence was used to support a model where serially arranged gap junctions of epithelial cells and connective tissue cells serve as a mechanism for recycling endolymphatic potassium ions that pass through cochlear hair cells during auditory transduction w15,32x. In vitro mutagenesis, immunohistological, and physiological studies revealed many aspects of Cx26 structure and expression before its association with hearing loss w4x. A mouse knock-out of Cx26 was embryonic-lethal, indicating that it has an essential role during an early stage of murine development w11x.
2. Discovery and prevalence of Cx26 mutations associated with hearing loss Kelsell et al. found a missense mutation ŽM34T. in Cx26 during the study of a family with autosomal dominant palmoplantar keratoderma ŽPPK. and hearing loss. This mutation was segregated as autosomal dominant with hearing loss but not the PPK w14x. Two Cx26 non-sense mutations ŽW24X and W77X. were found in three consanguineous families with autosomal recessive non-syndromic hearing loss ŽRNSHL. mapping to the same genetic region Žchromosome 13q11–12. of Cx26rGJB2 w14x. This discovery of mutations in Cx26 resulted in the detection of many RNSHL families from Italy Ž34r54., Spain Ž6r12., and Israel Ž2r4.; affected patients were homozygous for a single base deletion 35delG w34x. Carrasquillo et al. w5x found the same mutation in two inbred kindreds of Israeli–Arabs with hearing loss mapping to 13q11–12. One of the kindreds had two distinct mutations, 35delG and W77R. Denoyelle et al. w9x found 62 mutant alleles in 39 of 65 RNSHL families. About 70% of the Cx26 mutant alleles was 35delG. This population was all Caucasian. Most of the families were from the United Kingdom and New Zealand but included families from Tunisia, France, Lebanon, Algeria and one family from Portugal. Of 82 RNSHL families Ž51 Italian and 31 Spanish., Estivill et al. w10x found that 49% of them had mutations in Cx26. Most had the most common mutant allele, 35delG. The genotype of sporadic cases of hearing loss was also examined and found to have mutations in Cx26 in 27% of the cases. Isolated cases of Cx26-associated hearing loss have also been reported from North and Central Africa w3,20x. Among 58 American families diagnosed with RNSHL, 24 families were found to have mutations in both alleles of Cx26 w13x. The two most common alleles were the 35delG and the 167delT. Thirtythree 35delG mutant alleles and nine 167delT mutant alleles were found. Among 96 hearing controls, two indi-
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viduals were heterozygous for 35delG but no incidences of 167delT were noted. The frequency of the 35delG mutation in the American population is approximately 2.8%. In a study of an Ashkenazi Jewish population with RNSHL, both 35delG and 167delT mutations were found w21x. The frequency of the 167delT mutant allele in the sampled population was 4.03%, while the 35delG in this population was 0.73%. A survey of 32 Japanese RNSHL families found no incidence of the 35delG mutation but did find a new mutation, 235delC, that was relatively common in the Japanese population w30x.
3. Spectrum of Cx26 recessive mutations More than 40 mutations have been reported for connexin 26 Žsee Table 1.. A single splice site mutant has been described w29x. Four silent mutations have been discovered. Four mutations in unaffected individuals have been recognized. Because they have not yet been associated with any pathological condition, they are described as normal variants. Three single base insertionrframeshift mutants and 10 small deletions have been recognized. Seven non-sense mutants and 16 missense mutations have been discovered. One of the missense mutations occurs at codon 1, resulting in conversion of the initiating methionine codon to a valine codon. The three missense mutations found in the first transmembrane domain, M1, and the first extracellular domain, E1, have been characterized as dominant mutations. Codon 34 is generally a methionine though other connexins generally have a leucine at this position. The mutations, W44C and R75W, occur at invariant codons where no change is apparently tolerated.
4. Cx26 dominant mutations The M34T mutations described as causing autosomal dominant hearing loss ŽDFNA3. have also been found in individuals with normal hearing. Scott et al. w28x found the M34T heterozygote in a control population having normal hearing. Kelley et al. w13x found that three of 96 unrelated normal hearing individuals were heterozygous for M34T. Two RNSHL families with M34T were also reported. In one family, the mutation did not segregate with the hearing loss. In the second family, the mutation segregated as a recessive allele associated with hearing loss when coupled with a second missense mutation, V95M. To further complicate this puzzle, White et al. w31x have characterized the M34T mutation in an in vitro functional assay using Xenopus laeÕis oocyte system. In this assay, Cx26 with the M34T mutation acted as a dominant inhibitor of wild-type Cx26. The M34T allele appears to be present in human populations at a frequency where it would be a major cause of hearing loss. That has not been reported. Furthermore, no homozygous individuals with hearing loss have
P.M. Kelley et al.r Brain Research ReÕiews 32 (2000) 184–188
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Table 1 Mutations in GJB1, the gene encoding human connexin 26 Table includes mutations classified as variants. These are mutations that have been found in the normal population but have not been associated with a pathological state. Mutations are identified by as suggested by the Nomenclature Working Group w1x. Codon location
Nucleotide acid
Mutation type
IVS1 M1V 10 10 10 W24X V27I I30I M34T V37I W44C E47X 56 Q57X Y65X R75W W77R W77X 78 F83L V84L L89L 89 L90P V95M R98Q H100Y 104 105 111 S113R 120 K122I Q124X R127H I128I Y136X R143W G160S F161S 170 P173R V182V R184P 191 S199F 211
IVS1q1G ) A 1A )G 35delG 31–68delŽ38. 35insG 74G ) A 79G ) A 90T ) ArC 101T )C 109G ) A 132G )C 139G ) T 167delT 169C ) T 195C )G 223C ) T 229T )C 231G ) A 235delC 249C )G 250G )C 267C ) A 267insT 269T )C 283G ) A 293G ) A 298C ) T 310–323del14 314–327delŽ14. 333–334delŽAA. 339T )G 358delŽGAG. 365A ) T 370C ) T 380G ) A 384C ) T ? 427T )C 478G ) A 482T )C 510insA 482T )C 546G )C 551G )C 572delT 596C ) T 631–632delŽGT.
splice site w8,21x initiation codon missense w10x deletionrframeshift w5,9,34x deletionrframeshift w9x insertionrframeshift w10x non-sense w14x variant w13x silent w31x missense w14x variant w13x missense w7x non-sense w9,10x deletionrframeshift w31x non-sense non-sense w10x missense w24x missense w5x non-sense w14x deletionrframeshift w28x variant w27x missense w13x silent w31x insertionrframeshift w12x missense missense w13x missense w12x missense w12x deletionrframeshift w22x deletionrframeshift w13x deletionrframeshift w13x missense w28x deletionrin frame w9x missense w12x non-sense w27x missense w10x silent w31x non-sense w28x missense w28x variant w27x missense w24x insertionrframeshift w8x missense w24x silent w27x missense w24x deletionrframeshift w23x missense w12x deletionrframeshift w13x
been detected. If one considers the frequency of this allele in the normal population, the absence of homozygous individuals with hearing loss suggests that this allele may not even be a recessive allele but a normal variant. Alternatively, such an allele might be embryonic-lethal in the homozygous state. The variation in phenotype among individuals that are homozygous for the recessive allele, 35delG, and the paradox of the M34T phenotype in vivo
and in vitro suggest that the role of Cx26 is more complex. Several connexins are known to be expressed in the cochlea including Cx26, Cx30, and Cx31. The interaction of these different connexins might be key to unraveling this puzzle. Two additional dominant mutations in Cx26 have been reported. Denoyelle et al. w7x reported a missense mutation, W44C, in a French family that segregated with hearing loss as an autosomal dominant allele. No incidence of this mutation was found in 190 unrelated control individuals. Richard et al. w26x found a missense mutation, R75W, in an Egyptian family where both hearing loss and palmoplantar keratoderma co-segregated. This allele was present once in a set of 154 Egyptian controls that had no evidence of hereditary skin disorder. This allele was also tested in the X. laeÕis oocyte system and gave similar results as the M34T allele.
5. Mutational hot spot At least 80% of the incidence of Cx26rGJB1r DFNB1-induced hearing loss is due to the single base deletion mutation, 35delG. Haplotype analysis indicates that this mutation arose independently many times and is not the result of a single founder w5,21x. The 35delG mutation occurs at a site that, except for a single base, shares homology with a putative mutational hot spot w16x. This sequence TGŽArG.ŽArG.ŽGrT.ŽArC. is thought to be an arrest site for DNA polymerase alpha and be especially prone to frameshift mutations. A single base insertion, 35insG, and another small deletion, 31–68delŽ38., have also been found at this site Žsee Table 1..
6. Clinical phenotype The phenotype of Cx26-induced hearing loss is variable in the extent of hearing loss. Some individuals homozygous for the Cx26 35delG mutation have a mild hearing loss while others have a profound hearing loss. Part or all of this variability in hearing loss is due to the apparent progression of hearing loss that has been documented in some individuals and will certainly require further study. Carrasquillo et al. w5x noted that even in an isolated kindred, the severity of hearing loss was variable among siblings. Denoyelle et al. w9x reported a range in hearing loss from moderate to profound hearing loss in patients all who were homozygous for the 35delG mutation. In a study of Askenazi Jews with RNSHL, no mention of variable hearing loss was mentioned w21x. Individuals heterozygous for Cx26 mutations were found to have subtle differences in their otoacoustic emissions, suggesting a semidominant effect w21x. Cohn et al. w6x examined 46 individuals in 24 families that were either homozygous or compound heterozygous for Cx26 mutations. A subset of these patients
P.M. Kelley et al.r Brain Research ReÕiews 32 (2000) 184–188
was examined for vestibular function, otoacoustic emissions, auditory brainstem response, temporal bone computed tomography, electrocardiography, urinalyses, dysmorphology, and thyroid function. No other symptoms were associated with the hearing loss. Hearing loss varied in severity from mild–moderate to profound. About onethird of the individuals with Cx26 mutations had progressive hearing loss. Most of the patients studied were homozygous for Cx26 35delG. It is not clear whether specific mutations could be correlated with a specific phenotype. Denoyelle et al. w8x studied the clinical phenotype in 104 families having two affected children but with parents of normal hearing and 47 sporadic cases of hearing loss. As they had reported earlier, 35delG homozygotes had varying degrees of hearing loss. Combining the clinical data from Cohn and Denoyelle, the phenotype of Cx26-induced hearing loss becomes more clear. All individuals with Cx26 mutations in both alleles have a significant prelingual hearing loss. Hearing loss associated with the 35delG mutation is variable, ranging from mild–moderate to profound. The hearing loss is non-syndromic, as other symptoms including vestibular defects are not apparent. At least two-thirds with hearing loss are non-progressive.
Acknowledgements This work was supported in part by NIH-NIDCD P01 DC01813-05 ŽW.J.K... Submitted August 13, 1999.
References w1x S.E., Recommendations for a nomenclature system for human gene mutations. Nomenclature Working Group, Hum. Mutat. 11 Ž1998. 1–3. w2x M. Bahr, F. Andres, V. Timmerman, M.E. Nelis, C. Van Broeckhoven, J. Dichgans, Central visual, acoustic, and motor pathway involvement in a Charcot–Marie–Tooth family with an Asn205Ser mutation in the connexin 32 gene win process citationx, J. Neurol. Neurosurg. Psychiatry 66 Ž1999. 202–206. w3x G.W. Brobby, B. Muller-Myhsok, R.D. Horstmann, Connexin 26 R143W mutation associated with recessive non-syndromic sensorineural deafness in Africa wletterx, N. Engl. J. Med. 338 Ž1998. 548–550. w4x R. Bruzzone, T.W. White, D.L. Paul, Connections with connexins: the molecular basis of direct intercellular signaling, Eur. J. Biochem. 238 Ž1996. 1–27. w5x M.M. Carrasquillo, J. Zlotogora, S. Barges, A. Chakravarti, Two different connexin 26 mutations in an inbred kindred segregating non-syndromic recessive deafness: implications for genetic studies in isolated populations, Hum. Mol. Genet. 6 Ž1997. 2163–2172. w6x E.S. Cohn, P.M. Kelley, T.W. Fowler, M.P. Gorga, D.M. Lefkowitz, H.J. Kuehn, G.B. Schaefer, L.S. Gobar, F.J. Hahn, D.J. Harris, W.J. Kimberling, Clinical studies of families with hearing loss attributable to mutations in the connexin 26 gene ŽGJB2rDFNB1., Pediatrics 103 Ž1999. 546–550. w7x F. Denoyelle, G. Lina-Granade, H. Plauchu, R. Bruzzone, H. Chaib, F. Levi-Acobas, D. Weil, C. Petit, Connexin 26 gene linked to a dominant deafness wletterx, Nature 393 Ž1998. 319–320.
187
w8x F. Denoyelle, S. Marlin, D. Weil, L. Moatti, P. Chauvin, E.N. Garabedian, C. Petit, Clinical features of the prevalent form of childhood deafness, DFNB1, due to a connexin 26 gene defect: implications for genetic counselling win process citationx, Lancet 353 Ž1999. 1298–1303. w9x F. Denoyelle, D. Weil, M.A. Maw, S.A. Wilcox, N.J. Lench, D.R. Allen-Powell, A.H. Osborn, H.H. Dahl, A. Middleton, M.J. Houseman, C. Dode, S. Marlin, A. Boulila-ElGaied, M. Grati, H. Ayadi, S. BenArab, P. Bitoun, G. Lina-Granade, J. Godet, M. Mustapha, J. Loiselet, E. El-Zir, A. Aubois, A. Joannard, C. Petit, Prelingual deafness: high prevalence of a 30delG mutation in the connexin 26 gene, Hum. Mol. Genet. 6 Ž1997. 2173–2177. w10x X. Estivill, P. Fortina, S. Surrey, R. Rabionet, S. Melchionda, L. D’Agruma, E. Mansfield, E. Rappaport, N. Govea, M. Mila, L. Zelante, P. Gasparini, Connexin 26 mutations in sporadic and inherited sensorineural deafness, Lancet 351 Ž1998. 394–398. w11x H.D. Gabriel, D. Jung, C. Butzler, A. Temme, O. Traub, E. Winterhager, K. Willecke, Transplacental uptake of glucose is decreased in embryonic- lethal connexin 26-deficient mice, J. Cell Biol. 140 Ž1998. 1453–1461. w12x G.E. Green, D.A. Scott, J.M. McDonald, G.G. Woodworth, V.C. Sheffield, R.J. Smith, Carrier rates in the midwestern United States for GJB2 mutations causing inherited deafness, JAMA 281 Ž1999. 2211–2216. w13x P.M. Kelley, D.J. Harris, B.C. Comer, J.W. Askew, T. Fowler, S.D. Smith, W.J. Kimberling, Novel mutations in the connexin 26 gene ŽGJB2. that cause autosomal recessive ŽDFNB1. hearing loss, Am. J. Hum. Genet. 62 Ž1998. 792–799. w14x D.P. Kelsell, J. Dunlop, H.P. Stevens, N.J. Lench, J.N. Liang, G. Parry, R.F. Mueller, I.M. Leigh, Connexin 26 mutations in hereditary non-syndromic sensorineural deafness, Nature 387 Ž1997. 80– 83. w15x T. Kikuchi, R.S. Kimura, D.L. Paul, J.C. Adams, Gap junctions in the rat cochlea: immunohistochemical and ultrastructural analysis, Anat. Embryol. ŽBerlin. 191 Ž1995. 101–118. w16x M. Krawczak, D.N. Cooper, Gene deletions causing human genetic disease: mechanisms of mutagenesis and the role of the local DNA sequence environment, Hum. Genet. 86 Ž1991. 425–441. w17x J. Lautermann, W.F. ten Cate, P. Altenhoff Jr., O. Traub, H. Frank, K. Jahnke, E. Winterhager, Expression of the gap junction connexins 26 and 30 in the rat cochlea, Cell Tissue Res. 294 Ž1998. 415–420. w18x J. Lautermann, W.F. ten Cate, P. Altenhoff, K. Jahnke, E. Winterhager, Developmental expression of connexins 26 and 30 in the cochlea, The Association for Research in Otolaryngology Ž1999., Abstract. w19x S.W. Lee, C. Tomasetto, D. Paul, K. Keyomarsi, R. Sager, Transcriptional downregulation of gap junction proteins blocks junctional communication in human mammary tumor cell lines, J. Cell Biol. 118 Ž1992. 1213–1221. w20x N.J. Lench, A.F. Markham, R.F. Mueller, D.P. Kelsell, R.J. Smith, P.J. Willems, I. Schatteman, H. Capon, P.J. Van De Heyning, G. van Camp, A Moroccan family with autosomal recessive sensorineural hearing loss caused by a mutation in the gap junction protein gene connexin 26 ŽGJB2., J. Med. Genet. 35 Ž1998. 151–152. w21x R.J. Morell, H.J. Kim, L.J. Hood, L. Goforth, K. Friderici, R. Fisher, G. van Camp, C.I. Berlin, C. Oddoux, H. Ostrer, B. Keats, T.B. Friedman, Mutations in the connexin 26 gene ŽGJB2. among Ashkenazi Jews with non-syndromic recessive deafness, N. Engl. J. Med. 339 Ž1998. 1500–1505. w22x R.F. Mueller, A. Nehammer, A. Middleton, M.J. Houseman, N.J. Lench, Non-syndromal sensorineural hearing impairmentrdeafness: genotype–phenotype studies in autosomal recessive families, sibs and sporadically affected individuals analysed for connexin 26 mutations, Am. J. Hum. Genet. 63 Ž1998. A375, Abstr. w23x A. Murgia, R. Polli, E. Leonardi, M. Martella, C. Vinanzi, E. Orzan, Genotype–phenotype correlations in Cx26 deafness, Am. J. Hum. Genet. 63 Ž1998. A376, Abstr.
188
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w24x R. Rabionet, S. Melchionda, L. D’Agruma, L. Zelante, P. Gasparini, S. Estivill, Mutations in the connexin 26 gene ŽGJB2. in Italian and Spanish patients with congenital deafness, Am. J. Hum. Genet. 63 Ž1998. A381, Abstr. w25x G. Richard, L.E. Smith, R.A. Bailey, P. Itin, D. Hohl, E.H.J. Epstein, J.J. DiGiovanna, J.G. Compton, S.J. Bale, Mutations in the human connexin gene GJB3 cause erythrokeratodermia variabilis win process citationx, Nat. Genet. 20 Ž1998. 366–369. w26x G. Richard, T.W. White, L.E. Smith, R.A. Bailey, J.G. Compton, D.L. Paul, S.J. Bale, Functional defects of Cx26 resulting from a heterozygous missense mutation in a family with dominant deaf mutism and palmoplantar keratoderma, Hum. Genet. 103 Ž1998. 393–399. w27x D.A. Scott, M.L. Kraft, R. Carmi, A. Ramesh, K. Elbedour, Y. Yairi, C.R. Srisailapathy, S.S. Rosengren, A.F. Markham, R.F. Mueller, N.J. Lench, G. van Camp, R.J. Smith, V.C. Sheffield, Identification of mutations in the connexin 26 gene that cause autosomal recessive nonsyndromic hearing loss, Hum. Mutat. 11 Ž1998. 387–394. w28x D.A. Scott, M.L. Kraft, E.M. Stone, V.C. Sheffield, R.J. Smith, Connexin mutations and hearing loss wletterx, Nature 391 Ž1998. 32. w29x D.A. Scott, J.M. McDonald, M.L. Kraft, R. Carmi, K. Elbedour, E.M. Stone, V.C. Sheffield, R.J. Smith, Screening for connexin 26
w30x
w31x w32x w33x
w34x
mutations in U.S. individuals with non-syndromic hearing loss, Am. J. Hum. Genet. 63 Ž1998. A384, Abstr. S. Usami, S. Abe, H. Shinkawa, M.D. Weston, P.M. Kelley, W.J. Kimberling, Prevalent mutations in connexin 26 gene in sporadic and recessive non-syndromic deafness in Japanese population, Abstracts for the Association for Research in Otolaryngology Ž1999., Abstract. T.W. White, M.R. Deans, D.P. Kelsell, D.L. Paul, Connexin mutations in deafness wletterx, Nature 394 Ž1998. 630–631. T.W. White, D.L. Paul, Genetic diseases and gene knockouts reveal diverse connexin functions, Annu. Rev. Physiol. 61 Ž1999. 283–310. J.H. Xia, C.Y. Liu, B.S. Tang, Q. Pan, L. Huang, H.P. Dai, B.R. Zhang, W. Xie, D.X. Hu, D. Zheng, X.L. Shi, D.A. Wang, K. Xia, K.P. Yu, X.D. Liao, Y. Feng, Y.F. Yang, J.Y. Xiao, D.H. Xie, J.Z. Huang, Mutations in the gene encoding gap junction protein beta-3 associated with autosomal dominant hearing impairment, Nat. Genet. 20 Ž1998. 370–373. L. Zelante, P. Gasparini, X. Estivill, S. Melchionda, L. D’Agruma, N. Govea, M. Mila, M.D. Monica, J. Lutfi, M. Shohat, E. Mansfield, K. Delgrosso, E. Rappaport, S. Surrey, P. Fortina, Connexin 26 mutations associated with the most common form of non-syndromic neurosensory autosomal recessive deafness ŽDFNB1. in Mediterraneans, Hum. Mol. Genet. 6 Ž1997. 1605–1609.
Short review
Connexin 26: required for normal auditory function Philip M. Kelley ) , Edward Cohn, William J. Kimberling Center for Hereditary Communication Disorders, Boys Town National Research Hospital, Omaha, NE 68131, USA
Abstract A single base deletion mutation, 35delG, in the gene ŽGJB2rDFNB1.ŽOMIM 121011r220290. encoding the gap junction protein, connexin 26 is the most important single cause of genetic hearing loss in European and American populations. It is the cause of one of the most common human genetic disorders with a frequency similar to cystic fibrosis. Mutations in this connexin are associated with skin disorders. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Connexin 26; Hearing loss; Deafness; Mutation
Contents 1. Introduction .
.......................................................................
2. Discovery and prevalence of Cx26 mutations associated with hearing loss .
184
......................................
185
.........................................................
185
................................................................
185
5. Mutational hot spot
....................................................................
186
6. Clinical phenotype
....................................................................
186
.....................................................................
187
..........................................................................
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3. Spectrum of Cx26 recessive mutations . 4. Cx26 dominant mutations .
Acknowledgements . References
1. Introduction Connexin 26 ŽCx26., connexin 31 ŽCx31., and connexin 32 ŽCx32. are associated with hearing loss w2,14,33x. Connexin 30 ŽCx30., also a beta class connexin, is expressed in mammalian cochlea in many of the same cell types as Cx26 but hearing loss has not been associated with mutations in this gene w17,18x. Two of these connexins are associated with human skin disorders w25,26x. A single
) Corresponding [email protected]
author.
Fax:
q1-402-498-6331;
e-m ail:
base deletion mutation in Cx26rGJB2rDFNB1, 35delG, is responsible for 10% of all childhood hearing loss and for 20% of all childhood hereditary hearing loss w12x. It is the cause of one of the most common human genetic diseases with a frequency similar to cystic fibrosis w12x. Cx26 was discovered by subtractive hybridization, selecting for mRNAs expressed in normal human mammary epithelial cells but not in mammary tumor cell lines w19x. Kikuchi et al. w15x studied the expression of Cx26 by immunostaining the rat cochlea. He found staining in non-sensory epithelial cells, including the interdental cells of the spiral limbus, inner sulcus cells, organ of Corti supporting cells, outer sulcus cells, and cells within the
0165-0173r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 1 7 3 Ž 9 9 . 0 0 0 8 0 - 6
P.M. Kelley et al.r Brain Research ReÕiews 32 (2000) 184–188
root processes of the spiral ligament w15x. Kikuchi et al. w15x also detected staining in connective tissue cells of the cochlea including various fibrocyte types of the spiral limbus and spiral ligament, basal and intermediate cells of the stria vascularis, and mesenchymal cells which line the scala vestibuli. This evidence was used to support a model where serially arranged gap junctions of epithelial cells and connective tissue cells serve as a mechanism for recycling endolymphatic potassium ions that pass through cochlear hair cells during auditory transduction w15,32x. In vitro mutagenesis, immunohistological, and physiological studies revealed many aspects of Cx26 structure and expression before its association with hearing loss w4x. A mouse knock-out of Cx26 was embryonic-lethal, indicating that it has an essential role during an early stage of murine development w11x.
2. Discovery and prevalence of Cx26 mutations associated with hearing loss Kelsell et al. found a missense mutation ŽM34T. in Cx26 during the study of a family with autosomal dominant palmoplantar keratoderma ŽPPK. and hearing loss. This mutation was segregated as autosomal dominant with hearing loss but not the PPK w14x. Two Cx26 non-sense mutations ŽW24X and W77X. were found in three consanguineous families with autosomal recessive non-syndromic hearing loss ŽRNSHL. mapping to the same genetic region Žchromosome 13q11–12. of Cx26rGJB2 w14x. This discovery of mutations in Cx26 resulted in the detection of many RNSHL families from Italy Ž34r54., Spain Ž6r12., and Israel Ž2r4.; affected patients were homozygous for a single base deletion 35delG w34x. Carrasquillo et al. w5x found the same mutation in two inbred kindreds of Israeli–Arabs with hearing loss mapping to 13q11–12. One of the kindreds had two distinct mutations, 35delG and W77R. Denoyelle et al. w9x found 62 mutant alleles in 39 of 65 RNSHL families. About 70% of the Cx26 mutant alleles was 35delG. This population was all Caucasian. Most of the families were from the United Kingdom and New Zealand but included families from Tunisia, France, Lebanon, Algeria and one family from Portugal. Of 82 RNSHL families Ž51 Italian and 31 Spanish., Estivill et al. w10x found that 49% of them had mutations in Cx26. Most had the most common mutant allele, 35delG. The genotype of sporadic cases of hearing loss was also examined and found to have mutations in Cx26 in 27% of the cases. Isolated cases of Cx26-associated hearing loss have also been reported from North and Central Africa w3,20x. Among 58 American families diagnosed with RNSHL, 24 families were found to have mutations in both alleles of Cx26 w13x. The two most common alleles were the 35delG and the 167delT. Thirtythree 35delG mutant alleles and nine 167delT mutant alleles were found. Among 96 hearing controls, two indi-
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viduals were heterozygous for 35delG but no incidences of 167delT were noted. The frequency of the 35delG mutation in the American population is approximately 2.8%. In a study of an Ashkenazi Jewish population with RNSHL, both 35delG and 167delT mutations were found w21x. The frequency of the 167delT mutant allele in the sampled population was 4.03%, while the 35delG in this population was 0.73%. A survey of 32 Japanese RNSHL families found no incidence of the 35delG mutation but did find a new mutation, 235delC, that was relatively common in the Japanese population w30x.
3. Spectrum of Cx26 recessive mutations More than 40 mutations have been reported for connexin 26 Žsee Table 1.. A single splice site mutant has been described w29x. Four silent mutations have been discovered. Four mutations in unaffected individuals have been recognized. Because they have not yet been associated with any pathological condition, they are described as normal variants. Three single base insertionrframeshift mutants and 10 small deletions have been recognized. Seven non-sense mutants and 16 missense mutations have been discovered. One of the missense mutations occurs at codon 1, resulting in conversion of the initiating methionine codon to a valine codon. The three missense mutations found in the first transmembrane domain, M1, and the first extracellular domain, E1, have been characterized as dominant mutations. Codon 34 is generally a methionine though other connexins generally have a leucine at this position. The mutations, W44C and R75W, occur at invariant codons where no change is apparently tolerated.
4. Cx26 dominant mutations The M34T mutations described as causing autosomal dominant hearing loss ŽDFNA3. have also been found in individuals with normal hearing. Scott et al. w28x found the M34T heterozygote in a control population having normal hearing. Kelley et al. w13x found that three of 96 unrelated normal hearing individuals were heterozygous for M34T. Two RNSHL families with M34T were also reported. In one family, the mutation did not segregate with the hearing loss. In the second family, the mutation segregated as a recessive allele associated with hearing loss when coupled with a second missense mutation, V95M. To further complicate this puzzle, White et al. w31x have characterized the M34T mutation in an in vitro functional assay using Xenopus laeÕis oocyte system. In this assay, Cx26 with the M34T mutation acted as a dominant inhibitor of wild-type Cx26. The M34T allele appears to be present in human populations at a frequency where it would be a major cause of hearing loss. That has not been reported. Furthermore, no homozygous individuals with hearing loss have
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Table 1 Mutations in GJB1, the gene encoding human connexin 26 Table includes mutations classified as variants. These are mutations that have been found in the normal population but have not been associated with a pathological state. Mutations are identified by as suggested by the Nomenclature Working Group w1x. Codon location
Nucleotide acid
Mutation type
IVS1 M1V 10 10 10 W24X V27I I30I M34T V37I W44C E47X 56 Q57X Y65X R75W W77R W77X 78 F83L V84L L89L 89 L90P V95M R98Q H100Y 104 105 111 S113R 120 K122I Q124X R127H I128I Y136X R143W G160S F161S 170 P173R V182V R184P 191 S199F 211
IVS1q1G ) A 1A )G 35delG 31–68delŽ38. 35insG 74G ) A 79G ) A 90T ) ArC 101T )C 109G ) A 132G )C 139G ) T 167delT 169C ) T 195C )G 223C ) T 229T )C 231G ) A 235delC 249C )G 250G )C 267C ) A 267insT 269T )C 283G ) A 293G ) A 298C ) T 310–323del14 314–327delŽ14. 333–334delŽAA. 339T )G 358delŽGAG. 365A ) T 370C ) T 380G ) A 384C ) T ? 427T )C 478G ) A 482T )C 510insA 482T )C 546G )C 551G )C 572delT 596C ) T 631–632delŽGT.
splice site w8,21x initiation codon missense w10x deletionrframeshift w5,9,34x deletionrframeshift w9x insertionrframeshift w10x non-sense w14x variant w13x silent w31x missense w14x variant w13x missense w7x non-sense w9,10x deletionrframeshift w31x non-sense non-sense w10x missense w24x missense w5x non-sense w14x deletionrframeshift w28x variant w27x missense w13x silent w31x insertionrframeshift w12x missense missense w13x missense w12x missense w12x deletionrframeshift w22x deletionrframeshift w13x deletionrframeshift w13x missense w28x deletionrin frame w9x missense w12x non-sense w27x missense w10x silent w31x non-sense w28x missense w28x variant w27x missense w24x insertionrframeshift w8x missense w24x silent w27x missense w24x deletionrframeshift w23x missense w12x deletionrframeshift w13x
been detected. If one considers the frequency of this allele in the normal population, the absence of homozygous individuals with hearing loss suggests that this allele may not even be a recessive allele but a normal variant. Alternatively, such an allele might be embryonic-lethal in the homozygous state. The variation in phenotype among individuals that are homozygous for the recessive allele, 35delG, and the paradox of the M34T phenotype in vivo
and in vitro suggest that the role of Cx26 is more complex. Several connexins are known to be expressed in the cochlea including Cx26, Cx30, and Cx31. The interaction of these different connexins might be key to unraveling this puzzle. Two additional dominant mutations in Cx26 have been reported. Denoyelle et al. w7x reported a missense mutation, W44C, in a French family that segregated with hearing loss as an autosomal dominant allele. No incidence of this mutation was found in 190 unrelated control individuals. Richard et al. w26x found a missense mutation, R75W, in an Egyptian family where both hearing loss and palmoplantar keratoderma co-segregated. This allele was present once in a set of 154 Egyptian controls that had no evidence of hereditary skin disorder. This allele was also tested in the X. laeÕis oocyte system and gave similar results as the M34T allele.
5. Mutational hot spot At least 80% of the incidence of Cx26rGJB1r DFNB1-induced hearing loss is due to the single base deletion mutation, 35delG. Haplotype analysis indicates that this mutation arose independently many times and is not the result of a single founder w5,21x. The 35delG mutation occurs at a site that, except for a single base, shares homology with a putative mutational hot spot w16x. This sequence TGŽArG.ŽArG.ŽGrT.ŽArC. is thought to be an arrest site for DNA polymerase alpha and be especially prone to frameshift mutations. A single base insertion, 35insG, and another small deletion, 31–68delŽ38., have also been found at this site Žsee Table 1..
6. Clinical phenotype The phenotype of Cx26-induced hearing loss is variable in the extent of hearing loss. Some individuals homozygous for the Cx26 35delG mutation have a mild hearing loss while others have a profound hearing loss. Part or all of this variability in hearing loss is due to the apparent progression of hearing loss that has been documented in some individuals and will certainly require further study. Carrasquillo et al. w5x noted that even in an isolated kindred, the severity of hearing loss was variable among siblings. Denoyelle et al. w9x reported a range in hearing loss from moderate to profound hearing loss in patients all who were homozygous for the 35delG mutation. In a study of Askenazi Jews with RNSHL, no mention of variable hearing loss was mentioned w21x. Individuals heterozygous for Cx26 mutations were found to have subtle differences in their otoacoustic emissions, suggesting a semidominant effect w21x. Cohn et al. w6x examined 46 individuals in 24 families that were either homozygous or compound heterozygous for Cx26 mutations. A subset of these patients
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was examined for vestibular function, otoacoustic emissions, auditory brainstem response, temporal bone computed tomography, electrocardiography, urinalyses, dysmorphology, and thyroid function. No other symptoms were associated with the hearing loss. Hearing loss varied in severity from mild–moderate to profound. About onethird of the individuals with Cx26 mutations had progressive hearing loss. Most of the patients studied were homozygous for Cx26 35delG. It is not clear whether specific mutations could be correlated with a specific phenotype. Denoyelle et al. w8x studied the clinical phenotype in 104 families having two affected children but with parents of normal hearing and 47 sporadic cases of hearing loss. As they had reported earlier, 35delG homozygotes had varying degrees of hearing loss. Combining the clinical data from Cohn and Denoyelle, the phenotype of Cx26-induced hearing loss becomes more clear. All individuals with Cx26 mutations in both alleles have a significant prelingual hearing loss. Hearing loss associated with the 35delG mutation is variable, ranging from mild–moderate to profound. The hearing loss is non-syndromic, as other symptoms including vestibular defects are not apparent. At least two-thirds with hearing loss are non-progressive.
Acknowledgements This work was supported in part by NIH-NIDCD P01 DC01813-05 ŽW.J.K... Submitted August 13, 1999.
References w1x S.E., Recommendations for a nomenclature system for human gene mutations. Nomenclature Working Group, Hum. Mutat. 11 Ž1998. 1–3. w2x M. Bahr, F. Andres, V. Timmerman, M.E. Nelis, C. Van Broeckhoven, J. Dichgans, Central visual, acoustic, and motor pathway involvement in a Charcot–Marie–Tooth family with an Asn205Ser mutation in the connexin 32 gene win process citationx, J. Neurol. Neurosurg. Psychiatry 66 Ž1999. 202–206. w3x G.W. Brobby, B. Muller-Myhsok, R.D. Horstmann, Connexin 26 R143W mutation associated with recessive non-syndromic sensorineural deafness in Africa wletterx, N. Engl. J. Med. 338 Ž1998. 548–550. w4x R. Bruzzone, T.W. White, D.L. Paul, Connections with connexins: the molecular basis of direct intercellular signaling, Eur. J. Biochem. 238 Ž1996. 1–27. w5x M.M. Carrasquillo, J. Zlotogora, S. Barges, A. Chakravarti, Two different connexin 26 mutations in an inbred kindred segregating non-syndromic recessive deafness: implications for genetic studies in isolated populations, Hum. Mol. Genet. 6 Ž1997. 2163–2172. w6x E.S. Cohn, P.M. Kelley, T.W. Fowler, M.P. Gorga, D.M. Lefkowitz, H.J. Kuehn, G.B. Schaefer, L.S. Gobar, F.J. Hahn, D.J. Harris, W.J. Kimberling, Clinical studies of families with hearing loss attributable to mutations in the connexin 26 gene ŽGJB2rDFNB1., Pediatrics 103 Ž1999. 546–550. w7x F. Denoyelle, G. Lina-Granade, H. Plauchu, R. Bruzzone, H. Chaib, F. Levi-Acobas, D. Weil, C. Petit, Connexin 26 gene linked to a dominant deafness wletterx, Nature 393 Ž1998. 319–320.
187
w8x F. Denoyelle, S. Marlin, D. Weil, L. Moatti, P. Chauvin, E.N. Garabedian, C. Petit, Clinical features of the prevalent form of childhood deafness, DFNB1, due to a connexin 26 gene defect: implications for genetic counselling win process citationx, Lancet 353 Ž1999. 1298–1303. w9x F. Denoyelle, D. Weil, M.A. Maw, S.A. Wilcox, N.J. Lench, D.R. Allen-Powell, A.H. Osborn, H.H. Dahl, A. Middleton, M.J. Houseman, C. Dode, S. Marlin, A. Boulila-ElGaied, M. Grati, H. Ayadi, S. BenArab, P. Bitoun, G. Lina-Granade, J. Godet, M. Mustapha, J. Loiselet, E. El-Zir, A. Aubois, A. Joannard, C. Petit, Prelingual deafness: high prevalence of a 30delG mutation in the connexin 26 gene, Hum. Mol. Genet. 6 Ž1997. 2173–2177. w10x X. Estivill, P. Fortina, S. Surrey, R. Rabionet, S. Melchionda, L. D’Agruma, E. Mansfield, E. Rappaport, N. Govea, M. Mila, L. Zelante, P. Gasparini, Connexin 26 mutations in sporadic and inherited sensorineural deafness, Lancet 351 Ž1998. 394–398. w11x H.D. Gabriel, D. Jung, C. Butzler, A. Temme, O. Traub, E. Winterhager, K. Willecke, Transplacental uptake of glucose is decreased in embryonic- lethal connexin 26-deficient mice, J. Cell Biol. 140 Ž1998. 1453–1461. w12x G.E. Green, D.A. Scott, J.M. McDonald, G.G. Woodworth, V.C. Sheffield, R.J. Smith, Carrier rates in the midwestern United States for GJB2 mutations causing inherited deafness, JAMA 281 Ž1999. 2211–2216. w13x P.M. Kelley, D.J. Harris, B.C. Comer, J.W. Askew, T. Fowler, S.D. Smith, W.J. Kimberling, Novel mutations in the connexin 26 gene ŽGJB2. that cause autosomal recessive ŽDFNB1. hearing loss, Am. J. Hum. Genet. 62 Ž1998. 792–799. w14x D.P. Kelsell, J. Dunlop, H.P. Stevens, N.J. Lench, J.N. Liang, G. Parry, R.F. Mueller, I.M. Leigh, Connexin 26 mutations in hereditary non-syndromic sensorineural deafness, Nature 387 Ž1997. 80– 83. w15x T. Kikuchi, R.S. Kimura, D.L. Paul, J.C. Adams, Gap junctions in the rat cochlea: immunohistochemical and ultrastructural analysis, Anat. Embryol. ŽBerlin. 191 Ž1995. 101–118. w16x M. Krawczak, D.N. Cooper, Gene deletions causing human genetic disease: mechanisms of mutagenesis and the role of the local DNA sequence environment, Hum. Genet. 86 Ž1991. 425–441. w17x J. Lautermann, W.F. ten Cate, P. Altenhoff Jr., O. Traub, H. Frank, K. Jahnke, E. Winterhager, Expression of the gap junction connexins 26 and 30 in the rat cochlea, Cell Tissue Res. 294 Ž1998. 415–420. w18x J. Lautermann, W.F. ten Cate, P. Altenhoff, K. Jahnke, E. Winterhager, Developmental expression of connexins 26 and 30 in the cochlea, The Association for Research in Otolaryngology Ž1999., Abstract. w19x S.W. Lee, C. Tomasetto, D. Paul, K. Keyomarsi, R. Sager, Transcriptional downregulation of gap junction proteins blocks junctional communication in human mammary tumor cell lines, J. Cell Biol. 118 Ž1992. 1213–1221. w20x N.J. Lench, A.F. Markham, R.F. Mueller, D.P. Kelsell, R.J. Smith, P.J. Willems, I. Schatteman, H. Capon, P.J. Van De Heyning, G. van Camp, A Moroccan family with autosomal recessive sensorineural hearing loss caused by a mutation in the gap junction protein gene connexin 26 ŽGJB2., J. Med. Genet. 35 Ž1998. 151–152. w21x R.J. Morell, H.J. Kim, L.J. Hood, L. Goforth, K. Friderici, R. Fisher, G. van Camp, C.I. Berlin, C. Oddoux, H. Ostrer, B. Keats, T.B. Friedman, Mutations in the connexin 26 gene ŽGJB2. among Ashkenazi Jews with non-syndromic recessive deafness, N. Engl. J. Med. 339 Ž1998. 1500–1505. w22x R.F. Mueller, A. Nehammer, A. Middleton, M.J. Houseman, N.J. Lench, Non-syndromal sensorineural hearing impairmentrdeafness: genotype–phenotype studies in autosomal recessive families, sibs and sporadically affected individuals analysed for connexin 26 mutations, Am. J. Hum. Genet. 63 Ž1998. A375, Abstr. w23x A. Murgia, R. Polli, E. Leonardi, M. Martella, C. Vinanzi, E. Orzan, Genotype–phenotype correlations in Cx26 deafness, Am. J. Hum. Genet. 63 Ž1998. A376, Abstr.
188
P.M. Kelley et al.r Brain Research ReÕiews 32 (2000) 184–188
w24x R. Rabionet, S. Melchionda, L. D’Agruma, L. Zelante, P. Gasparini, S. Estivill, Mutations in the connexin 26 gene ŽGJB2. in Italian and Spanish patients with congenital deafness, Am. J. Hum. Genet. 63 Ž1998. A381, Abstr. w25x G. Richard, L.E. Smith, R.A. Bailey, P. Itin, D. Hohl, E.H.J. Epstein, J.J. DiGiovanna, J.G. Compton, S.J. Bale, Mutations in the human connexin gene GJB3 cause erythrokeratodermia variabilis win process citationx, Nat. Genet. 20 Ž1998. 366–369. w26x G. Richard, T.W. White, L.E. Smith, R.A. Bailey, J.G. Compton, D.L. Paul, S.J. Bale, Functional defects of Cx26 resulting from a heterozygous missense mutation in a family with dominant deaf mutism and palmoplantar keratoderma, Hum. Genet. 103 Ž1998. 393–399. w27x D.A. Scott, M.L. Kraft, R. Carmi, A. Ramesh, K. Elbedour, Y. Yairi, C.R. Srisailapathy, S.S. Rosengren, A.F. Markham, R.F. Mueller, N.J. Lench, G. van Camp, R.J. Smith, V.C. Sheffield, Identification of mutations in the connexin 26 gene that cause autosomal recessive nonsyndromic hearing loss, Hum. Mutat. 11 Ž1998. 387–394. w28x D.A. Scott, M.L. Kraft, E.M. Stone, V.C. Sheffield, R.J. Smith, Connexin mutations and hearing loss wletterx, Nature 391 Ž1998. 32. w29x D.A. Scott, J.M. McDonald, M.L. Kraft, R. Carmi, K. Elbedour, E.M. Stone, V.C. Sheffield, R.J. Smith, Screening for connexin 26
w30x
w31x w32x w33x
w34x
mutations in U.S. individuals with non-syndromic hearing loss, Am. J. Hum. Genet. 63 Ž1998. A384, Abstr. S. Usami, S. Abe, H. Shinkawa, M.D. Weston, P.M. Kelley, W.J. Kimberling, Prevalent mutations in connexin 26 gene in sporadic and recessive non-syndromic deafness in Japanese population, Abstracts for the Association for Research in Otolaryngology Ž1999., Abstract. T.W. White, M.R. Deans, D.P. Kelsell, D.L. Paul, Connexin mutations in deafness wletterx, Nature 394 Ž1998. 630–631. T.W. White, D.L. Paul, Genetic diseases and gene knockouts reveal diverse connexin functions, Annu. Rev. Physiol. 61 Ž1999. 283–310. J.H. Xia, C.Y. Liu, B.S. Tang, Q. Pan, L. Huang, H.P. Dai, B.R. Zhang, W. Xie, D.X. Hu, D. Zheng, X.L. Shi, D.A. Wang, K. Xia, K.P. Yu, X.D. Liao, Y. Feng, Y.F. Yang, J.Y. Xiao, D.H. Xie, J.Z. Huang, Mutations in the gene encoding gap junction protein beta-3 associated with autosomal dominant hearing impairment, Nat. Genet. 20 Ž1998. 370–373. L. Zelante, P. Gasparini, X. Estivill, S. Melchionda, L. D’Agruma, N. Govea, M. Mila, M.D. Monica, J. Lutfi, M. Shohat, E. Mansfield, K. Delgrosso, E. Rappaport, S. Surrey, P. Fortina, Connexin 26 mutations associated with the most common form of non-syndromic neurosensory autosomal recessive deafness ŽDFNB1. in Mediterraneans, Hum. Mol. Genet. 6 Ž1997. 1605–1609.