Hearing loss is mainly due to geneticfactors1. Mutations in the connexin-

26 gene (GJB2), located on 13q12, areresponsible for non-syndromic recessiveand dominant forms of deafness2–4. Con-nexin-31 and connexin-32 have also beenimplicated in deafness5,6. The identifica-tion of deaf families linked to 13q12 butnegative for mutations in GJB2 (ref. 7) sug-gested the presence of other deafness genesin this region. Recently, the mouse con-nexin-30 gene (Gjb6), which is expressedin cochlea, has been mapped to a regionwith syntenic homology to human chro-

mosome 13q12 (refs 8,9). To verify ifhuman GJB6 is involved in deafness, wecloned a 1,799-bp cDNA fragment con-taining an ORF of 261 amino acids (EMBLHSA005585). CX30 protein has a structuresimilar to that of other connexins10 andshares 93% homology with mouse Cx30and 76% identity with human CX26. GJB6is not interrupted by introns and maps tochromosome 13q12, approximately 800 kbcentromeric to GJB2.

SSCP mutational analysis in 198 deafpatients, including 38 families linked to13q12, revealed a threonine-to-methion-

ine change at position 5 (T5M) in an Itali-an family affected by bilateral middle/high-frequency hearing loss (Fig. 1a–c).Audiograms in T5M family membersshowed a 20–50-dB decrease at frequen-cies of 2,000–8,000 Hz (I-2), a progressiveimpaired threshold above 500 Hz (II-1)and a profound sensorineural deafness(II-2). This variability of hearing impair-ment can be explained by a differentexpressivity of the disease, which is almostthe rule for dominant deafness.

Northern blots, RT-PCR and in situhybridization on mouse embryos revealedGjb6 expression in trachea, thyroid, thy-mus, brain and cochlea, confirmingreported expression patterns (refs 8,9,11).

The threonine residue at position 5 isevolutionarily conserved and also presentin human connexin 26 (Fig. 1d). TheT5M substitution abolishes a hydrophilicresidue possibly involved in inter- or

Mutations in GJB6 causenonsyndromic autosomal dominantdeafness at DFNA3 locus

correspondence

16 nature genetics • volume 23 • september 1999

light of speculation that some form ofDNA rearrangement is involved duringthe selective expression of individualodorant receptor genes13.

Our studies demonstrate the advantageof combining artificial chromosometransgenesis with zebrafish embryology todecipher the complexity of developmentalgene regulation. This technique should beuseful for the identification and analysisof mutated zebrafish genes created bygenetic screens14,15.

Jason R. Jessen1, Catherine E. Willett2

& Shuo Lin1

Fig. 2 Zebrafish rag1 isexpressed in olfactory tissues.Several of the rag1 reporterconstructs, including the ragPAC, directed GFP expressionin embryonic olfactory neu-rons and adult olfactoryepithelium of germline trans-genic zebrafish. We detectedGFP expression, beginning atapproximately 30 h post-fer-tilization, in individual neu-rons at the apical surface ofthe two olfactory placodes(a, dorsal view of a day 5embryo transgenic with therag PAC; b, frontal view withdorsal to the top). As theolfactory placodes differenti-ated into adult epithelium,GFP expression became restricted to the sensory epithelium of adult olfactory rosettes (c). We detected endogenous rag1 expression in the olfactory placodesusing whole-mount RNA in situ hybridization (d, dorsal view of a day 5 albino embryo; e, frontal view with dorsal to the top). At higher magnification, weobserved rag1 expression in individual neurons of the olfactory placodes (f, dorsal view of day 5 embryo, arrow denotes an extended axon). The digoxigenin-labelled, anti-sense rag1 RNA probe used in this study corresponded to the third coding exon. To assess rag1 expression in adult olfactory tissues, we dissectedolfactory bulbs from three-month-old transgenic zebrafish containing the rag PAC and performed RT-PCR on total RNA (1 µg). PCR primers were specific to thefirst and second coding exons, assuring amplification of correctly spliced mRNA. After 25 cycles of amplification, PCR products were analysed using Southernhybridization (the amount of rag1 PCR product from thymus was diluted 1/4 before gel loading). g, We detected rag1 transcripts in adult olfactory bulbs,mesonephros (kidney) and thymus, but not in heart or trunk (muscle) samples.

1Institute of Molecular Medicine and Geneticsand Department of Biochemistry and MolecularBiology, Medical College of Georgia, Augusta,Georgia 30912, USA. 2Department of Biology,Massachusetts Institute of Technology,Cambridge, Massachusetts 02139, USA.Correspondence should be addressed to S.L.(e-mail: slin@immag.mcg.edu).

AcknowledgementsWe thank members of our laboratories fordiscussions and assistance, and L. Steiner forhelpful discussions and support. This work wassupported by grants from NIH to S.L. (R01RR13227-01) and L. Steiner (2RO1 AI08054).C.E.W. supported by NIH grant 9T32 AI07463.

1. Tonegawa, S. Nature 302, 575–581 (1983).2. Oettinger, M.A., Schatz, D.G., Gorka, C. & Baltimore,

D. Science 248, 1517–1523 (1990).3. Mombaerts, P. et al. Cell 68, 869–877 (1992).4. Shinkai, Y. et al. Cell 68, 855–867 (1992).5. Brown, S.T. et al. J. Immunol. 158, 5071–5074 (1997).6. Fuller, K. & Storb, U. Mol. Immunol. 34, 939–954

(1997).7. Kurioka, H. et al. Mol. Immunol. 33, 1059–1066

(1996).8. Zarrin, A.A., Fong, I., Malkin, L., Marsden, P.A. &

Berinstein, N.L. J. Immunol. 159, 4382–4394 (1997).9. Willett, C.E., Zapata, A.G., Hopkins, N. & Steiner, L.A.

Dev. Biol. 182, 331–341 (1997).10. Willett, C.E., Cherry, J.J. & Steiner, L.A.

Immunogenetics 45, 394–404 (1997).11. Jessen, J.R. et al. Proc. Natl Acad. Sci. USA 95,

5121–5126 (1998).12. Barth, A.L., Justice, N.J. & Ngai, J. Neuron 16, 23–34

(1996).13. Chess, A. Adv. Immunol. 69, 437–447 (1998).14. Haffter, P. et al. Development 123, 1–36 (1996).15. Driever, W. et al. Development 123, 37–46 (1996).

b c

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nature genetics • volume 23 • september 1999 17

intramolecular interactions with a largerhydrophobic residue. Moreover, whenthe secondary structure probabilitieswere computed, this substitution in-creased the probability of residue 5 being

in a helix (from 0.58 to 0.8, maximum 1).We assessed the role of T5M by electro-physiological studies on Xenopus laevisoocytes10,12,13 by injection with in vitro-transcribed complementary RNA enco-

ding human wild-type or mutant CX30,or combinations of both. Paired oocytesinjected with water, CX30, mutantT5MCX30, or CX30 and T5MCX30 hadsimilar membrane potentials (41.5±1.9mV, 42.0±1.5 mV, –39.5±1.9 and–39.3±4.2 mV, respectively). We obtainedgap junctional currents with wild-typeCX30, indicating high levels of intercellu-lar channel activity (Fig. 2a,c), in agree-ment with mouse data8. On the contrary,both the T5MCX30 mutant and the con-trol (water-injected oocytes) gave nega-tive results that did not induce couplingbetween paired oocytes and gave onlysmall currents due to the endogenousCX38 (Fig. 2c). To test a possible domi-nant-negative effect of T5M, we co-expressed wild-type and mutant CX30.T5MCX30 suppressed the wild-typetransjunctional conductance in a domi-nant-negative manner (Fig. 2b,c), result-ing in inhibition of intercellularcoupling. These data further support thehypothesis that T5M mutation causeshearing loss by dominant inhibition ofthe activity of wild-type CX30 channels.A similar dominant-negative effect hasbeen demonstrated for mutation M34Tof connexin-26 (ref. 14). This dominant-negative inhibitory effect is likely to bedue to direct interaction of the intact anddefective connexins at the plasma mem-brane level, as has been shown for otherdominant-negative inhibitory ion chan-nel subunits15. Other mechanisms suchas defective trafficking, however, can notbe excluded. We have shown here thatGJB6 encodes the fourth connexinmutated in deafness and is the secondDFNA3 gene.

Anna Grifa1, Carsten A. Wagner2,Lucrezia D’Ambrosio1, Salvatore Melchionda1,Francesco Bernardi3, Nuria Lopez-Bigas4,Raquel Rabionet4, Mariona Arbones4,Matteo Della Monica5, Xavier Estivill4,Leopoldo Zelante1, Florian Lang2

& Paolo Gasparini11Servizio di Genetica Medica, IRCCS-Ospedale“CSS”, San Giovanni Rotondo, Italy. 2Instituteof Physiology, University of Tübingen,Gmelinstr. 5, 72076 Tübingen, Germany.3Dipartimento di Biochimica, Università degliStudi di Ferrara, Italy. 4Departament deGenetica Molecular, IRO-Hospital Duran iReynals, Barcelona, Spain. 5Servizio di GeneticaMedica, Ospedale “G.Moscati”, Avellino, Italy.Correspondence should be addressed to P.G. (e-mail: genetcss@fg.nettuno.it).

AcknowledgementsThis study was supported by grants from Telethon,Italian Ministry of Health and CNR-PF.Biotecnologie (to P.G.); EC (CT 98-3514),Federal Ministry of Education, Science, Research and

Fig. 1 GJB6 mutational results.a, Sequencing chromographs for T5Mmutation. b, Pedigree harbouringGJB6 mutation. c, RsaI digestion afterPCR obtained with a mutagenesisprimer designed to create a restric-tion site in wild type but not themutant. T5M mutant allele, 266 bp;normal allele, 245 bp. T5M was notfound in 120 normal chromosomes.d, Alignment of the first nine codons of human, gibbon and mouse CX30, as well as human CX26,sequences. A high degree of cross-species homology is seen, including the threonine 5 residue, which isalso conserved in human CX26.

a bnormal

human CX30 mutant

human CX30

gibbon CX30

mouse CX30

human CX26

mutant

245 bp226 bp

d

Oocyte injection Mean conductance s.e.m. number of pairs

oocyte 1/oocyte 2 (µS) (±)H2O/H2O 0.05* 0.02 10CX30/CX30 2.45 0.18 8T5MCX30/T5MCX30 0.11* 0.05 4T5MCX30+CX30/T5MCX30+CX30 0.07* 0.06 4

Fig. 2 Electrophysiological studies on paired X. laevis oocytes. a, Original tracing from a wild-typeCX30/CX30-expressing oocyte pair. Currents were measured in oocyte 2 while both oocytes were held at –60mV and oocyte 1 was stepped in 10 mV increments to –120 mV (left) and +60 mV (right). b, Original tracingfrom an oocyte pair co-expressing wild-type and mutant CX30. Currents were measured as above. c, Con-ductance results for wild-type and mutant CX30 channels. We obtained gap junctional initial conductancegi from the peak current, and the steady-state conductance gss after the decaying current reached a steadystate. All data are means±s.e.m. Asterisks indicate results that are significantly different from CX30/CX30.

a

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connexin 30 wild type

connexin 30 wild type + mutant

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Technology (Fö. 01KS9602) and InterdisciplinaryCenter for Clinical Research (to C.A.W. and F.L.);and Fondo de Investigaciones Sanitarias de laSeguridad Social (to N.L.-B., R.R. and X.E.).

1. Cohen, M.M. Jr & Gorlin, R.J. Hereditary HearingLoss and its Syndromes 9–21 (Oxford UniversityPress, Oxford, 1995).

2. Kelsell, D.P. et al. Nature 387, 80–83 (1997).3. Zelante, L. et al. Hum. Mol. Genet. 9, 1605–1609

(1997).4. Denoyelle, F. et al. Nature 393, 319–320 (1998).5. Xia, J.H. et al. Nature Genet. 20, 370–373 (1998).6. Stojkovic, T., Latour, P., Vandenberghe, A.,

Hurtevent, J.F. & Vermersch, P. Neurology 52,1010–1014 (1999).

7. Estivill, X. et al. Lancet 351, 394–398 (1998).8. Dahl, E. et al. J. Biol. Chem. 271, 17903–17910

(1996).9. Lautermann, J. et al. Cell Tissue Res. 294, 415–420

(1998).

10. Kunzelman, P. et al. Glia 25, 111–119 (1999).11. Ebihara, L. Methods Enzymol. 207, 376–380

(1992).12. Harris, A.L., Spray, D.C. & Bennett, M.V.L. J. Gen.

Physiol. 77, 95–117 (1981).13. Spray, D.C., Harris, A.L. & Bennett, M.V.L. J. Gen.

Physiol. 77, 77–93 (1981).14. White, W.T., Deans, M., Kelsell, D. & Paul, D. Nature

394, 630 (1998).15. Pusch, M., Steinmeyer, K., Koch, M.C. & Jentsch, T.

Neuron 15, 1455–1463 (1995).

correspondence

Fibroblast growth factor receptor 3(FGFR3) belongs to a family of struc-

turally related tyrosine kinase receptorsencoded by four different genes. Thesereceptors are glycoproteins composed oftwo to three extracellular immunoglobu-lin (Ig)-like domains, a transmembranedomain and a split tyrosine-kinasedomain. Specific point mutations in dif-ferent domains of FGFR3 are associatedwith autosomal dominant human skeletaldisorders such as hypochondroplasia,achondroplasia, severe achondroplasiawith developmental delay and acanthosisnigricans (SADDAN), and thanatophoricdysplasia1,2. Several reports have demon-strated that these mutations lead to con-stitutive activation of the receptor1–3.Introduction of activating mutations intothe mouse gene Fgfr3 using ‘knock-in’approaches4,5 and the targeting of acti-vated Fgfr3 to growth plate cartilage inmice6 result in dwarfism. Conversely, thetargeted disruption of Fgfr3 in miceresults in the overgrowth of long bonesand vertebrae7,8. These observations sug-gest that Fgfr3 is a negative regulator ofbone growth. In contrast with this

inhibitory role, an oncogenic role hasbeen proposed for FGFR3 in multiplemyeloma (MM). In this lymphoid neo-plasm, a t(4;14)(p16.3;q32.3) chromoso-mal translocation with breakpoints on4p16 located 50–100 kb centromeric toFGFR3 is present in 20–25% of tumours,and associated with overexpression ofFGFR3 (refs 9,10). In rare cases (2/12 MMcell lines and 1/85 primary MMtumours), activating mutations of FGFR3previously identified in human skeletaldisorders have been found, always accom-panied by the t(4;14)(p16.3;q32.3) trans-location9–11. The real contribution ofFGFR3 in MM oncogenesis is not clearlyestablished, as it has recently been shownthat the 4p16 breakpoints in MM occur ina novel gene (MMEST/WHSC1), creatingfusion transcripts between the new geneand the immunoglobulin heavy chain(IgH) locus at 14q32.3, disrupted in aswitch region by the translocation12,13.Nothing is known about the role ofFGFR3 in epithelial cancers (carcinomas),which account for 90% of malignant neo-plasms. Having detected FGFR3 expres-sion in normal bladder and cervix

epithelia (data not shown), we examinedthe expression and mutational status ofFGFR3 in a series of bladder and cervixcarcinomas to determine whether FGFR3is involved in epithelial tumorigenesis.

We assessed transcript levels of the twoFGFR3 variants14, FGFR3b and FGFR3c,by semi-quantitative RT-PCR (ref. 15) in76 primary bladder carcinomas, 6 normalurothelia, 29 primary cervical carcinomasand 6 normal cervical epithelia. Asexpected, the FGFR3b variant, character-istic of the epithelial lineage14, was theonly form expressed in both malignantand non-malignant epithelial tissues. Itwas detected in 70 of 76 (92%) bladdercarcinomas and 27 of 29 (93%) cervicalcarcinomas. We performed PCR-SSCPanalysis of the coding region of FGFR3 onreverse-transcribed RNA from a repre-sentative subset of 26 bladder and 12cervix tumour samples. The sequences ofabnormally migrating bands revealed sin-gle nucleotide substitutions in 9 of 26bladder carcinomas (35%) and 3 of 12(25%) cervix carcinomas (Table 1). Thesesame changes were observed in corre-sponding tumour genomic sequences.Matched constitutional DNA, available inthe nine bladder tumour cases with muta-tions, contained wild-type sequences,demonstrating the somatic nature ofthese mutations (Fig. 1a, and data notshown). All FGFR3 missense somaticmutations identified (R248C, S249C,G372C and K652E) were identical to the

Frequent activating mutationsof FGFR3 in human bladderand cervix carcinomas

Table 1 • FGFR3 mutations in primary bladder and cervix cancers

Sample Stage/grade Codon nt position Mutation Predicted effect

1447, bladder Ta/G2 249 746 TCC→TGC Ser→Cys342, bladder T1a/G1 249 746 TCC→TGC Ser→Cys813, bladder T1a/G1 372 1,114 GGC→TGC Gly→Cys1393.1, bladder T1a/G3 249 746 TCC→TGC Ser→Cys506, bladder T1b/G2 372 1,114 GGC→TGC Gly→Cys1084, bladder T1b/G3 652 1,954 AAG→GAG Lys→Glu745.1, bladder T2/G3 248 742 CGC→TGC Arg→Cys1077, bladder T3/G2 249 746 TCC→TGC Ser→Cys1210, bladder T3/G2 249 746 TCC→TGC Ser→Cys6.96.1, cervix Ib 249 746 TCC→TGC Ser→Cys4.139, cervix IIa 249 746 TCC→TGC Ser→Cys4.13, cervix IIb 249 746 TCC→TGC Ser→Cys

Tumour stage was defined using TNM and FIGO classifications for bladder and cervix carcinomas, respectively; Mostofi’s histopathological grading system wasused for bladder cancer; codon and mutated nucleotide (nt position) are numbered according to FGFR3b cDNA ORF. All mutations were identified in both cDNAand genomic DNA from bladder and cervix tumours. For all bladder tumour cases, non-tumour genomic DNA was tested and found to be wild type. Correspon-ding normal DNAs were not available for cervix tumours.

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