American Journal of Medical Genetics 117A:89–91 (2003)

Research Letter

De Novo Mutation in the Gene EncodingConnexin-26 (GJB2) in a Sporadic Case ofKeratitis-Ichthyosis-Deafness (KID) Syndrome

To the Editor:

Keratitis-ichthyosis-deafness (KID) syndrome (MIM148210) is a congenital ectodermal dysplasia character-ized by the association of hyperkeratotic skin lesionswith vascularizing keratitis andprofound sensorineuralhearing loss [Burns, 1915; Langer et al., 1990; Wilsonet al., 1991; Caceres-Rios et al., 1996]. The termichthyosis is misleading, because the skin lesions aremore properly classified as erythrokeratoderma. Otherfeatures of this syndrome include scarring alopecia,dystrophic nails, dental abnormalities, increased risk ofdeveloping squamous cell carcinoma, and increasedsusceptibility to bacterial and mycotic skin infections[Langer et al., 1990; Wilson et al., 1991; Caceres-Rioset al., 1996]. Most cases of KID syndrome are sporadic,but the existence of familial cases suggested a geneticetiology [Legrand et al., 1982; Grobb et al., 1987;Tuppurainen et al., 1988; Nazzaro et al., 1990]. Herewe report the finding of a de novo mutation in the geneencoding connexin-26 in a Spanish patient diagnosedwith KID syndrome.

The proband, a 17-year-old female, is the third child ofnonconsanguineous healthy parents. Her two siblingsare unaffected. Pregnancy and delivery were unevent-ful. At birth, skin was dry, scaly, and pruriginous allover the body, but palms and soles were more severelyaffected. She had also total scalp alopecia (scalp hairbegan to grow at 2 years of age) and dystrophic, brittlenails, particularly in the toes. Photophobia was presentfrom the first months of life. At age 8 months, otoscopicexamination was normal, but testing of the auditorybrainstem responses (ABR), obtained with rarefactionclicks, showed only wave V at 100 dBHL bilaterally.Tympanometry values for static compliance and middleear pressure were normal in both ears. Ipsilateral andcontralateral acoustic reflexes were absent in left

and right stimulation. The diagnosis of bilateral, pro-found sensorineural hearing loss was confirmed at age9 months. Then, the patient started rehabilitation forlanguage development, with hearing aids and speechtherapy (cued speech). At age 3 years, visual reinforce-mentaudiometryshowedresponses to soundat90dBHLfor 250 Hz and at 100 dBHL for 500 Hz in both ears; noresponse was obtained for all the other frequencies. Atage 6 years, pure-tone audiometry showed bilateral andsymmetric sensorineural hearing loss (thresholds of 85dbHL at 250 Hz, 95 dBHL at 500 Hz, and 100 dBHL at1,000 Hz, with no response at higher frequencies).During childhood, the patient had repeated episodes ofexternal otitis and media otitis with effusion. At age14 years, the vestibular response of the patient wasevaluated.No spontaneous or positional nystagmuswasdetected.The rotatoryandbithermal caloric stimulationtests showed significant bilateral vestibular paresis.The saccadic and smooth pursuit eye movements werewithin normal limits. These results suggest a bilateralperipheral vestibular lesion.

Physical examination at age 17 years revealed thatthe scalp had sparse, brittle hair, as well as keratoticscalingplaques.Eyelidswerexerodermic,withacrackedaspect. All the body surface had a diffuse ichthyosiformaspect,with largehyperkeratotic plaques in the face andlimbs. These plaques, well demarcated, were scaly anderythematous, with itching. There were erythematousand verrucous plaques, with well-delineated borders, inhands and feet. Palmoplantar keratoderma was alsopresent, with a typical reticulated pattern. Finger nailswere almost normal, but toe nails were brittle, withsevere dyskeratosis. Teeth were normal but apparentlythere had been a delay in their eruption. Ophthalmolo-gic examination showed active keratitis and blepharitis,with photophobia. Fundi were normal. Altogether,the ophthalmologic, auditory, and dermatologic find-ings reported above supported the diagnosis of KIDsyndrome.

In the last 4 years, mutations in several genes encod-ing members of the connexin family of gap-junctionproteins have been shown to be responsible for hearingimpairment and skin disorders: GJB2 (connexin-26),GJB3 (connexin-31), andGJB6 (connexin-30) [Richard,2000; Kelsell et al., 2001]. We investigated these genesin our patient with KID syndrome.

*Correspondence to: Dr. Felipe Moreno, Unidad de GeneticaMolecular, Hospital Ramon y Cajal, Carretera de Colmenar, km 9,28034 Madrid, Spain. E-mail: fmoreno@hrc.insalud.es

Received 20 May 2002; Accepted 20 June 2002

DOI 10.1002/ajmg.a.10851

� 2002 Wiley-Liss, Inc.

Informed consent was obtained from all the indivi-duals included in the study. DNA was extracted fromperipheral blood samples by standard procedures. Thecomplete open reading frame (ORF) of each gene wasPCR-amplified, and the PCR products were sequenced.No sequence alteration was found in the GJB6 (con-nexin-30) gene. For GJB3 (connexin-31), the analysisrevealed the existence of a C-to-T transition at nucleo-tide 798 of the ORF, a silent mutation in the codonencoding the asparagine residue at position 266 of thepolypeptide (N266N). This DNA polymorphism hasalready been reported [Richard et al., 2000]. On theother hand, DNA sequencing of theGJB2 (connexin-26)ORF did reveal a significant change: a G-to-A transitionwas found at nucleotide 148, which results in thesubstitution of the aspartic acid residue at position50 by an asparagine residue (D50N; Fig. 1A). We devel-oped a specific test for this missense mutation based onthe fact that it destroys a restriction site for theendonuclease AspI (Roche). A 220 bp DNA fragmentwas PCR-amplified (forward primer, 50-CAAACCGCC-CAGAGTAGAAG-30; reverse primer, 50-GTGATCG-

TAGCACACGTTCTTG-30) and digested with AspI.Digestion of the PCR product from the wild-type alleleproduces two fragments, of 170 and 50 bp, whereas thePCRproduct from themutant allele remains undigested(220 bp). Testing of the family under study showed thatboth parents and both healthy siblings did not carry themutation, which was only present, in heterozygosity, inthe patient (Fig. 1B). The mutation was not found in117 healthy control individuals.

Both parents and their three children were thengenotyped for five polymorphic genetic markers closeto the GJB2 gene on 13q12, whose order is as follows:cen-D13S1835-D13S141-GJB2-D13S175-D13S1831-D13S1275-tel. Allele segregation showed no inconsis-tency. For all the tested markers, the three siblingsshared the samematernal allele; on the otherhand, bothof the healthy siblings shared the same paternal allele,whereas the proband had inherited the other one, foreach tested marker (Fig. 1C). Altogether, our geneticresults indicate that the D50N mutation appeared denovo in the proband, a conclusion consistent with thefact thatmost of the cases ofKIDsyndrome are sporadic.

Connexins are protein components of the intercellularchannels termed gap junctions. They are membraneproteins with four transmembrane domains, one cyto-plasmic and two extracellular loops, and both theN- andC-termini facing the cytoplasm. Six monomers of con-nexin bind each other to form a hexamer (connexon),which is located in the plasma membrane. Each con-nexonbindsanother connexon inanadjacent cell to forman intercellular channel [Goodenough et al., 1996;Kumar and Gilula, 1996]. The aspartic acid residue atposition 50 of the connexin-26 polypeptide (D50) islocated in extracellular loop 1 (E1), a domain that seemsto be involved in the interaction between connexons andin the control of voltage gating of the channel [Good-enough et al., 1996; Kumar and Gilula, 1996]. Thisresidue is conserved in a majority of connexins (Fig. 2).Interestingly, the four GJB2 mutations hithertoreported to be responsible for the association of skindisease and sensorineural hearing impairment alter theE1 domain. These include G59A in palmoplantarkeratoderma with high-frequency hearing loss (MIM148350) [Heathcote et al., 2000],DE42 [Bale et al., 1999],and R75W [Richard et al., 1998] in palmoplantarkeratoderma with profound hearing loss, and D66H inmutilating keratoderma with moderate hearing loss(Vohwinkel syndrome; MIM 124500) [Maestrini et al.,1999; Kelsell et al., 2000]. On the other hand, missensemutations causing nonsyndromic hearing impairmentare distributed all along theGJB2 gene [Rabionet et al.,2002]. Therefore, the clustering in the E1 domain of themutations responsible for the association of dermatolo-gic and auditory disorders suggests that the role playedby this domain is essential for the normal functioning ofthe connexin-26 channels in skin. Further progress instructural modeling of connexins, as well as construc-tion of transgenic mice with the reported mutations,shouldhelp to elucidate themolecular basis of this groupof disorders affecting both the skin and the inner ear.

During the preparation of this article, we learned ofthe work by van Steensel et al. [2002]. This research

Fig. 1. De novo mutation in the GJB2 gene in a sporadic case of KIDsyndrome.A:Direct sequencing of the coding strand of exon 2 from a controlindividual (left) and from the proband (right), which is heterozygous for theD50N mutation. Positions in the nucleotide sequence of the GJB2 openreading frameare indicated bynumbers.B:Haplotype analysis of the familyunder study for five microsatellite markers from the 13q12 region. Allelesare numbered and haplotypes are indicated by vertical bars. The position ofthe GJB2 gene is marked by an arrow. C: Specific test for the D50Nmutation.AspI digests of PCR products from all familymembers resolved ina1.5%agarose gel. Fragment sizes inbasepairs (bp) are indicated on the left.The undigested 220 bp band reveals the presence of the mutation.

90 Alvarez et al.

team has recently identified the D50N mutation in aDutch sporadic case of KID syndrome. The coincidenceof the samemutation in two different patients with KIDsyndrome provides further support to the hypothesisthat it is in fact the causative mutation of this disorderand suggests that this mutation may be frequentlyfound in other affected individuals. A general screeningof the reported cases of KID syndrome for thismutation,and for the putative existence of other mutations in theGJB2 gene, should contribute to the development of themolecular diagnosis and to progress in the pathophy-siological understanding of the syndrome.

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Araceli AlvarezIgnacio del CastilloAlejandra PeraManuela VillamarMiguel A. Moreno-PelayoFelipe Moreno*Unidad de Genetica MolecularHospital Ramon y CajalMadrid, Spain

Ramon MorenoServicio de DermatologıaHospital Ramon y CajalMadrid, Spain

M. Cruz TapiaServicio de ORLHospital Clınico San Carlos–Pabellon

Madrid, Spain

Fig. 2. Comparison of sequences of the first extracellular loop (E1) for 11members of the family of human connexins. Residues equivalent to aspartic acid50 (connexin-26) in other connexins are boxed. Residue numbers are referred to connexin-26 sequence.

GJB2 (Connexin-26) Mutation in KID Syndrome 91