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A Novel Recessive 31 (GJB3) Mutation in a Case of Erythrokeratodermia Variabilis

To the Editor: multicellular organisms. The coordinated exchange of mole- cules is used in a wide spectrum of cellular activities, Erythrokeratodermia variabilis (EKV, MIM 133200) is part such as transmission of neuronal signals, cell contraction, of the clinically and genetically heterogeneous group of cell growth control, organ homeostasis, and differentiation erythrokeratodermias. It is characterized by migrating red (Goodenough et al, 1996; White and Paul, 1999). Reflecting patches resembling a geographic map, and by localized or their communicating functions, Cxs are expressed in a wide generalized hyperkeratosis with scaling of the skin. The variety of tissues (Krutovskikh and Yamasaki, 2000). hyperkeratotic areas often show well-demarcated, geogra- Eleven human Cx have been described, several phically outlined, and fixed plaques, in a strikingly symme- of which have been associated with diseases such as trical distribution. Scales on the skin vary in appearance, Charcot–Marie–Tooth (GJB1, MIM #304040), hearing loss from fine and yellow to thick and brown, leading to a hystrix- (GJB2, GJB3, and GJB6; MIM #121011 and 603324), like appearance. The erythematous patches seem to occur cataract (GJA3 and GJA8; MIM #121015 and #600897), independently from the hyperkeratosis. In some cases, EKV (GJB3, GJB4), a variant of Vohwinkel syndrome (GJB2, the patients also show non-epidermolytic palmo-plantar MIM #148350), KID syndrome (GJB2, MIM #148210), and keratoderma. The study was conducted according to the Oculodentodigital dysplasia (MIM #164200). Declaration of Helsinki guidelines. We also obtained patient We examined a 16-y-old female presenting hyperker- consent to use biopsy and blood samples for genetic atosis of palms and soles (Fig 1a,b), diffuse erythematous analysis. patches of body skin, changing in size and shape (Fig 1d), The hyperkeratosis and erythema can be triggered by and a diffuse ichthyotic hyperkeratosis, characterized by stress, such as temperature changes, mechanical irritation, thick dark brown scales (Fig 1a), sparing only small areas of or sun exposure. The onset is usually at birth or during the body. The patient’s parents reported the presence of infancy, and the disease persists throughout life. EKV is erythema localized only on the limbs at birth, which disa- mainly inherited as an autosomal dominant disease, ppeared within 1 wk. The erythematous lesions reappeared although recessive transmission has occasionally been at 1 mo of age, extended to the entire skin surface, and they reported (Gottfried et al, 2002). Mutations associated with were changing location over the span of a day. During the EKV have been identified in connexin (Cx) genes, Cx31 and first decade of life, the erythema decreased in intensity, 30.3 (Richard et al, 1998, 2000, 2003; Wilgoss et al, 1999; while the diffusion of hyperkeratotic areas increased. The Macari et al, 2000). other family members were not affected by the disease, nor Cxs are a group of that share a common pattern by any other dermatological pathology. The histological of structural motifs or domains, including four trans- examination (Fig 1c) showed ortho-hyperkeratosis, papillo- membrane domains (M1–4), two extracellular domains matosis, and light superficial lymphocyte infiltration in the (E1–2), and three cytoplasmic domains. The cytoplasmic dermis. Audiometric analysis of the proband and her domains are the amino-terminus (NT), the cytoplasmic loop parents did not reveal hearing impairment. (CL), and the carboxy-terminus (CT). The CL and CT domains As mutations in GJB3 and GJB4 genes have been are characteristic of each connexin, while the membrane correlated to EKV, we investigated for mutations in these spanning and the extracellular domains are highly con- two genes. We obtained the consensus for skin biopsy and served. genetic analysis from patient and parents. Amplification and Homomeric or heteromeric interaction of six Cxs leads to sequence analysis of the Cx31 (amplified from RNA the formation of the connexon or hemichannel (Kumar and extracted from patient palmar epidermis) revealed a novel Silula, 1996), localized on the plasma membrane surface. homozygous mutation, a G to A transversion at The extracellular interaction of two opposing connexons 829 (according to Genebank sequence AF099730). The generates a channel. The clustering of a large number of mutation leads to the substitution of a conserved glutamic channels forms the (Yeager et al, 1996). acid in position 100 (ConSurf analysis in Gottfried et al, 2002 Gap junctional channels show selective permeability and ConSurf, University of Tel Aviv, Israel), localized in the CL voltage sensitivity (Bruzzone et al, 1996; Goodenough et al, domain of the , with a lysine (Fig 2b). The complete 1996; Kumar and Giulia, 1996). They play a role in the analysis of the Cx31 and Cx30.3 coding region did not show response to environmental stimuli in almost all cells of any other mutation. Sequence analysis of PCR fragments amplified from genomic DNA of other family members showed the presence of the mutated allele, in heterozyg- Abbreviations: Cx, connexin; EKV, erythrokeratodermia variabilis osity (Fig 2c), in both parents and in other unaffected

Copyright r 2004 by The Society for Investigative Dermatology, Inc. 837 838 LETTER TO THE EDITOR THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 1 Clinical presentation and histopathology. (a) Diffuse ichthyotic hyperkeratosis, charac- terized by thick dark brown scales and erythematous patches of body skin, changing in size and shape in the course of weeks, leaving some areas completely unaffected. (b) Hyperkeratosis of palms. (c) Hematoxylin– eosin staining of patient skin sections, show- ing orthohyperkeratosis, papillomatosis and light superficial dermal lymphocytic infiltra- tion. (d) Clinical presentation of the patient after Acitretin treatment. The dark brown scales have almost disappeared, while er- ythematous patches are still present.

Figure 2 Pedigree, mutation analysis and enzymatic digestion for confirmation analysis. (a) Pedigree of the patient’s maternal and paternal family shows the presence of a common ancestor. Asterisks indicate the individuals for whom the DNA was available for mutational analysis. (b–d) Sequence analysis of the Cx31 gene, performed on patient cDNA (RT-PCR) and genomic DNA (PCR) and only on genomic DNA (PCR) for all the other members. The primers used are ACTCAGAACACTGCCTGGTACATA ( þ ) and GGCACTGGGCCTGTGGGTTAC (), designed from the published sequence (AF099030). Wild-type guanine 829 is represented by a single black peak in the chromatogram (black arrow in panel d). The green peak at position 829 indicates a Timine residue (black arrow in panel b), which leads to the amino acid substitution Glu100Lys. In panel c, the presence of two peaks (black/green indicated by the arrow) reveals the presence of both alleles in heterozygous individuals. (e) The G to A transversion ablates an existing BseR-I restriction site. The digestion with this enzyme of the 1117 bp amplicon produces two bands (703 bp, 414 bp, lane 7) for the wild- type allele, three bands in the presence of the heterozygous mutation (1117 bp, 703 bp, 414 bp, lanes 1–2–4–5–6–8) and one band when both alleles are mutated (1117 bp, lane 3). The results indicate that the patient’s parents and most other relatives (with one exception) are heterozygous for the mutation. 122 : 3 MARCH 2004 LETTER TO THE EDITOR 839 members of the family. The mutation deletes a BseR-I DOI: 10.1111/j.0022-202X.2004.22311.x restriction site; therefore, restriction analysis has been used Manuscript received April 30, 2003; revised October 15, 2003; to confirm the mutation and to exclude polymorphisms. We accepted for publication October 17, 2003 generated a 1117 bp PCR amplicon normally containing the enzyme recognition site, which upon digestion resulted in Address correspondence to: Gerry Melino, Via Montpellier 1, 00133 Rome, Italy. Email: gerry.Melino@uniroma2 two bands. The patient homozygous for the mutation showed only an uncut band of 1117 bp (Fig 2e). The patient’s parents were heterozygous, suggesting that the References mutation has been inherited from a common ancestor (Fig 2a). Bruzzone R, White TW, Paul DL: Connections with : The molecular basis of direct intercellular signalling. Eur J Biochem 238:1–27, 1996 The patient was treated for 2 mo with a 20 mg daily dose Di WL, Monypenny J, Common JE, et al: Defective trafficking and cell death is of Acitretin (retinoic acid derivative, Graham-Brown and characteristic of skin disease-associated connexin 31 mutations. Hum Chave, 2002), and showed almost complete remission of hy- Mol Genet 15 11:2005–2014, 2002 perkeratotic phenotype, without improvement of the Diestel S, Richard G, Doring B, Traub O: Expression of a connexin31 mutation causing erythrokeratodermia variabilis is lethal for HeLa cells. Biochem erythema (Fig 1d). The effect on the hyperkeratosis might Biophys Res Commun 23 296:721–728, 2002 be due to the downregulation of the mutated gene by Goodenough D, Goliger J, Paul DL: Connexins, connexons and intercellular retinoic acid, since we (unpublished data) and others communication. Annu Rev Biochem 65:475–502, 1996 (Grummer et al 1996) have shown the ability of retinoic Gottfried I, Landau M, Glaser F, et al: A mutation in GJB3 is associated with recessive erythrokeratodermia variabilis (EKV) and leads to defective acid to downregulate Cx genes. Functional studies on Cxs trafficking of the connexin 31 protein. Hum Mol Genet 15 11:1311–1316, mutations (Di et al, 2002; Gottfried et al, 2002) demon- 2002 strated an accumulation in the cytoplasm of the mutant Graham-Brown RA, Chave TA: Acitretin for erythrokeratodermia variabilis in a 9- proteins, suggesting that they cannot be properly trans- year-old girl. Pediatr Dermatol 19:510–512, 2002 ported to the plasma membrane, probably due to defective Grummer R, Hellmann P, Traub O, Soares MJ, el-Sabban ME, Winterhager E: Regulation of connexin31 upon retinoic acid treatment in folding, hemichannel assembly or membrane targeting. rat choriocarcinoma cells. Exp Cell Res 25 227:23–32, 1996 Moreover, induction of cell death mediated by the mutated Krutovskikh V, Yamasaki H: Connexin gene mutations in human genetic diseases. Cx31 has been reported (Diestel et al, 2002). It has also Mutat Res 462:197–207, 2000 been shown that Cx31 mutations affect calcium metabolism Kumar N, Giulia N: The Gap Junction communication channel. Cell 84:381–388, 1996 (Rouan et al, 2003). Cx31 is also expressed in peripheral Liu XZ, Xia XJ, Xu LR, et al: Mutations in connexin31 underlie recessive as well as and auditory nerves, and mutations causing dominant dominant non-syndromic hearing loss. Hum Mol Genet 1 9:63–67, 2000 (Xia et al, 1998) and recessive (Liu et al, 2000) hearing Lopez-Bigas N, Olive M, Rabionet R, et al: Connexin 31 (GJB3) is expressed in impairment and neuropathies have been described (Lopez- the peripheral and auditory nerves and causes neuropathy and hearing impairment. Hum Mol Genet 15 10:947–952, 2001 Bigas et al, 2001). Since the patient studied showed only Macari F, Landau M, Cousin P, et al: Mutation in the gene for connexin 30.3 in a dermatologic symptoms, we can conclude that the amino family with erythrokeratodermia variabilis. Am J Hum Genet 67:1296– acid substitution and the specific domain affected by the 1301, 2000 mutation play an important role in determining the clinical Richard G, Brown N, Rouan F, et al: Genetic heterogeneity in erythrokeratodermia phenotype, confirming that mutations involving different variabilis: Novel mutations in the connexin gene GJB4 (Cx30.3) and genotype–phenotype correlations. J Invest Dermatol 120:601–609, 2003 regions of these proteins give rise to different diseases. Richard G, Brown N, Smith LE, et al: The spectrum of mutations in erythro- The data reported clearly indicate that the homozygous keratodermias—Novel and de novo mutations in GJB3. Hum Genet expression of the E100K mutation is responsible for the EKV 106:321–329, 2000 phenotype, by a loss of function mechanism rather than a Richard G, Smith LE, Bailey RA, et al: Mutations in the human connexin gene GJB3 cause erythrokeratodermia variabilis. Nat Genet 20:366–369, 1998 dominant-negative one, described in the majority of Cx31 Rouan F, Lo CW, Fertala A, et al: Divergent effects of two sequence variants of mutations. Further studies on connexin gene mutations will GJB3 (G12D and R32W) on the function of connexin 31 in vitro. Exp help to define their biochemical and physiological role in Dermatol 12 (2):191–197, 2003 tissues where they are expressed. White TW, Paul DL: Genetic diseases and gene knockouts reveal diverse connexin functions. Annu Rev Physiol 61:283–310, 1999 Wilgoss A, Leigh IM, Barnes MR, et al: Identification of a novel mutation R42P in the beta-3 associated with autosomal dominant Alessandro Terrinoni,Ã Aida Leta,w Cristina Pedicelli,z Eleonora Candi,y Marco Ranalli,y Pietro Puddu,z Mauro Paradisi,z Corrado Angelo,z erythrokeratoderma variabilis. J Invest Dermatol 113:1119–1122, 1999 Giacinto Baggetta,w and Gerry MelinoÃy# Xia JH, Liu CY, Tang BS, et al: Mutations in the gene encoding gap junction ÃBiochemistry Laboratory, IDI-IRCCS, c/o Department of Experimental protein beta-3 associated with autosomal dominant hearing impairment. Medicine and Biochemical Sciences, University of Rome ‘‘Tor Vergata’’, Nat Genet 20:370–373, 1998 Rome, Italy; wDepartement of Pharmaco-Biology, University of Calab- Yeager M, Nicholson BJ: Structure of gap junction intercellular channels. Current ria, 87036 Cosenza, Italy; zVII Pediatric Division, IDI-IRCCS, Via dei Opin Strutt Biol 6:183–192, 1996 Monti di Creta, Rome, Italy; yDepartment of Experimental Medicine and Biochemical Sciences, University of Rome ‘‘Tor Vergata’’, Rome, Italy; zV Dermatological Division, IDI-IRCCS, Via dei Monti di Creta, Rome, Italy; #Medical Research Council, Toxicology Unit, Hodgkin Building, Leicester University, Lancaster Road, Leicester, UK

This work was partially supported by a grant from Telethon (GGP02251 to EC), and by AIRC, EU (QLG1-1999-00739, QLK-CT-2002-01956), MIUR, MinSan to G.M.