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See related article on pg 1540 translocated into the plasma membrane. Once expressed on the cell surface, the hemichannel docks with a connexon of an adjacent cell to form a channel that Variabilis et is termed gap junction. Connexons can form either homotypic (docking of two Progressiva Allelic to Oculo-Dento- identical connexons), heterotypic (docking of two dissimilar homomeric Digital Dysplasia connexons), or heteromeric (docking of two heteromeric connexons) channels Sabine Duchatelet1,2 and Alain Hovnanian1,2,3 (Mese et al., 2007). These diverse Erythrokeratodermia variabilis et progressiva (EKVP) is a with combinations of create clinical and genetic heterogeneity, most often transmitted in an autosomal different types of channels, each having dominant manner, caused by mutations in GJB3 and GJB4 genes encoding unique properties (ionic conductance, connexins (Cx)31 and 30.3, respectively. In this issue, Boyden et al. (2015) report permeability, sensitivity to voltage, or for the first time de novo dominant mutations in GJA1 encoding the ubiquitous pH). Of note, several connexins may also Cx43 in patients with EKVP. These results expand the genetic heterogeneity of form functional nonjunctional hemi- EKVP and the human disease phenotypes associated with GJA1 mutations. They channels, although their physiological disclose that EKVP is allelic to oculo-dento-digital dysplasia, a rare syndrome relevance remains uncertain (Pfenniger previously known to be caused by dominant GJA1 mutations. et al., 2010). Mutations in 11 genes cause a variety of genetic dis- Journal of Investigative (2015) 135, 1475–1478. doi:10.1038/jid.2014.535 orders, implicating a key role in tissue homeostasis (Lai-Cheong et al., 2007). Mutations in connexins have been Clinical features of EKVP clinical heterogeneity of the disease. associated with deafness, neuropathy, EKVP (MIM 133200) is characterized by EKVP is usually inherited as an auto- and various skin phenotypes. Several the co-existence of localized or general- somal dominant trait, but rare auto- genodermatosis have been shown to be ized hyperkeratotic plaques back- somal recessive transmission has also caused by mutations in genes encoding ground and transient stationary or been reported (Gottfried et al., 2002; connexin (Cx)26, 30, 30.3, 31, and more migratory erythematous plaques Fuchs-Telem et al., 2011). rarely Cx43 (Mese et al., 2007). Speci- (Macfarlane et al., 1991). The hallmark fically, mutations in GJB2 encoding of EKVP is the continual occurrence of Connexins and genetic skin diseases Cx26 can lead to the Vohwinkel transient, sharply outlined, figurate red Gap junctions are transmembrane com- syndrome (mutilating PPK associated patches of variable intensity that fade plexes that permit the rapid exchange of with honeycomb-like and within a few hours or days. Approxi- ions, secondary messengers, and small starfish-like keratoses and deafness, MIM matively 50% of EKVP patients have metabolites between neighboring cells 124500), the keratitis-ichtyosis-deafness (PPK). (Mese et al., 2007). They are formed by (KID) syndrome (MIM 148210), the Initially, two separate clinical entities connexins, a multigen family of at least hystrix-like with deafness syn- were distinguished according to the 21 members. Connexin proteins are drome (MIM 602540), PPK associated presence of migratory or stationary named after their molecular mass, and with deafness (MIM 148350), and the erythematous patches, defining erythro- their genes have been classified by Bart-Pumphrey (knuckle pads, leukony- keratodermia variabilis (Mendes da sequence similarities, defining three chia and sensorineural deafness) syn- Costa, 1925) and progressive sym- main groups––gap junction α (GJA), β γ drome (MIM 149200). Mutations in GJB6 metric erythrokeratodermia (Gottron, (GJB), and (GJC). Connexins consist of encoding Cx30 cause the Clouston 1922), respectively. The observation of four transmembrane domains with syndrome (Hidrotic ectodermal dyspla- both phenotypes in two sisters disclosed intracellular N- and C-termini. sia, MIM 129500), and mutations in that these two conditions represented, Connexin monomers oligomerize to GJB3 and GJB4, encoding Cx31 and in fact, variable expression of a single hexameric homomeric or heteromeric 30.3, respectively, have been identified inherited clinical entity (Macfarlane hemichannels (also called connexons) in EKVP (Common et al., 2005). et al., 1991). Thus, the designation in the endoplasmic reticulum (ER) and EKVP was proposed to account for the the Golgi apparatus, which are then Clinical and genetic heterogeneity of EKVP 1INSERM UMR 1163, Laboratory of Genetic Skin Diseases, Imagine Institute for Genetic Diseases, Paris, EKVP shows considerable intra- and 2 3 France; University Paris Descartes, Sorbonne Paris Cité, Paris, France and Department of Genetics, Necker interfamilial clinical heterogeneity Enfants Malades Hospital, Paris, France (Common et al., 2005). The clinical Correspondence: Alain Hovnanian, INSERM UMR 1163, Department of Genetics, Laboratory of Genetic Skin Diseases, Imagine Institute for Genetic Diseases, 2nd Floor, 24 bld du Montparnasse, 75015 Paris, variability observed, even in patients France. E-mail: [email protected] from the same family, suggests the

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Clinical Implications rarely, GJA1 mutations have been reported in craniometaphyseal dysplasia ● Erythrokeratodermia variabilis et progressiva (EKVP) has been known to and sudden infant death (Laird, 2014). be caused by mutations in GJB3 and GJB4 genes encoding connexin (Cx) Most ODDD mutations are dominant 31 and 30.3, respectively. Boyden et al. (2015) provide evidence that EKVP missense mutations spread through the can also be caused by mutations in GJA1 encoding the ubiquitous first two-thirds of the protein, and more expressed Cx43. rarely in the C-terminal domain (see ● Aside from enlarged porcelain-white lunulae found in the three patients Figure 4 by Boyden et al. 2015) (Laird, with GJA1 mutations, no obvious genotype–phenotype correlation can be 2014). Rare nonsense or small found in EKVP. In particular, EKVP shares no clinical features with oculo- frameshift or inframe indels have also dento-digital dysplasia and shows limited overlap with the keratoderma- been reported. In particular, ODDD hypotrichosis-leukonychia totalis syndrome, both conditions which are mutations associated with palmoplantar also caused by GJA1 mutations. were initially identified as ● The co-existence of hyperkeratotic plaques with transient erythematous truncating mutations in the C-terminal fi part of the protein. Subsequent reports plaques and no extra-cutaneous ndings remains the main clinical fi features of EKVP, which allows one to distinguish it from these two allelic described missense mutations in the rst conditions. cytoplasmic loop and the N-terminal part of the molecule. Thus, there is no correlation between the localization or nature of the mutation and the effects of modifier genes, epigenetics, channel with Cx37, 40, and 43 (White development of keratoderma. and/or environmental factors. and Bruzzone, 1996; He et al., 1999; More generally, no specific geno- Heterozygous mutations in GJB3 and Abrams et al., 2006). This expands the type–phenotype correlation has been GJB4 were reported in EKVP patients possibility of distinct functional conse- evidenced in ODDD as individuals (Richard et al., 1998; Macari et al., quences resulting in different hetero- carrying mutations in the same Cx43 2000). They are mainly missense muta- typic or heteromeric gap junctions. domain can exhibit significant differ- tions. Recessive mutations in GJB3 have In this issue, Boyden et al. (2015) ences in clinical features, although rare also been identified in EKVP patients reported the identification, by exome reported recessive mutations lead to (Gottfried et al., 2002; Fuchs-Telem sequencing, dominant de novo muta- more severe phenotypes. et al., 2011). However, some patients tions in GJA1 encoding Cx43 in three Very recently, a GJA1 mutation was do not carry mutations in either gene patients with EKVP. These patients identified in two familial cases and (Richard et al., 2003; Common et al., were not phenotypically different from one sporadic case affected with the 2005). Of note, mutations in GJB3, patients with GJB3 or GJB4 mutations, keratoderma-hypotrichosis-leukonychia expressed in the skin, cochlea, and the although enlarged porcelain-white totalis syndrome (KHLS), a rare disorder peripheral nervous system, also cause lunulae has not been previously characterized by severe skin hyperker- hearing loss or neuropathy associated observed in EKVP. This result expands atosis, congenital alopecia, and leuko- with deafness (Rabionet et al., 2000). EKVP genetic heterogeneity and the nychia totalis, with no additional extra- There is no clear correlation between disease phenotypes associated with cutaneous features (Wang et al., 2014). phenotype and the nature and/or locali- GJA1 mutations, which have been Thus, mutations in GJA1 underlie zation of GJB3 mutations as missense previously identified in oculo-dento- severe skin phenotypes such as KHLS mutations responsible for EKVP or deaf- digital dysplasia (ODDD; see below). and EKVP, as well as, in a less consis- ness co-localize in the same domains. tent manner, rare and moderate cuta- Previous studies have revealed that GJA1 mutations in ODDD and other rare neous features in ODDD. EKVP patients harboring GJB3 or GJB4 diseases mutations show no clinical differences, Mutations in GJA1, encoding the ubi- Connexin mutations show no genotype– although circinate or gyrate lesions quitously expressed connexin 43, were phenotype correlation and have different were initially suggested as a specific first reported in nonsyndromic deaf- effects on gap junction biology feature of GJB4 mutations (Richard ness and subsequently in ODDD Connexin molecules have distinct func- et al., 2003; Common et al., 2005). (MIM164200). ODDD is a rare pleio- tional domains required for proper gap The molecular and functional inter- tropic genetic developmental disorder junction formation and functionality. action between Cx31 and Cx30.3 in that associates craniofacial and limb The N-terminal domain is important in gap junctions may explain the absence dysmorphisms, ocular, and dental physiological proprieties of gap junc- of genotype–phenotype correlation and anomalies (Laird, 2014). PPK and focal tion and oligomerization (see Figure 4 the similarity in clinical phenotypes hyperkeratosis have also been by Boyden et al. 2015). The trans- between patients with GJB3 or GJB4 described in rare cases of ODDD membrane domains form the pore of mutations (Plantard et al., 2003). Cx31 (Paznekas et al., 2003; van Steensel the gap junction and also regulate can also interact with Cx26, 30, 32, 43, et al., 2005; Kelly et al., 2006; Vreeburg heteromeric channel compatibility. and 45, whereas Cx30.3 can form a et al., 2007; Kogame et al., 2014). More The extracellular loops are important

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for gap junction formation (connexon- overlapping with conditions caused by Boyden LM, Craiglow BG, Zhou J et al. (2015) connexon interaction) and the docking GJB2 mutations (Wang et al., 2014). Dominant de novo mutations in GJA1 cause erythrokeratodermia variabilis et progressiva, compatibility in heterotypic chan- Thus, a transdominant effect of KHLS without features of oculodentodigital dyspla- nels. The cytoplasmic loop and the Cx43 mutations on Cx26 may also sia. JInvestDermatol135:1540–7 C-terminal domain have a role in contribute to KHLS pathogenesis. Churko JM, Langlois S, Pan X et al. (2010) The channel gating but also contain motifs EKVP mutations in GJA1 reported by potency of the fs260 connexin43 mutant to for binding with other partners (Laird, Boyden et al. (2015) cause Cx43 impair keratinocyte differentiation is distinct from other disease-linked connexin43 2014). The two GJA1 missense muta- retention in the Golgi apparatus pre- mutants. Biochem J 429:473–83 tions underlying EKVP (transmembrane venting gap junction formation and Common JE, O'Toole EA, Leigh IM et al. (2005) domains 1 and 4) described by Boyden therefore lead to loss of channel Clinical and genetic heterogeneity of erythro- et al. (2015) and the missense mutation function as ODDD mutations. Interest- keratoderma variabilis. J Invest Dermatol 125: causing KHLS (N-terminal domain) are ingly, functional interaction between 920–7 not restricted to a particular Cx43 Cx43 and Cx31 or Cx30.3 has been Dobrowolski R, Sommershof A, Willecke K (2007) domain, and their localization does suggested (He et al., 1999; Schnichels Some oculodentodigital dysplasia-associated fi Cx43 mutations cause increased hemichannel not differ from those identi ed in et al., 2007), and Cx43 has been shown activity in addition to deficient gap junction ODDD. Thus, the functional conse- to interact with a number of important channels. J Membr Biol 219:9–17 quences of the Cx43 mutations on gap structural and signaling molecules Fuchs-Telem D, Pessach Y, Mevorah B et al. (2011) junction formation and activity, rather (Herve et al., 2007). Thus, reported Erythrokeratoderma variabilis caused by a than the nature or the location of the recessive mutation in GJB3. Clin Exp Dermatol EKVP Cx43 mutations may have 36:406–11 mutation in a specific domain, would transdominant action on co-expressed Gong XQ, Shao Q, Langlois S et al. (2007) appear to determine the associated wild-type Cx31 or 30.3, leading to a Differential potency of dominant negative clinical phenotype. similar clinical phenotype as GJB3 or connexin43 mutants in oculodentodigital dys- Biological steps possibly involved in GJB4 mutations. Cx43 can also interact plasia. JBiolChem282:19190–202 defective gap junction function include with Cx37, 40, 45, and 56 (Laird, 2014), Gottfried I, Landau M, Glaser F et al. (2002) A impaired trafficking with cytoplasmic as well as with different partners such mutation in GJB3 is associated with recessive erythrokeratodermia variabilis (EKV) and leads retention (in the ER and/or the Golgi as tight or adherens junction proteins, to defective trafficking of the connexin 31 apparatus), reduced or abolished capa- kinases, and phosphatases (Herve protein. Hum Mol Genet 11:1311–6 city to assemble into hemichannels, et al., 2007). Disruption of mutated Gottron H (1922) Erythrokeratodermia progressiva and loss of gap junction activity or Cx43 interaction with these partners symmetrica. Zentbl Haut-Geschl-Krankh 4:493 enhanced channel function. In addition, may also account for variations in the He DS, Jiang JX, Taffet SM et al. (1999) Formation of disease mechanisms may also involve disease phenotypes associated with heteromeric gap junction channels by connex- fi ins 40 and 43 in vascular smooth muscle cells. transdominant effects on other co- speci c mutations. Proc Natl Acad Sci USA 96:6495–500 expressed connexin isoforms because Thus, discrepancies between func- Herve JC, Bourmeyster N, Sarrouilhe D et al. (2007) of heteromeric connexons and hetero- tional consequences could explain the Gap junctional complexes: from partners to typic channel formation, or impaired different phenotypes associated with functions. Prog Biophys Mol Biol 94:29–65 interaction with other partners. A direct these mutations. Kelly SC, Ratajczak P, Keller M et al. (2006) A novel role of hemichannel dysfunction in Identification of the functional con- GJA 1 mutation in oculo-dento-digital dyspla- sia with curly hair and hyperkeratosis. Eur J diseases has also been suggested, although sequences of mutations on hemichan- Dermatol 16:241–5 the physiological function of hemichan- nel and gap junction function on their Kogame T, Dainichi T, Shimomura Y et al. (2014) nels remains elusive (Dobrowolski et al., interactions with other connexins and Palmoplantar in oculodentodigital 2007). Interestingly, Cx43 mutations in partners is essential to understanding dysplasia with a GJA1 point mutation out of ODDD have been shown to result most the organ specificities and specific the C-terminal region of connexin 43. J Dermatol 41:1095–7 often in loss of gap junction function, phenotypes associated with different Lai-Cheong JE, Arita K, McGrath JA (2007) Genetic despite normal localization, with impaired mutations within the same connexin diseases of junctions. JInvestDermatol127: or increased hemichannel activity and gene. This information is also crucial 2713–25 fi more rarely impaired traf cking with ER when considering specific pharmacolo- Lai A, Le DN, Paznekas WA et al. (2006) or Golgi retention (Shibayama et al., 2005; gical treatments, as described recently Oculodentodigital dysplasia connexin43 Lai et al., 2006; Dobrowolski et al., 2007; for Cx26 mutations in the KID syndrome mutations result in non-functional connexin hemichannels and gap junctions in C6 Gong et al., 2007; Churko et al., 2010; (Levit et al., 2014). glioma cells. JCellSci119:532–41 Shao et al., 2012; Laird, 2014). These Laird DW (2014) Syndromic and non-syndromic mutations have a dominant negative effect CONFLICT OF INTEREST disease-linked Cx43 mutations. FEBS Lett 588: fl on the wild-type counterpart (Gong et al., The authors state no con ict of interest. 1339–48 2007; Shao et al., 2012). Levit NA, Sellitto C, Wang HZ et al. (2014) Aberrant In contrast, the Cx43 mutation caus- REFERENCES connexin26 hemichannels underlying keratitis- ing KHLS does not alter gap junction Abrams CK, Freidin MM, Verselis VK et al. (2006) ichthyosis-deafness syndrome are potently inhibited by mefloquine. J Invest Dermatol; function but causes a gain-of-function Properties of human connexin 31, which is implicated in hereditary dermatological dis- e-pub ahead of print 17 September 2014 in hemichannel activity (Wang et al., ease and deafness. Proc Natl Acad Sci USA Macari F, Landau M, Cousin P et al. (2000) 2014). KHLS presents phenotypic 103:5213–8 Mutation in the gene for connexin 30.3 in a

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family with erythrokeratodermia variabilis. Am See related article on pg 1649 J Hum Genet 67:1296–301 Macfarlane AW, Chapman SJ, Verbov JL (1991) Is erythrokeratoderma one disorder? A clinical and ultrastructural study of two siblings. Br J Dermatol 124:487–91 FICZ: A Messenger of Light in Mendes da Costa S (1925) Erythro- et keratodermia variabilis in a mother and daughter. Acta Human Skin Derm Venereol 6:255–61 1 1 Mese G, Richard G, White TW (2007) Gap Deeba N. Syed and Hasan Mukhtar junctions: basic structure and function. J Invest Dermatol 127:2516–24 Photosensitization, subsequent to photon absorption by chromophores present Paznekas WA, Boyadjiev SA, Shapiro RE et al. in the human skin, appears to be a key mechanism of UV-induced oxidative (2003) Connexin 43 (GJA1) mutations cause stress. The tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ), an the pleiotropic phenotype of oculodentodigital dysplasia. Am J Hum Genet 72:408–18 aryl hydrocarbon receptor ligand, has been found to be a potent UVA Pfenniger A, Wohlwend A, Kwak BR (2010) photosensitizer, effective at nanomolar concentrations. A novel addition to the Mutations in connexin genes and disease. family of endogenous photosensitizers, the precise mechanism(s) through which Eur J Clin Invest 41:103–16 it mediates oxidative stress in UVA exposed skin and its response to the UVB Plantard L, Huber M, Macari F et al. (2003) spectrum of the solar UV flux remains unexplored. Further studies related to its Molecular interaction of connexin 30.3 and functionality in the human skin, its utility as a tool against UV-induced adverse connexin 31 suggests a dominant-negative fl mechanism associated with erythrokeratoder- effects, and its role in in ammatory skin diseases will have the potential to open mia variabilis. Hum Mol Genet 12:3287–94 up new avenues in the realms of human skin photobiology. Rabionet R, Gasparini P, Estivill X (2000) Molecular Journal of Investigative Dermatology (2015) 135, 1478–1481. doi:10.1038/jid.2015.52 genetics of hearing impairment due to muta- tions in gap junction genes encoding beta – connexins. Hum Mutat 16:190 202 A study published in this issue of the by endogenous non-DNA chromo- Richard G, Brown N, Rouan F et al. (2003) Genetic journal sheds light on the previously phores present in the human skin heterogeneity in erythrokeratodermia variabi- fi lis: novel mutations in the connexin gene GJB4 unexplored photosensitizing ef cacy of subsequent to photon absorption (Cx30.3) and genotype-phenotype correla- 6-formylindolo[3,2-b]carbazole (FICZ) appears to be an important mechanism tions. JInvestDermatol120:601–9 in epidermal keratinocytes. Greater of UV-induced ROS production Richard G, Smith LE, Bailey RA et al. (1998) than 95% of the UV energy derived (Wondrak et al., 2006). The sensitizer Mutations in the human connexin gene GJB3 from solar radiation incident on the chromophores after initial photon cause erythrokeratodermia variabilis. Nat Genet 20:366–9 human skin comprises the deeply pene- absorption either revert to the ground – Schnichels M, Worsdorfer P, Dobrowolski R et al. trating longer wavelength UVA (320 state by energy dissipation or form (2007) The connexin31 F137L mutant mouse 400 nm), whereas the UVB (290– highly reactive bi-radical triplet states. as a model for the human skin disease 320 nm) content on the skin varies The long lived triplet state of the erythrokeratodermia variabilis (EKV). Hum Mol Genet 16:1216–24 between 2 and 5% (Wondrak et al., sensitizer serves as a key photoexcited Shao Q, Liu Q, Lorentz R et al. (2012) Structure and 2006). Understandably, there exists a intermediate that induces skin photo- functional studies of N-terminal Cx43 mutants positive correlation between wave- damage through direct interaction with linked to oculodentodigital dysplasia. Mol Biol lengths and skin photon pene- substrate molecules such as DNA bases – Cell 23:3312 21 tration so that 100-fold higher photon (type I reaction) or activation of mole- Shibayama J, Paznekas W, Seki A et al. (2005) energy is delivered to the lower epider- cular oxygen via electron or energy Functional characterization of connexin43 mutations found in patients with oculodento- mis and upper dermis in the UVA as transfer reactions (type II reaction) digital dysplasia. Circ Res 96:e83–91 compared with the UVB region resulting in ROS generation (Castano van Steensel MA, Spruijt L, van der Burgt I et al. (Wondrak et al., 2006). The sub- et al., 2004). The superoxide radical (2005) A 2-bp deletion in the GJA1 gene is sequent generation of reactive oxygen anions derived from these type I or II associated with oculo-dento-digital dysplasia species (ROS), responsible for UV photosensitization reactions result in with palmoplantar keratoderma. Am J Med Genet A 132A:171–4 photodamage, occurs through various the formation of hydrogen peroxide Vreeburg M, de Zwart-Storm EA, Schouten MI et al. mechanisms including UV-enhanced through spontaneous dismutation (2007) Skin changes in oculo-dento-digital electron leakage from the mitochondrial (Castano et al., 2004). UVA has been dysplasia are correlated with C-terminal trun- respiratory chain and remodeling of shown to induce the formation of cyclo- cations of connexin 43. Am J Med Genet A 143:360–3 cholesterol-rich plasma membrane rafts butane pyrimidine dimers subsequent to Wang H, Cao X, Lin Z et al. (2014) Exome of keratinocytes (Gniadecki et al., triplet energy transfer from excited sequencing reveals mutation in GJA1 as a cause 2002). Nonetheless, photosensitization photosensitizers to pyrimidine bases. of keratoderma-hypotrichosis-leukonychia tota- lis syndrome. Hum Mol Genet 24:243–50 1Department of Dermatology, University of Wisconsin, Madison Medical Sciences Center, Madison, White TW, Bruzzone R (1996) Multiple connexin Wisconsin, USA proteins in single intercellular channels: con- Correspondence: Hasan Mukhtar, Department of Dermatology, University of Wisconsin, Madison nexin compatibility and functional conse- Medical Sciences Center, Room 4385, 1300 University Avenue, Madison, Wisconsin 53706, USA. quences. J Bioenerg Biomembr 28:339–50 E-mail: [email protected]

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