Erythrokeratodermia Variabilis Research Articles

Intracellular Distribution, Assembly and Effect of Disease-associated Connexin 31 Mutants in HeLa Cells

Mutations in connexin 31 (Cx31) are associated with erythrokeratodermia variabilis (EKV), hearing impairment and peripheral neuropathy; however, the pathological mechanism of Cx31 mutants remains unknown. This study analyzed 11 disease-associated Cx31 variants and one non-disease-associated Cx31 variant and compared their intracellular distribution and assembly in HeLa cells and their effect on these cells. The fluorescent localization assay showed no gap junction plaque formation in the cells expressing the recessive EKV-associated mutant (L34P) and four hearing impairment-associated mutants (66delD, 141delI, R180X and E183K), significantly reduced plaque formation in the cells with five EKV-associated dominant mutants (G12R, G12D, R42P, C86S and F137L) and no obvious change in the cells with two other mutants (I141V and 652del12). Immunoblotting analysis showed that 12 mutated Cx31s, like WT-Cx31, are able to form the Triton X-100 insoluble complex; however, the quantity of Triton X-100 insoluble complex in the transfected HeLa cells varied among different Cx31 mutants. Additionally, the expression of five EKV-associated dominant mutants (G12R, G12D, R42P, C86S and F137L) caused cell death in HeLa cells. However, the five hearing impairment-associated mutants did not induce cell death. The above results suggest that disease-associated mutants gain deleterious functions differentially. In summary, disease-associated Cx31 mutants impair the formation of normal gap junctions at different levels, and the diseases associated with Cx31 mutations may result from the abnormal assembly, trafficking and metabolism of the Cx31 mutants.

Erythrokeratodermas: A classification in a state of flux?

The term 'erythrokeratodermas' or 'erythrokeratodermias' has been applied to a group of inherited disorders characterized by well-demarcated erythematous lesions and hyperkeratotic plaques. Connexin mutations have been demonstrated to be responsible for most cases of erythrokeratoderma variabilis but there remain some cases without demonstrated connexin mutations, suggesting genetic heterogeneity. The position of progressive symmetric erythrokeratoderma has become rather unclear. Loricin mutations have been found in some cases that clinically resemble variant Vohwinkel syndrome and other cases have features that overlap with those of erythrokeratoderma variablis. Whether progressive symmetric erythrokeratoderma exists as a distinct entity is under question.

A new, recurrent mutation of GJB3 (Cx31) in erythrokeratodermia variabilis

Erythrokeratodermia variabilis (EKV) is an autosomal dominant or recessive genodermatosis characterized by the coexistence of randomly occurring, transient, erythematous patches and hyperkeratosis of the skin. The disorder has been mapped to chromosome 1p35.1 but is genetically heterogeneous. EKV may be caused by pathogenic mutations in one of two neighbouring connexin genes, GJB3 and GJB4, encoding the gap junction proteins Cx31 and Cx30.3, respectively. Twelve distinct mutations identified to date cluster either at the cytoplasmic amino-terminus or in the four transmembrane domains. To report a large family with EKV and an unrelated sporadic case. DNA amplification and mutation analysis, followed by denaturing high-performance liquid chromatography to confirm the segregation of the mutations in the two families with EKV. A novel, recurrent GJB3 mutation (625C-->T; L209F) was identified in the family with EKV and in the unrelated sporadic case. This mutation is the first to affect a conserved residue in the cytoplasmic carboxy-terminus of any connexin gene with a cutaneous phenotype, emphasizing its structural and/or functional importance.

Cx31 is assembled and trafficked to cell surface by ER-Golgi pathway and degraded by proteasomal or lysosomal pathways

Gap junctions, consisting of connexins, allow the exchange of small molecules (less than 1 KD) between adjacent cells, thus providing a mechanism for synchronizing the responses of groups of cells to environmental stimuli. Connexin 31 is a member of the connexin family. Mutations on connexin 31 are associated with erythrokeratodermia variabilis, hearing impairment and peripheral neuropathy. However, the pathological mechanism for connexin 31 mutants in these diseases are still unknown. In this study, we analyzed the assembly, trafficking and metabolism of connexin 31 in HeLa cells stably expressing connexin 31. Calcein transfer assay showed that calcein transfer was inhibited when cells were treated with Brefeldin A or cytochalasin D, but not when treated with nocodazole or a-glycyrrhetinic acid, suggesting that Golgi apparatus and actin filaments, but not microtubules, are crucial to the trafficking and assembly of connexin 31, as well as the formation of gap junction intercellular communication by connexin 31. Additionally, a-glycyrrhetinic acid did not effectively inhibit gap junctional intercellular communication formed by connexin 31. Pulse-chase assay revealed that connexin 31 had a half-life of about 6 h. Moreover, Western blotting and fluorescent staining demonstrated that in HeLa cells stably expressing connexin 31, the amount of connexin 31 was significantly increased after these cells were treated with proteasomal or lysosomal inhibitors. These findings indicate that connexin 31 was rapidly renewed, and possibly degraded by both proteasomal and lysosomal pathways.

Erythrokeratodermia variabilis (EKV) – eine Störung der epidermalen Expression von Gap‐Junction‐Proteinen

ZusammenfassungDie Erythrokeratodermia variabilis (EKV) ist eine seltene autosomal‐dominant vererbte Genodermatose mit einer epidermalen Verhornungsstörung. Das klinische Bild der EKV wird von zwei Morphen geprägt: transiente, schnell wandernde Erytheme und persistierende braune Hyperkeratosen. Vor kurzem wurde der zugrunde liegende Gendefekt der EKV auf dem kurzen Arm von Chromosom 1 lokalisiert, der für das Gap‐Junction‐Protein Connexin 31 kodiert. Wir stellen einen 48jährigen Patienten vor, bei dem seit dem dreißigsten Lebensjahr großflächige, scharf begrenzte, randständig schuppende Erytheme an den Extremitäten, Gesäß und Rumpf auftreten. Histologisch fand sich eine orthokeratotische Hyperkeratose mit fokaler Parakeratose bei Akanthose der Epidermis. Immunhistochemisch konnte eine vermindert Expression des Gap‐Junction‐Proteins Connexin 31 sowie vermehrte Expression von Connexin 43 dargestellt werden. Ultrastrukturell zeigten sich erweiterte Interzellularräume in der oberen Epidermis mit unauffälligen Desmosomen, Adherensjunktionen und regulären Gap Junctions.In der Epidermis wird u. a. die Zellproliferation und ‐differenzierung über die Gap Junctions reguliert. Die Mutation im Connexin 31 wird daher als ursächlich für das klinische Bild der EKV angesehen. Die Überexpression von Connexin 43, die hier erstmals beschrieben wird, entsteht möglicherweise reaktiv als Folge der CX31‐Mutation und kompensiert vorübergehend den Defekt.

An atypical form of erythrokeratodermia variabilis maps to chromosome 7q22

Erythrokeratodermia variabilis 3 (Kamouraska type) or EKV3 is a newly described autosomal recessive disorder observed in patients from the Bas St-Laurent region of Quebec. It has similar skin lesions as observed for EKV, including congenital hyperkeratosis and red patches of variable sizes, shapes, and duration. EKV3 is also characterized by ichthyosis, sensorineural hearing loss, peripheral neuropathy, psychomotor retardation, congenital chronic diarrhea, and an elevation of very long chain fatty acids (VLCFAs). To map the disease locus, we performed candidate gene analysis and a genomewide scan to identify a common homozygous region in affected individuals from three non-consanguineous families. Mutations in connexin 31 (GJB3) and connexin 30.3 (GJB4), implicated in previous reports of EKV, and connexin 26 (GJB2), implicated in palmoplantar keratoderma, were unlikely given the lack of shared homozygous haplotypes in the regions surrounding these genes. The most promising region of common homozygosity observed in a 4,600 single-nucleotide polymorphism genome scan was further characterized by using microsatellites. A 6.8-Mb region on chromosome 7 between D7S2539 and rs727708 was found to be homozygous for the same haplotype in all affected individuals but not in the parents or an unaffected sibling. This region contains connexin 31.3 (GJE1), and although no mutation have been observed in the coding region of this gene, further analyses are required in order to exclude it. Identification of the gene responsible for this disorder will provide insights into the etiology of this multisystemic disorder.

Gap junction diseases of the skin.

Gap junctions are intercellular channels that allow the passage of water, ions, and small molecules. They are involved in quick, short-range messaging between cells and are found in skin, nervous tissue, heart, and muscle. An increasing number of hereditary skin disorders appear to be caused by mutations in one of the genes coding for the constituent proteins of gap junctions, known as connexins. In this review, the currently known connexin disorders that feature skin abnormalities are described: keratitis-ichthyosis deafness syndrome, erythrokeratoderma variabilis, Vohwinkel's syndrome, and a novel disorder called hypotrichosis-deafness syndrome. What is known about the pathogenesis of these disorders is discussed and related to gap junction physiology.

Connexin mutations in human disease.

Connexin mutations in human disease.

The Ichthyosis Registry—A Resource Ready for Use

The National Registry for Ichthyosis and Related Disorders was funded by NIAMS in 1994 to improve understanding of the pathophysiology, diagnosis, and treatment of the ichthyoses. Individuals with ichthyosis enroll in order to obtain and provide information about their disorder. A list of the disorders enrolled in the Registry can be found at www.skinregistry.com Diagnosis is confirmed by strict clinical, histologic, and in the case of recessive X-linked ichthyosis, biochemical criteria. In appropriate cases, mutation detection is offered to individuals in whom known mutations have been shown to underlie their disorder. Extensive identifying, historical, clinical, and quality of life data are obtained during an interview by the Registry coordinator and are entered into a secure database. Over 600 subjects have been enrolled. In most cases (>97%), enrollees ask to be informed of pertinent studies. Thus, a large group of affected individuals with well-characterized disorders interested in participating in studies of their disease is available for industrious investigators. The Registry is available for “mining” at many levels. For example, a recent review of the quality-of-life data of enrollees was presented at the International Investigative Dermatology meeting 1Fleckman P, Hamill G, Weinstock MA: The national registry for ichthyosis and related disorders—Health related quality of life and patient reported outcomes. J Invest Dermatol 121:420, 2003 (abstr)1Fleckman P, Hamill G, Weinstock MA: The national registry for ichthyosis and related disorders—Health related quality of life and patient reported outcomes. J Invest Dermatol 121:420, 2003 (abstr) and has been submitted for publication. Other investigators have used the Registry to identify and inform subjects about studies of specific clinical disorders such as erythrokeratodermia variabilis, X-linked recessive ichthyosis, CIE, and KID Syndrome (Taubenheim, 1999Taubenheim A. Use of the Internet to search for information on arthritis and musculoskeletal and skin diseases.1999Google Scholar;Richard et al., 1998Richard G. Smith L.E. Bailey R.A. et al.Mutations in the human connexin gene GJB3 cause erythrokeratodermia variabilis.Nat Genet. 1998; 20: 366-369Crossref PubMed Scopus (301) Google Scholar;Zettersten et al., 1998Zettersten E. Man M.Q. Sato J. et al.Recessive x-linked ichthyosis: Role of cholesterol–sulfate accumulation in the barrier abnormality.J Invest Dermatol. 1998; 111: 784-790Crossref PubMed Scopus (94) Google Scholar;Fowler et al., 2004Fowler A.J. Moskowitz D.G. Wong A. Cohen S.P. Williams M.L. Heyman M.B. Nutritional status and gastrointestinal structure and function in children with ichthyosis and growth failure.J Pediatr Gastroenterol Nutr. 2004; 38: 164-169Crossref PubMed Scopus (21) Google Scholar). In addition, a discussion group for investigators with clinical interests is maintained on the Registry web site. Investigators from within or outside of the US interested in pursuing this valuable resource are encouraged to contact the Registry for more complete information.

Molecular interaction of connexin 30.3 and connexin 31 suggests a dominant-negative mechanism associated with erythrokeratodermia variabilis.

Connexins are homologous four-transmembrane-domain proteins and major components of gap junctions. We recently identified mutations in either GJB3 or GJB4 genes, encoding respectively connexin 31 (Cx31) or 30.3 (Cx30.3), as causally involved in erythrokeratodermia variabilis (EKV), a mostly autosomal dominant disorder of keratinization. Despite slight differences, phenotypes of EKV Mendes Da Costa (Cx31) and EKV Cram-Mevorah (Cx30.3) show major clinical overlap and both Cx30.3 and Cx31 are expressed in the upper epidermal layers. These similarities suggested to us that Cx30.3 and Cx31 may interact at a molecular level. Indeed, expression of wild-type Cx30.3 in HeLa cell resulted only in minor amounts of protein addressed to the plasma membrane. Mutant Cx30.3 was hardly detectable and disturbed intercellular coupling. In sharp contrast, co-expression of both wild-type proteins led to a gigantic increase of stabilized heteromeric gap junctions. Furthermore, co-expressed wild-type Cx30.3 and Cx31 coprecipitate, which demonstrates a physical interaction. Inhibitor experiments revealed that this interaction begins in the endoplasmic reticulum. These results not only provide new insights into epidermal connexin synthesis and polymerization, but also allow a novel molecular explanation for the similarity of EKV phenotypes.

Eritroqueratodermia simétrica progressiva: relato de caso

A eritroqueratodermia simétrica progressiva (EQSP) é genodermatose rara, caracterizada por placas eritematosas e hiperceratóticas fixas, de distribuição simétrica nas extremidades. A primeira descrição da doença foi feita por Darier em 1911, e desde então existem poucas publicações a respeito do assunto. Os autores relatam um caso de EQSP em uma menina de nove anos de idade, com lesões localizadas nos cotovelos e joelhos, de aspecto típico. Os achados histológicos foram inespecíficos, e o tratamento com produtos de uso tópico, insatisfatório. A descrição do caso visa correlacionar os achados clínicos, histológicos, e a evolução do quadro, dentro dos conhecimentos atuais sobre a doença.

Genetic Heterogeneity in Erythrokeratodermia Variabilis: Novel Mutations in the Connexin Gene GJB4 (Cx30.3) and Genotype-Phenotype Correlations

Erythrokeratodermia variabilis is an autosomal dominant genodermatosis characterized by persistent plaque-like or generalized hyperkeratosis and transient red patches of variable size, shape, and location. The disorder maps to a cluster of connexin genes on chromosome 1p34-p35.1 and, in a subset of families, results from mutations in the gene GJB3 encoding the gap junction protein connexin-31 (Cx31). A recent report suggested the involvement of another connexin gene (GJB4) in the etiology of erythrokeratodermia variabilis. In this study, we sequenced the coding region of GJB4 in 13 unrelated erythrokeratodermia variabilis families without detectable mutations in GJB3. Mutation analysis revealed six distinct missense mutations in five families and a sporadic case of erythrokeratodermia variabilis, all of which were not found in controls. Mutation G12D, identified in an extended Dutch family, lies in the predicted amino-terminus and may interfere with the flexibility of this domain, connexin selectivity, or gating polarity of gap junction channels. Other mutations (R22H, T85P, F137L, F189Y) were located in the transmembrane domains of Cx30.3, and are predicted to hinder regulation of voltage gating or alter the kinetics of channel closure. Affected individuals of two unrelated families harbored point mutations leading to amino acid substitution F137L, which was also reported in GJB3, yet the extent and severity of hyperkeratosis was milder compared to the corresponding mutation in GJB3. Two mutations (T85P, F137L) were associated with the occurrence of rapidly changing erythematous patches with prominent, circinate, or gyrate borders in affected children but not in adults, supporting the notion that this feature is specific to Cx30.3 defects. Nevertheless, we observed highly variable intrafamilial phenotypes, suggesting the strong influence of modifying genetic and epigenetic factors. In addition to pathogenic mutations, we identified several missense mutations and a 4 bp deletion within the GJB4 coding region, which might represent either inconsequential polymorphisms or recessive mutations. In conclusion, our results demonstrate genetic heterogeneity in erythrokeratodermia variabilis, and emphasize that intercellular communication mediated by both Cx31 and Cx30.3 is crucial for epidermal differentiation.

Divergent effects of two sequence variants of GJB3 (G12D and R32W) on the function of connexin 31 in vitro.

Recently, we identified several missense mutations of the connexin gene GJB3 encoding connexin 31 (Cx31) in erythrokeratodermia variabilis (EKV), an autosomal dominant skin disorder. These mutations include G12D, which replaces a conserved glycine residue in the amino-terminus of Cx31 and is associated with a severe EKV phenotype. In contrast, the biologic relevance of the GJB3 sequence variant R32W located in the first transmembrane domain of Cx31 is disputed. To examine the effects of these sequence variants on Cx31 biogenesis and gap junction activity we expressed wild type and mutant Cx31-Flag constructs in HeLa cells. Using immunostaining, all expression variants were detected in the cytoplasm and in a punctate pattern at the cell surface, indicating that G12D and R32W did not interfere with either protein synthesis or transport to the cell membrane. Similarly, oligomerization into hemichannels appeared not impaired when expressing either Cx31 mutant as assessed by size exclusion chromatography, immunoblotting and immunostaining. However, dye transfer experiments and monitoring of intracellular calcium levels in response to serum stimulation revealed that G12D-Cx31 did not form functional gap junction channels, probably due to incorrect assembly or altered properties of Cx31 channels. In contrast, intercellular coupling between cells expressing R32W-Cx31 was comparable to that of wtCx31, suggesting that R32W is a functionally inconsequential polymorphism of Cx31.

Reticular erythrokeratoderma: a new disorder of cornification.

Heritable disorders of cornification form a large, clinically and genetically heterogeneous group. Recent advances in molecular genetics provide for the first time the opportunity to reliably classify some of these disorders based on their underlying etiology. Many rare phenotypes, however, still remain unclassified and do not fit into established classification schemes. We report here a 12-year-old girl who developed an ichthyosis vulgaris-like skin disorder 6 months after birth. Several years later, the clinical features had changed considerably. The patient had developed streaks of hyperkeratotic, slightly scaling skin with underlying erythema distributed in a reticulate, occasionally annular pattern on the trunk and extremities. The lesions were stable and had not changed significantly in size or distribution over the ensuing years. Histopathologic and ultrastructural findings were nonspecific and there was no evidence for metabolic disorders. The partial clinical overlap with erythrokeratodermia variabilis prompted us to screen several connexin genes but no pathogenic mutations were identified. We believe that this disorder belongs to the group of erythrokeratodermas and represents a novel, previously unrecognized entity.

Ichthyosis: etiology, diagnosis, and management.

The ichthyoses are a heterogeneous group of disorders with both inherited and acquired forms. Clinical presentation, pattern of inheritance, and laboratory evaluation may establish a precise diagnosis, which can assist in prognosis and genetic counseling. Congenital autosomal recessive ichthyosis (CARI) usually presents at birth, often as a collodion baby. CARI can progress into any one of a spectrum of disorders. Lamellar ichthyosis is characterized by dark, plate (armor)-like scale. This disease is often caused by mutations in the gene encoding the enzyme transglutaminase 1. Congenital ichthyosiform erythroderma is another phenotype within CARI, marked by generalized redness and fine white scale. Epidermolytic hyperkeratosis is an autosomal dominant disorder characterized by hyperkeratosis and blistering, and at least six clinical phenotypes have been described. It may be due to mutations in the gene encoding the intermediate filament proteins keratin 1 and 10. Ichthyosis vulgaris is the most common ichthyosis, and is inherited in an autosomal dominant pattern. Involvement is generally mild and may vary greatly with climate and humidity. X-linked ichthyosis, due to a defect in the enzyme steroid sulfatase, affects males with generalized scaling that usually begins soon after birth. There may be associated corneal opacities that do not affect vision. Sjögren-Larsson syndrome is an autosomal recessive ichthyosis associated with progressive spastic paralysis and mental retardation. This condition is caused by mutations in the gene for fatty aldehyde dehydrogenase. Refsum's disease, due to accumulation of phytanic acid, results in ichthyosis and progressive neurologic dysfunction. The erythrokeratodermas are characterized by hyperkeratosis and localized erythema. Erythrokeratodermia variabilis is autosomal dominant and characterized by generalized or localized hyperkeratosis and migratory red patches. Mutations in the genes encoding the gap junction proteins, connexins, underlie this disorder. Netherton's syndrome is an autosomal recessive disorder characterized by ichthyosis, a hair shaft abnormality and atopy. The ichthyosis may present at birth with erythroderma or in some cases a collodion presentation. However, a frequent characteristic skin manifestation is ichthyosis linearis circumflexa. Netherton's syndrome has been found to be due to an abnormality in a serum protease inhibitor. Acquired ichthyosis can have a variety of underlying causes including neoplastic, infectious, drugs, endocrine, metabolic, autoimmune, malabsorptive states, and hereditary. Topical, and in more severe cases, systemic, therapy are useful in managing this array of disorders of cornification.

Defective trafficking and cell death is characteristic of skin disease-associated connexin 31 mutations.

Distinct germline mutations in the gene (GJB3) encoding connexin 31 (Cx31) underlie the skin disease erythrokeratoderma variabilis (EKV) or sensorineural hearing loss with/without peripheral neuropathy. Here we describe a number of functional analyses to investigate the effect of these different disease-associated Cx31 mutants on connexon trafficking and intercellular communication. Immunostaining of a biopsy taken from an EKV patient harbouring the R42P mutation revealed sparse epidermal staining of Cx31, and, when present, it had a perinuclear localization. Transfection and microinjection studies in both keratinocytes and fibroblast cell lines also demonstrated that R42P and four other EKV-associated mutant Cx31 proteins displayed defective trafficking to the plasma membrane. The deafness/neuropathy only mutant 66delD had primarily a cytoplasmic localization, but some protein was visualized at the plasma membrane in a few transfected cells. Both 66delD- and R32W-Cx31/EGFP proteins had significantly impaired dye transfer rates compared to wild-type Cx31/EGFP protein. A striking characteristic feature observed with the dominant skin disease Cx31 mutations was a high incidence of cell death. This was not observed with wild-type, R32W 66delD Cx31 proteins. In conclusion, we have identified some key cellular phenotypic differences with respect to disease-associated Cx31 mutations.

Expression of a connexin31 mutation causing erythrokeratodermia variabilis is lethal for HeLa cells

The autosomal dominant skin disorder erythrokeratodermia variabilis (EKV) has been linked to mutations in the human connexin31 (hCx31) gene, which is expressed in the epidermis. We characterized and compared a pathogenic mutation resulting in replacement of amino acid glycine 12 with arginine (G12R) with wild-type hCx31 protein. HeLa cells were transfected with wild-type and mutant hCx31 cDNA, respectively, using different—constitutive and inducible—vector systems. Independent of the expression vector, wild-type and mutant hCx31 were expressed at comparative levels and localized at the plasma membranes. Mutated channels (hCx31G12R) showed higher conductance in dye coupling studies than wild type channels. Furthermore, HeLa cells died within 5 days after constitutive expression of the mutant protein. Using an inducible expression system, we demonstrated a direct correlation between survival/life span of transfected HeLa cells and expression level of the mutant protein, indicating a gain-of-function mechanism due to a defective channel closure mechanism.

A mutation in GJB3 is associated with recessive erythrokeratodermia variabilis (EKV) and leads to defective trafficking of the connexin 31 protein

Erythrokeratodermia variabilis (EKV) is a skin disorder characterized by variable (transient) erythemas and fixed keratosis. The disorder maps to chromosome 1p34-35, a location that contains the GJB3 gene encoding the gap junction protein connexin 31. Until now, only heterozygote mutations in the form of dominant inheritance have been described in this gene associated with EKV. We report here a homozygote mutation in the connexin 31 gene, found in a family that shows recessive inheritance of the disorder, thus providing the first molecular support for a recessive variant of EKV. The entire GJB3 coding sequence was scanned for mutations by sequencing. We detected a T-->C transition at position 101 of the coding sequence, which replaces a leucine with a proline at residue 34 of the protein (L34P). Evolutionary analysis shows that this mutation is located at a highly conserved region of connexin in the first putative transmembrane helix (TMH). In transfected keratinocytes, L34P connexin 31 had a cytoplasmic distribution, suggesting that the mutant form of this protein will not form normal gap junctions between adjacent cells. The change of leucine to proline is likely to alter the structure of the first TMH of connexin by inducing a kink, thus influencing connexon structure and function.

A common frameshift mutation and other variants in GJB4 (connexin 30.3): Analysis of hearing impairment families

Mutations in GJB1, GJB2, GJB3 and GJB6 are involved in hearing impairment. GJB2, GJB3 and GJB6 are also mutated in patients with hyperproliferative skin disorders. The human GJB4 gene has been deduced in silico and a mutation in a family with erythrokeratodermia variabilis has been reported. We describe here the analysis of the GJB4 gene in hearing impairment patients and control subjects. We have identified a common (4%) frameshift mutation (154del4) in GJB4 in both affected and hearing subjects, one patient being homozygous for the mutation. We have also detected five amino acid variants (R103C, R124Q, R160C, C169W and E204A) in individuals that have not skin disorders. While mutation 154del4 is not associated with hearing impairment the involvement of some of the amino acid variants detected here is uncertain. These GJB4 variants should help to define the putative role of connexin 30.3 in both skin disorders and hearing impairment.

Multiple Epidermal Connexins are Expressed in Different Keratinocyte Subpopulations Including Connexin 31

Recent genetic studies have demonstrated the importance of epidermal gap junctions with mutations in four beta-connexins associated with autosomal dominant epidermal disease. One of these disorders, erythrokeratoderma variabilis, is associated with germline mutations in the genes encoding connexins (Cx) Cx31 and Cx30.3. Towards understanding the functional mechanism of Cx31 mutations in epidermal disease, we have developed and characterized a polyclonal antibody raised against human Cx31. Using this antibody to immunostain normal epidermis, Cx31 protein was found to be expressed predominately in the stratum granulosum with a punctate pattern of staining at the plasma membrane. In addition, we used reverse transcriptase polymerase chain reaction and, where reagents were available, immunocytochemistry to investigate which other connexins are expressed in the epidermis. Surprisingly, this analysis revealed that there are at least 10 connexins expressed with an overlapping distribution and localization to distinct keratinocyte subpopulations. These data provide additional evidence for multiple gap junction channel types in the human epidermis. Elucidation of this complexity of channel types with respect to specific permeabilities and function of each wildtype and mutant channel type in epidermal biology will require further investigations.