Erythrokeratodermia variabilis: Report of two cases and a novel missense variant in GJB4 encoding connexin 30.3

ARTICLE

ejd.2011.1617

Auteur(s) : Haris Kokotas¹ hkokotas@yahoo.gr, Konstantina Papagiannaki², Maria Grigoriadou¹, Michael B. Petersen¹, Alexandra Katsarou²

¹ Department of Genetics, Institute of Child Health, “Aghia Sophia” Children's Hospital, Athens 11527, Greece

² 1^st Department of Dermatology & Venereology, “Andreas Sygros” Hospital, Athens, Greece

Reprints: H. Kokotas

Erythrokeratodermia variabilis (EKV) (MIM 133200) is a rare genodermatosis with two distinctive morphologic components. The first is the occurrence of bizarre, figurate, sharply demarcated erythematous patches that change their shape and distribution within minutes, hours or days. The second is the occurrence of more persistent plaques of hyperkeratosis with striking geographic outlines. Such plaques may arise independently on previously normal skin or on areas of persistent erythema [1]. Often, one of the features may be more dominant than the other, and rarely, one may be absent, with either the erythema or hyperkeratotic plaques as the only manifestation [2]. The erythematous and hyperkeratotic lesions may occur anywhere on the body but the most common sites of involvement are the face, buttocks and limbs [3]. Mucous membranes are not involved and there is no disturbance of teeth, nails or hairs but there may be varying degrees of palmoplantar keratoderma in association with the disease [4]. Both internal and external events may trigger the disease. These include trauma, psychological stress, temperature changes, and sun exposure [2]. Hormonal influences have been suggested with reports of resolution of lesions at menopause and deterioration during pregnancy or with estrogen-containing contraceptive preparations [5]. The age of onset is birth to within the first year of life for the majority of the patients and lesions persist for the patient's entire life [2].

The genetic defect and pathogenesis underlying EKV remain unclear. The vast majority of reported cases suggest a monogenic autosomal dominant mode of inheritance with variability of expression [6-8], although a recessively inherited form of EKV has also been suggested [7]. EKV has been mapped to chromosome 1p34-35 where a cluster of several Cx genes is located [8]. Connexins are a family of proteins that form the subunits of gap junction channels. The latter are of paramount importance for intercellular communication, by allowing the transfer of ions and second messenger molecules between adjacent cells [9]. They share a common pattern of structural motifs or domains, including four transmembrane domains, two extracellular domains, and three cytoplasmic domains. The cytoplasmic domains are the amino-terminus, the cytoplasmic loop, and the carboxy-terminus. The cytoplasmic loop and carboxy-terminus domains are characteristic of each Cx, while the membrane spanning and extracellular domains are highly conserved. Chromosomal mapping studies have localized heterozygous mutations involved in EKV to GJB3 (GenBank, NM_001005752.1), the gene encoding Cx31. Several cases with EKV harboring GJB3 mutations have been previously reported [10-17]. However, patients with EKV have been described without mutations in GJB3, but positive for mutations within the GJB4 gene (GenBank, NM_153212.2) encoding Cx30.3 [18-20].

Materials and methods

A five-year-old patient presenting with general scaling migratory erythematous areas on the trunk and extremities and a 31-year-old patient with migratory erythematous areas and fixed hyperkeratotic plaques on the trunk and extremities were referred to our Departments for clinical, histological, and molecular genetic evaluation. Both patients were female and of Greek origin. Written informed consent was provided in both cases. The clinical and molecular examinations were approved by the Ethics Committees of the “Andreas Sygros” Hospital and the Institute of Child Health. All procedures followed were in accordance with the Helsinki Declaration of 1975, as amended in 1983.

A complete clinical examination was performed on both subjects and a detailed medical history of their families was obtained with the use of a standard questionnaire. Furthermore, a skin biopsy was taken from the right lower leg of the first patient and from the abdomen of the second patient and genomic DNA was extracted from venous blood using a standard protocol [21]. The DNA samples were subsequently subjected to molecular analysis of the GJB3 and GJB4 genes by direct sequencing following amplification of the coding region of both genes by polymerase chain reaction (PCR). We used previously published primer sets and PCR protocols [22]. PCR-amplified samples were purified and sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the same primers used for PCR. Sequencing products were separated by capillary electrophoresis on an ABI 310 genetic analyzer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. The evaluation of the chromatograms was performed using the Sequencing Analysis Software Version 5.2, and the alignment of the obtained sequences with the reference sequences of the two genes was done with the SeqScape V2.5 software (Applied Biosystems, Foster City, CA, USA). In cases where novel variants are identified, in silico tools, including PolyPhen2 [23], SIFT [24] and ClustalW2 [25], are being recruited in order to evaluate the possible pathogenicity of each variant.

We recruited the amplification refractory mutation system (ARMS) PCR technique in order to investigate the existence of the novel finding of the second patient in ethnically matched controls. Occasionally, in ARMS-PCR, a single-base mismatch at the 3’ terminus of a primer is insufficient to achieve the desired level of discrimination between the mutant and the normal allele. A 3’-terminal mismatch coupled with an additional mismatch usually one, two, or three bases from the 3’ terminus can increase discrimination. A ‘strong’ mismatch (G/A or C/T) at the 3’-terminus of an allele-specific primer will likely require a ‘weak’ second mismatch (C/A or G/T) and vice versa, whereas a ‘medium’ mismatch (A/A, C/C, G/G or T/T) at the 3’-terminus will likely require a ‘medium’ second mismatch. We designed forward primer GJB4mut: 5’-CAT GCA CGT GGC CTA CC T C A-3’ with two mismatches, one at the 3’ end and a second mismatch at the third nucleotide position from the 3’ end, in order to detect mutant alleles, and forward primer GJB4nor: 5’-CAT GCA CGT GGC CTA CCG CG–3’ for the detection of normal alleles. Primer GJB4-R: 5’-AGC AAG TAC GTC CAC CAC AGT C-3’ served as a reverse primer for the detection of both the normal and mutant alleles. The PCR was performed according to the following protocol: 95 ^̊C for 3 min, followed by 30 cycles of 94 ^̊C for 1 min, 63 ^̊C for 1 min, 72 ^̊C for 1 min 30 s, and a final step of extension for 6 min at 72 ^̊C.

One hundred unrelated Greek patients with phenylketonuria or hyperphenylalaninemia who had previously undergone clinical examination served as controls in this study.

Results

Clinical evaluation

The five-year-old girl presented with general scaling migratory erythematous areas on the trunk and extremities. Physical examination revealed a few sharply demarcated erythematous patches on the four extremities and the buttocks. Mild scaling was noted on areas of erythema and normal skin. No skin lesions were noted at birth but the eruption had been present since she was three years old. The parents stated that the lesions changed shape and distribution over the course of hours and were exacerbated by hot weather and emotional stress. She was otherwise healthy and her skin condition did not appear to be symptomatic. A detailed family history revealed no members with a similar condition, however, her mother suffered from osteogenesis imperfecta.

The 31-year-old patient presented with migratory erythematous areas and fixed hyperkeratotic plaques (figure 1) on the trunk and extremities. Some of these erythematous lesions appeared as erythema gyratum repens, characterized by rapidly migrating figurate erythema in annular or garland arrangements. The migratory erythema had been present since she was one year old. From the age of two years, hyperkeratotic lesions gradually presented on her trunk and extremities. Physical examination revealed diffuse geographic, well-demarcated erythematous scaly keratotic plaques involving the buttocks, axillae and folds. There was no involvement of the scalp, palms and soles. The patient reported an exacerbation of the disease during pregnancy. Topical (corticosteroid, retinoid) as well as systemic therapies were applied but none succeeded in maintaining a long term clearance of the disease. No further symptoms were observed in this patient. Although her parents were reported to be third cousins originating from close Greek villages, a detailed family history revealed no similar cutaneous condition in her relatives.

Histological and molecular analyses

Both biopsy specimens revealed similar histological changes concerning basket weave hyperkeratosis with parakeratosis, mild acanthosis and minimal lymphocytic infiltration around the superficial blood vessels (figure 2).

Molecular analysis of the GJB3 and GJB4 genes failed to identify any alteration in the coding sequences of either gene in the first patient. Although the coding region of GJB3 was normal in the second patient, we identified a novel c.295G>A missense variant in homozygosity in the GJB4 gene (figure 3), resulting in a p.E99K (p.Glu99Lys) change of the Cx30.3 protein sequence. Evaluation of the pathogenicity of the c.295G>A variant using PolyPhen2 and SIFT (figures 4 and 5) in silico tools demonstrated that it is likely to be a benign polymorphism rather than a pathogenic mutation. The use of ClustalW2 showed that the position 99 of the amino acid chain of the Cx30.3 protein demonstrated a medium conservation among ten species (figure 6), a result that further supported the findings of PolyPhen2 and SIFT. However, the c.295G>A (p.Glu99Lys) variant was not detected in any of the 100 controls tested.

Discussion

Gap junction proteins are molecules that form intercellular channels connecting adjacent cells, and are widely expressed in the human body. However, a mutation in a specific Cx gene will usually affect only one organ (i.e. skin, inner ear), probably because of its specific role [26]. At least ten different types of connexins are expressed in different keratinocyte populations of human skin [27]. Mutations in four connexins have been associated with skin disorders: Cx31 with EKV [10], Cx30.3 with EKV associated with erythema gyratum repens-like features [18], Cx26 with Vohwinkel's syndrome [28, 29], Keratitis-Ichthyosis-Deafness (KID) and hystrix-like ichthyosis with deafness (HID) syndromes [30], and Cx30 with hidrotic ectodermal dysplasia [31].

Connexins share a common structure of four transmembrane helices connected by two extracellular loops and one cytoplasmic loop; thus, both their C and N termini are cytoplasmic. Different functions are predicted for each domain of the protein. Initially, it had been suggested that the location of a dominant mutation in Cx31 could determine which organ would be affected – skin or inner ear. Mutations in the extracellular loops, implicated in the specificity of Cx-Cx interactions, would result in hearing loss, whereas mutations in other domains, such as those associated with voltage gating, would result in EKV [32]. However, as the number of EKV-associated mutations has risen, this correlation has proven to be untrue [12].

In this study we performed clinical, histological and molecular genetic examinations in two unrelated Greek female subjects suffering from EKV. The patients presented with general scaling migratory erythematous areas on the trunk and extremities, and migratory erythematous areas and fixed hyperkeratotic plaques on the trunk and extremities, respectively. Skin biopsies showed similar results in both patients. The patients were tested for mutations in the GJB3 and GJB4 genes, previously associated with EKV. No mutation was found in the GJB3 and GJB4 genes in the first patient and the GJB3 gene of the second patient. Screening of the GJB4 gene in the latter showed a novel homozygous c.295G>A (p.E99K) transition in the coding sequence of the gene. The mutated glutamic acid residue (p.Glu99Lys) lies in the second cytoplasmic region of the Cx30.3 protein, at position 99, which was found to be non-conserved. Although the evaluation of the novel finding with in silico tools demonstrated that the variant probably represents a benign polymorphism, we failed to detect it in 100 Greek controls. Our data suggest that the c.295G>A substitution in the GJB4 gene is a variant of unknown significance, most probably not associated with EKV, and that the clinical manifestations of both patients could be due to other genetic and non-genetic factors. It appears that homozygosity of the c.295G>A (p.E99K) variant in the second patient is due to the fact that her parents originate from the same region and were reported to be third cousins.

Acknowledgements: The authors would like to thank the two EKV patients and their families for participating in this study. Financial support: none. Conflict of interest: none.

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