Dominant De Novo Mutations in GJA1 Cause Erythrokeratodermia Variabilis et Progressiva, without Features of Oculodentodigital Dysplasia

Abstract

Genetic investigation of inherited skin disorders has informed the understanding of skin self-renewal, differentiation, and barrier function. Erythrokeratodermia variabilis et progressiva (EKVP) is a rare, inherited skin disease that is characterized by transient figurate patches of erythema, localized or generalized scaling, and frequent palmoplantar keratoderma. By using exome sequencing, we show that de novo missense mutations in GJA1 (gap junction protein alpha 1) cause EKVP. The severe, progressive skin disease in EKVP subjects with GJA1 mutations is distinct from limited cutaneous findings rarely found in the systemic disorder oculodentodigital dysplasia, also caused by dominant GJA1 mutations. GJA1 encodes connexin 43 (Cx43), the most widely expressed gap junction protein. We show that the GJA1 mutations in EKVP subjects lead to disruption of Cx43 membrane localization and aggregation within the Golgi. These findings reveal a critical role for Cx43 in epidermal homeostasis, and they provide evidence of organ-specific pathobiology resulting from different mutations within GJA1.

Figure 1. Clinical and histologic features of EKVP due to GJA1 mutation

(a) The knee of 101-1 was normal at birth, but became thickened and scaly when he began to crawl; shown at age 30 months. (b) The knee of 6-year-old 102-1 shows progression to corrugated, thickened, scaly skin which has extended beyond sites of friction. (c) The knee of 30-year-old 103-1 shows marked hyperkeratosis with peeling scale; a patch of figurate erythema is present inferiorly. (d) Histology of affected leg skin of 102-1 shows a compact, thickened stratum corneum (SC) with retained nuclei (arrow), papillomatosis, a thickened granular layer (G), and a perivascular lymphocytic infiltrate (asterisk).

Figure 2. Prominent white nails and progressive keratoderma in EKVP due to GJA1 mutation

All subjects show prominent porcelain white proximal nails without dystrophy. Honeycombed thick hyperkeratosis is present on the palms, and similar thick hyperkeratosis is present on the feet. Keratoderma is progressive with 30-month-old 101-1 (a), showing less hyperkeratosis than 6-year-old 102-1 (b) or 30-year-old 103-1 (c). Subject 103-1 is on a systemic retinoid, accounting for peeling seen at lateral aspects of hand and foot and less exuberant hyperkeratosis.

Figure 3. Figurate erythema and darkening of the skin in EKVP due to GJA1 mutation

All subjects report figurate erythema and have experienced progressive skin darkening. (a) In subject 101-1, figurate erythema is present on the back, flank, arm, and upper thigh. (b) Subject 102-1 shows thick, corrugated keratoderma with darkening prominent on the neck, forearm, axilla, and abdomen. There is a patch of erythema on the wrist. (c) In subject 103-1, figurate erythema is present on the upper and lower back. Notably, subject 103-1 reports that figurate erythema became more frequent and prominent at age 10 years but was present throughout childhood. Treatment with acitretin has reduced scaling, but hyperpigmentation is prominent on the flanks, shoulders, and neck. In all cases, erythema is induced by stress and warm conditions and can be accompanied by a stinging or burning sensation.

Figure 4. GJA1 mutations in EKVP kindreds

(a) Affected and unaffected subjects are denoted with black and white symbols, respectively; adoption is shown with a dashed line. GJA1 alleles determined by sequencing of genomic DNA are denoted as ‘+’ (wild-type) or by the amino acid substitution in red (mutant). To the right of each pedigree, Sanger sequence traces at GJA1 mutation sites are shown for each subject and parents from whom DNA was available (mutant bases indicated with *). Amino acid sequences are shown at the top of each trace (mutant residues in red). Mutations are p.E227D, c.A681T (subjects 101-1 and 102-1) and p.A44V, c.C131T (subject 103-1) (NCBI RefSeq NM_000165). (b) Schematic model of Cx43 shows locations of mutations in EKVP patients reported here (red), and those reported in ODDD (blue), ODDD with palmoplantar hyperkeratosis (purple), and ODDD with Clouston syndrome (green).

Figure 5. Cx43 and paralogs

Transmembrane domains are outlined; conserved residues are shaded gray. Disease-causing Cx43 mutations are shown above the alignment: EKVP reported here (red shade), ODDD (blue), ODDD with palmoplantar hyperkeratosis (purple), ODDD with Clouston syndrome (green shade); frameshifts (‘fs’), deletions (‘d’), insertions (‘i’). Mutation sites in paralogs reported to cause skin disease are in orange.

Figure 6. Cx43 is mis-localized in EKVP

(a–c) Cx43 immunolocalization was performed on tissue sections from wild-type (wt) and affected patient skin (102-1: E227D, 103-1: A44V), with Cx43 antibody in red and DAPI nuclear counterstain in blue. (a) Wild-type tissue shows primarily intercellular membrane localization of Cx43. (b–c) Affected skin shows primarily cytoplasmic localization. (d–f) Wild-type (wt) and mutant Cx43 (E227D, A44V) were HA-tagged and expressed in HeLa cells. Immunolocalization of Cx43 is red, cis-Golgi marker GM130 is green, and DAPI nuclear counterstain is blue. (d) Wild-type Cx43 localizes to intercellular junctions and does not co-localize with GM130. (e and f) The E227D and A44V mutants do not localize to intercellular junctions and accumulate in a subcellular compartment, partly co-localizing with GM130. Scale bars are 20 μm in all panels.