Plectin defects in epidermolysis bullosa simplex with muscular dystrophy.

Epidermolysis bullosa simplex with muscular dystrophy (EBS-MD, MIM 226670) is caused by plectin defects. We performed mutational analysis and immunohistochemistry using EBS-MD (n = 3 cases) and control skeletal muscle to determine pathogenesis. Mutational analysis revealed a novel homozygous plectin-exon32 rod domain mutation (R2465X). All plectin/HD1-121 antibodies stained the control skeletal muscle membrane. However, plectin antibodies stained the cytoplasm of type II control muscle fibers (as confirmed by ATPase staining), whereas HD1-121 stained the cytoplasm of type I fibers. EBS-MD samples lacked membrane (n = 3) but retained cytoplasmic HD1-121 (n = 1) and plectin staining in type II fibers (n = 3). Ultrastructurally, EBS-MD demonstrated widening and vacuolization adjacent to the membrane and disorganization of Z-lines (n = 2 of 3) compared to controls (n = 5). Control muscle immunogold labeling colocalized plectin and desmin to filamentous bridges between Z-lines and the membrane that were disrupted in EBS-MD muscle. We conclude that fiber-specific plectin expression is associated with the desmin-cytoskeleton, Z-lines, and crucially myocyte membrane linkage, analogous to hemidesmosomes in skin.

Progress in epidermolysis bullosa: the phenotypic spectrum of plectin mutations.

Plectin, a large multidomain adhesive protein with versatile binding functions, is expressed in a number of tissues and cell types. In the skin, plectin is a critical component of hemidesmosomes, interacting with keratin intermediate filaments and beta4 integrin. Mutations in the plectin gene (PLEC1) result in fragility of skin, demonstrating blister formation at the level of hemidesmosomes. These blistering disorders belong to the spectrum of epidermolysis bullosa (EB) phenotypes, and three distinct variants because of plectin mutations have been identified. First, EB with muscular dystrophy, an autosomal recessive syndrome, is frequently caused by premature termination codon-causing mutations leading to the absence of plectin both in the skin and in the muscle. Second, a heterozygous missense mutation (R2110W) in PLEC1 has been documented in patients with EB simplex of the Ogna type, a rare autosomal dominant disorder. Finally, recent studies have disclosed plectin mutations in patients with EB with pyloric atresia, an autosomal recessive syndrome, frequently with lethal consequences. Collectively, these observations attest to the phenotypic spectrum of plectin mutations, and provide the basis for accurate genetic counselling with prognostic implications, as well as for prenatal diagnosis in families at the risk of recurrence of the disease.

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.

trans-dominant inhibition of connexin-43 by mutant connexin-26: implications for dominant connexin disorders affecting epidermal differentiation.

Dominant mutations of GJB2-encoding connexin-26 (Cx26) have pleiotropic effects, causing either hearing impairment (HI) alone or in association with palmoplantar keratoderma (PPK/HI). We examined a British family with the latter phenotype and identified a new dominant GJB2 mutation predicted to eliminate the amino acid residue E42 (DeltaE42) in Cx26. To dissect the pathomechanisms that result in diverse phenotypes of dominant GJB2 mutations, we studied the effect of three Cx26 mutants (DeltaE42, D66H and R75W) identified in individuals with PPK/HI, and another (W44C) present in individuals with non-syndromic HI on gap junctional intercellular communication. We expressed mutant Cx26 alone and together with the epidermal connexins Cx26, Cx37 and Cx43 in paired Xenopus oocytes, and measured the intercellular coupling by dual voltage clamping. Homotypic expression of each connexin as well as co-expression of wild-type (wt) Cx26/wtCx43 and wtCx26/wtCx37 yielded variable, yet robust, levels of channel activity. However, all four Cx26 mutants were functionally impaired and failed to induce intercellular coupling. When co-expressed with wtCx26, all four mutants suppressed the wtCx26 channel activity consistent with a dominant inhibitory effect. However, only those Cx26 mutants associated with a skin phenotype also significantly (P<0.05) inhibited intercellular conductance of co-expressed wtCx43, indicating a direct interaction of mutant Cx26 units with wtCx43. These results demonstrate, for the first time, a trans-dominant negative effect of Cx26 mutants in vitro. Furthermore, they support a novel concept suggesting that the principal mechanism for manifestation of dominant GJB2 mutations in the skin is their dominant interference with the function of wtCx43. This assumption is further corroborated by our finding that Cx26 and Cx43 focally colocalize at gap junctional plaques in affected skin tissue of two carriers of DeltaE42.

A compound heterozygous one amino-acid insertion/nonsense mutation in the plectin gene causes epidermolysis bullosa simplex with plectin deficiency.

Plectin is a cytoskeleton linker protein expressed in a variety of tissues including skin, muscle, and nerves. Mutations in its gene are associated with epidermolysis bullosa simplex with late-onset muscular dystrophy. Whereas in most of these patients the pathogenic events are mediated by nonsense-mediated mRNA decay, the consequences of an in-frame mutation are less clear. We analyzed a patient with compound heterozygosity for a 3-bp insertion at position 1287 leading to the insertion of leucine as well as the missense mutation Q1518X leading to a stop codon. The presence of plectin mRNA was demonstrated by a RNase protection assay. However, a marked reduction of plectin protein was found using immunofluorescence microscopy of the patient's skin and Western blot analysis of the patient's cultured keratinocytes. The loss of plectin protein was associated with morphological alterations in plectin-containing structures of the dermo-epidermal junction, in skeletal muscle, and in nerves as detected by electron microscopy. In an in vitro overlay assay using recombinant plectin peptides spanning exons 2 to 15 the insertion of leucine resulted in markedly increased self-aggregation of plectin peptides. These results describe for the first time the functional consequences of an in-frame insertion mutation in humans.

Mutation reports: epidermolysis bullosa simplex associated with severe mucous membrane involvement and novel mutations in the plectin gene.

We report a novel case of epidermolysis bullosa simplex with severe mucous membrane involvement and mutations in the plectin gene (PLEC1). The patient suffered from extensive blistering of the skin and oral and laryngeal mucous membranes. Electron microscopy of a lesional skin biopsy showed cleft formation within the basal cell layer of the epidermis. Antigen mapping displayed entirely negative staining for plectin, a large (>500 kDa) multifunctional adhesion protein present in hemidesmosomes of the basal keratinocytes. Mutation analysis revealed compound heterozygous, previously undisclosed nonsense mutations, Q1713X and R2351X, of paternal and maternal origin, respectively, within exon 32 of PLEC1. Based on earlier reports, plectin deficiency is associated with late onset muscular dystrophy in patients with epidermolysis bullosa. No signs of muscle weakness have been observed during the 4 y follow-up of our patient. This case illustrates the fact that molecular pathological analyses have prognostic implications in identification and evaluation of patients who appear to be at risk for development of muscular dystrophy later in life.

Epidermolysis bullosa: novel and de novo premature termination codon and deletion mutations in the plectin gene predict late-onset muscular dystrophy.

Epidermolysis bullosa (EB) with late-onset muscular dystrophy (EB-MD) is a hemidesmosomal variant of EB due to mutations in the plectin gene (PLEC1). The age of onset of muscle involvement has been noted to vary from infancy to the fourth decade of life. Immunofluorescence of the patients' skin and muscle biopsies is usually negative for staining with antibodies recognizing plectin, a large cytoskeleton-associated anchorage protein. In this study we report novel plectin mutations in two families with EB. In both families, the proband was a newborn with neonatal blistering with no evidence for muscle weakness as yet. Peripheral blood DNA was isolated and examined by heteroduplex scanning strategy, protein truncation test (PTT), and/or direct sequencing of the plectin gene. One of the probands was compound heterozygote for nonsense mutations E2005X/K4460X, and the proband in the second family was compound heterozygote for deletion mutations 5083delG/2745-9del21, the latter mutation extending from -9 to +12 at the intron 22/exon 23 border. The mutations K4460X and 5083delG were not present in either one of the parents, thus being de novo events. In both cases, nonpaternity was excluded by microsatellite marker analysis. The stop codon mutations are predicted to result in the synthesis of a truncated protein lacking the carboxy-terminal globular domain of the protein and possibly causing nonsense-mediated decay of the corresponding mRNA. The 2745-9del21 deletion mutation abolishes the splice site at the intron 22/exon 23 junction, predicting abnormal splicing events. Because plectin deficiency is associated with muscular dystrophy, molecular diagnostics of the plectin gene provides prognostic value in evaluation of these patients who appear to be at risk to develop muscular dystrophy.

Dominant dystrophic epidermolysis bullosa (Pasini) caused by a novel glycine substitution mutation in the type VII collagen gene (COL7A1).

A 12 y old girl with the albopapuloid variant (Pasini) of dominant dystrophic epidermolysis bullosa is studied. The albopapuloid lesions developed within the first year of life, contained milia and were associated with pruritus. Mutation detection of the COL7A1 gene revealed a G-->A transition at nucleotide position 6110 in the mutant allele converting a glycine to glutamic acid (G2037E). This report adds to the expanding database on COL7A1 mutations in dystrophic epidermolysis bullosa.

Four novel plectin gene mutations in Japanese patients with epidermolysis bullosa with muscular dystrophy disclosed by heteroduplex scanning and protein truncation tests.

Epidermolysis bullosa with muscular dystrophy (EB-MD) is a distinct variant of EB caused by mutations in the plectin gene (PLEC1). In this study, we have examined two Japanese patients with EB-MD using heteroduplex scanning or a protein truncation test for mutation detection analysis. The results revealed that both patients were compound heterozygotes for novel PLEC1 mutations (Q1936X/Q1053X and R2421X/12633ins4), which all caused premature termination of translation of the corresponding polypeptides. These cases, which demonstrate the utility of two complementary mutation detection strategies, add to the repertoire of plectin mutations in EB-MD.

Novel and de novo glycine substitution mutations in the type VII collagen gene (COL7A1) in dystrophic epidermolysis bullosa: implications for genetic counseling.

The dystrophic forms of epidermolysis bullosa (DEB) are due to mutations in the type VII collagen gene (COL7A1). In dominant DEB, a characteristic genetic lesion is a glycine substitution mutation within the collagenous domain of the protein. In this study, we have examined the molecular basis of six new families in which the proband has clinical features and/or ultrastructural findings consistent with DEB. The results revealed a glycine substitution mutation in all six families, four of which are novel and previously unpublished. In three families with clinically unaffected parents, de novo mutations G2043R and G2040V were found. These results emphasize the predominance of glycine substitution mutations in dominant DEB, and indicate that in some cases the phenotype is due to de novo dominant mutations.

Novel ITGB4 mutations in lethal and nonlethal variants of epidermolysis bullosa with pyloric atresia: missense versus nonsense.

Epidermolysis bullosa with pyloric atresia (EB-PA), an autosomal recessive genodermatosis, manifests with neonatal cutaneous blistering associated with congenital pyloric atresia. The disease is frequently lethal, but nonlethal cases have also been reported. Expression of the alpha6 beta4 integrin is altered at the dermal-epidermal basement-membrane zone; recently, mutations in the corresponding genes (ITGA6 and ITGB4) have been disclosed in a limited number of patients, premature termination codons in both alleles being characteristic of lethal variants. In this study, we have examined the molecular basis of EB-PA in five families, two of them with lethal and three of them with nonlethal variants of the disease. Mutation analysis disclosed novel lesions in both ITGB4 alleles of each proband. One of the patients with lethal EB-PA was a compound heterozygote for premature termination-codon mutations (C738X/4791delCA), whereas the other patient with a lethal variant was homozygous for a missense mutation involving a cysteine residue (C61Y). The three nonlethal cases had missense mutations in both alleles (C562R/C562R, R1281W/R252C, and R1281W/R1281W). Immunofluorescence staining of skin in two of the nonlethal patients and in one of the lethal cases was positive, yet attenuated, for alpha6 and beta4 integrins. These results confirm that ITGB4 mutations underlie EB-PA and show that missense mutations may lead to nonlethal phenotypes.