Identical glycine substitution mutations in type VII collagen may underlie both dominant and recessive forms of dystrophic epidermolysis bullosa.

Autosomal dominant and recessive forms of dystrophic epidermolysis bullosa (DEB) result from mutations in the type VII collagen gene (COL7A1). Although paradigms have emerged for genotype/phenotype correlation in DEB, some pathogenic mutations in COL7A1, notably glycine substitutions within the type VII collagen triple helix, may lead to diagnostic difficulties, since certain glycine substitutions can result in either dominant or recessive mutant alleles. Delineation of glycine substitution mutations into two discrete groups, however, is made difficult by observations that, for some particular glycine substitutions in type VII collagen, the same mutation can result in both dominant and recessive disease. In this report we describe four further glycine missense mutations: p.Gly1483Asp, p.Gly1770Ser, p.Gly2213Arg and p.Gly2369Ser, which can lead to either dominant or recessive DEB, and which result in a spectrum of clinical abnormalities. We also identify a further 30 new glycine substitution mutations that cause either dominant or recessive DEB, but not both. In screening the COL7A1 gene for mutations in individuals with DEB our data highlight that delineation of glycine substitutions in type VII collagen has important implications for genetic counselling.

The inversa type of recessive dystrophic epidermolysis bullosa is caused by specific arginine and glycine substitutions in type VII collagen.

BACKGROUND: The inversa type of recessive dystrophic epidermolysis bullosa (RDEB-I) is a rare variant of dystrophic epidermolysis bullosa, characterised by blistering in the body flexures, trunk, and mucosa. The cause of this specific distribution is unknown. So far, 20 COL7A1 genotypes have been described in RDEB-I and genotype-phenotype correlations have not been studied extensively. The aim of the study was to gain more insight into the pathophysiology of this intriguing RDEB-I phenotype. METHODS: Twenty Dutch and British RDEB-I patients, and full genotypes in 18 of them, were identified. The literature on RDEB-I genotypes was reviewed and an extensive genotype-phenotype correlation study for RDEB-I was conducted. RESULTS: All 20 patients had generalised blistering at birth and during early infancy. In most patients, the age of transition from generalised to inversa distribution was before the age of 4 years. A spectrum of disease severity, ranging from the mildest 'mucosal only' phenotype to the severest phenotype with limited acral involvement, was noted. The 29 genotypes of these RDEB-I patients and those reported in the literature revealed that RDEB-I is associated with specific recessive arginine and glycine substitutions in the triple helix domain of type VII collagen. DISCUSSION AND CONCLUSION: Why these specific arginine and glycine substitutions cause the inversa distribution remains unknown. It was not possible to identify clear differences in location and nature of substituting amino acids between these mutations and missense mutations causing other RDEB phenotypes. It is hypothesised that the higher skin temperature in the affected areas plays an important role in the pathophysiology of RDEB-I.

Congenital muscular dystrophy, myasthenic symptoms and epidermolysis bullosa simplex (EBS) associated with mutations in the PLEC1 gene encoding plectin.

Mutations in the PLEC1 gene encoding plectin have been reported in neonatal epidermolysis bullosa simplex with muscular dystrophy of later-onset (EBS-MD). A neuromuscular transmission defect has been reported in one previous patient. We report a boy presenting from birth with features of a congenital muscular dystrophy and late-onset myasthenic symptoms. Repetitive nerve stimulation showed significant decrement, and strength improved with pyridostigmine. Subtle blistering noticed only retrospectively prompted further genetic testing, revealing recessive PLEC1 mutations. We conclude that PLEC1 should be considered in the differential diagnosis of congenital muscular dystrophies and myasthenic syndromes, even in the absence of prominent skin involvement.

Transmission electron microscopy for the diagnosis of epidermolysis bullosa.

Transmission electron microscopy (TEM) has long been the best available method for the diagnosis of epidermolysis bullosa. Today, TEM is largely superseded by immunofluorescence microscopy mapping, which is generally more available. This article discusses its continuing role in confirming or refining results obtained by other methods, or in establishing the diagnosis where other techniques have been unsuitable or have failed. It covers key steps for optimizing tissue preparation, features of analysis, recently classified epidermolysis bullosa disorders, and strengths and weaknesses of TEM.

Homozygous mutations in the 5' region of the JUP gene result in cutaneous disease but normal heart development in children.

Desmosomes are intercellular adhesive junctions and attachment sites for the intermediate filament (IF) cytoskeleton, prominent in tissues subject to high levels of mechanical stress such as the epidermis and heart. The obligate desmosomal constituent, plakoglobin (PG), is involved in coupling transmembrane desmosomal components with IFs. PG also contributes to intercellular adhesion through adherens junctions and has additional signaling roles. To date, two mutations in the gene encoding PG, JUP, have been described, and in both instances, patients harboring pathogenic mutations suffered from arrhythmogenic right ventricular cardiomyopathy with or without skin abnormalities. We describe homozygous nonsense mutation, p.S24X, and homozygous splice site mutation, c.468G>A, in the JUP gene that results in skin fragility, diffuse palmoplantar keratoderma, and woolly hair with no symptoms of cardiomyopathy. We show barely detectable levels of PG immunostaining in skin sections from patients harboring these mutations and show that an alternative AUG codon in p.S24X mRNA translates a 42-amino-acid N-terminal truncation. We conclude that PG is required for correct maintenance of skin integrity, and the absence of heart phenotype in patients suggests that aberrant PG expression does not compromise normal human heart development in children. Our findings provide new insight into the distinct roles that PG has in the epidermis and heart.

Potential of fibroblast cell therapy for recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin-blistering disorder caused by mutations in the COL7A1 gene that lead to reduced type-VII collagen and defective anchoring fibrils at the dermal-epidermal junction (DEJ). Presently there are no effective treatments for this disorder. Recent mouse studies have shown that intradermal injections of normal human fibroblasts can generate new human type-VII collagen and anchoring fibrils at the DEJ. To assess potential clinical benefits in humans, we gave single intradermal injections of allogeneic fibroblasts to five subjects with RDEB. We noted increased type-VII collagen at the DEJ at 2 weeks and at 3 months following injection and increased anchoring fibrils, although none of these had normal morphology. No adverse effects, clinical or immunopathologic, were noted. We believe the major effect of allogeneic fibroblasts is to increase the recipients' own COL7A1 mRNA levels with greater deposition of mutant type-VII collagen at the DEJ and formation of additional rudimentary anchoring fibrils. Nevertheless, this mutant protein may be partially functional and capable of increasing adhesion at the DEJ. This is the first study demonstrating that intradermal injections of allogeneic fibroblasts have therapeutic potential in human subjects with RDEB.

Complete maternal isodisomy of chromosome 3 in a child with recessive dystrophic epidermolysis bullosa but no other phenotypic abnormalities.

The mechanobullous disease Hallopeau-Siemens recessive dystrophic epidermolysis bullosa (HS-RDEB) results from mutations in the type VII collagen gene (COL7A1) on chromosome 3p21.31. Typically, there are frameshift, splice site, or nonsense mutations on both alleles. In this report, we describe a patient with HS-RDEB, who was homozygous for a new frameshift mutation, 345insG, in exon 3 of COL7A1. However, sequencing of parental DNA showed that although the patient's mother was a heterozygous carrier of this mutation, the father's DNA contained only wild-type sequence. Microsatellite marker analysis confirmed paternity and genotyping of 28 microsatellites spanning chromosome 3 revealed that the affected child was homozygous for every marker tested with all alleles originating from a single maternal chromosome 3. Thus, the HS-RDEB phenotype in this patient is due to complete maternal isodisomy of chromosome 3 and reduction to homozygosity of the mutant COL7A1 gene locus. To our knowledge, there are no published reports of uniparental disomy (UPD) in HS-RDEB; moreover, this case represents only the third example of UPD of chromosome 3 to be reported. The severity of the HS-RDEB in this case was similar to other affected individuals and no additional phenotypic abnormalities were observed, suggesting an absence of maternally imprinted genes on chromosome 3.

LEKTI is localized in lamellar granules, separated from KLK5 and KLK7, and is secreted in the extracellular spaces of the superficial stratum granulosum.

Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a putative serine protease inhibitor encoded by serine protease inhibitor Kazal-type 5 (SPINK5). It is strongly expressed in differentiated keratinocytes in normal skin but expression is markedly reduced or absent in Netherton syndrome (NS), a severe ichthyosis caused by SPINK5 mutations. At present, however, both the precise intracellular localization and biological roles of LEKTI are not known. To understand the functional role of LEKTI, we examined the localization of LEKTI together with kallikrein (KLK)7 and KLK5, possible targets of LEKTI, in the human epidermis, by confocal laser scanning microscopy and immunoelectron microscopy. In normal skin, LEKTI, KLK7, and KLK5 were all found in the lamellar granule (LG) system, but were separately localized. LEKTI was expressed earlier than KLK7 and KLK5. In NS skin, LEKTI was absent and an abnormal split in the superficial stratum granulosum was seen in three of four cases. Collectively, these results suggest that in normal skin the LG system transports and secretes LEKTI earlier than KLK7 and KLK5 preventing premature loss of stratum corneum integrity/cohesion. Our data provide new insights into the biological functions of LG and the pathogenesis of NS.

Desmosomes exhibit site-specific features in human palm skin.

Hereditary skin disorders resulting from desmosome gene pathology may preferentially involve the palms and soles. Why this is so is not clear. Moreover, even in normal control skin it is unknown whether there are differences in desmosome number, size or structural organization in palmoplantar sites compared with skin from other body regions. Therefore, we sought evidence for such differences by examining desmosome expression in relation to epidermal differentiation in both epidermis and cultured keratinocytes from normal human palm and breast skin samples. Confocal microscopy of skin biopsy material showed relative differences in the expression profiles of several desmosomal proteins (desmogleins, desmocollins, desmoplakin, plakoglobin and plakophilin 1) between the two sites. Western blotting revealed a higher expression level of all five proteins in palm compared with breastcultured keratinocytes. Staining for the differentiation-associated component, involucrin, suggested an earlier onset of synthesis of this protein in palm epidermis, and a suspension-induced differentiation assay showed that involucrin synthesis began earlier in palm keratinocytes than in breast cells. At 4-8 h, the number of involucrin-positive cells in palm keratinocytes was almost twice that in breast. Morphometric analysis showed that, overall, desmosomes were larger but of similar population density in the palm compared with breast skin. These findings demonstrate differences in desmosome structure and protein expression between the two sites, possibly reflecting the needs of palms and soles to withstand constant mechanical stress. They may also help to explain the preferential involvement of this region in certain hereditary disorders (palmoplantar keratodermas), associated with mutations in desmoplakin or desmoglein 1.

Lack of plakophilin 1 increases keratinocyte migration and reduces desmosome stability.

Ablation of the desmosomal plaque component plakophilin 1 underlies the autosomal recessive genodermatosis, skin fragility-ectodermal dysplasia syndrome (OMIM 604536). Skin from affected patients is thickened with increased scale, and there is loss of adhesion between adjacent keratinocytes, which exhibit few small, poorly formed desmosomes. To investigate further the influence of plakophilin 1 on keratinocyte adhesion and desmosome morphology, we compared plakophilin 1-deficient keratinocytes (vector controls) with those expressing recombinant plakophilin 1 introduced by retroviral transduction. We found that plakophilin 1 increases desmosomal protein content within the cell rather than enhancing transcriptional levels of desmosomal genes. Re-expression of plakophilin 1 in null cells retards cell migration but does not alter keratinocyte cell growth. Confluent sheets of plakophilin 1-deficient keratinocytes display fewer calcium-independent desmosomes than do plakophilin 1-deficient keratinocytes expressing recombinant plakophilin 1 or keratinocytes expressing endogenous plakophilin 1. In addition electron microscopy studies show that re-expression of plakophilin 1 affects desmosome size and number. Collectively, these results demonstrate that restoration of plakophilin 1 function in our culture system influences the transition of desmosomes from a calcium-dependent to a calcium-independent state and this correlates with altered keratinocyte migration in response to wounding. Thus, plakophilin 1 has a key role in increasing desmosomal protein content, in desmosome assembly, and in regulating cell migration.

Lipoid proteinosis maps to 1q21 and is caused by mutations in the extracellular matrix protein 1 gene (ECM1).

Lipoid proteinosis (LP), also known as hyalinosis cutis et mucosae or Urbach-Wiethe disease (OMIM 247100) is a rare, autosomal recessive disorder typified by generalized thickening of skin, mucosae and certain viscera. Classical features include beaded eyelid papules and laryngeal infiltration leading to hoarseness. Histologically, there is widespread deposition of hyaline (glycoprotein) material and disruption/reduplication of basement membrane. The aetiology of LP is currently unknown. Using DNA from three affected siblings in a consanguineous Saudi Arabian family we performed genome-wide linkage and mapped the disorder to 1q21 (marker D1S498) with a two-point LOD score of 3.45 at theta = 0. A further 28 affected individuals from five other unrelated consanguineous family groups from different geographical regions also showed complete linkage and resulted in a maximum two-point LOD score of 21.85 at theta = 0. Using available markers in the interval between D1S442 and D1S305, the observed recombinants placed the gene in a 2.3 cM critical interval between D1S2344 and D1S2343 (Marshfield genetic map) corresponding to an approximately 6.5 Mb region on the UCSC physical map. Using a candidate gene approach (comparison of control versus LP gene expression in cultured fibroblasts) and subsequent direct sequencing of genomic DNA, we identified six different homozygous loss-of-function mutations in the extracellular matrix protein 1 gene (ECM1). Although the precise function of ECM1 is not known, our findings provide the first clinical indication of its relevance to skin adhesion, epidermal differentiation, wound healing, scarring, angiogenesis/angiopathy and basement membrane physiology, as well as defining the molecular basis of this inherited disorder.