Takuji Masunaga

Epidermal Basement Membrane: Its Molecular Organization and Blistering Disorders

The epidermal basement membrane is a specialized structure localized between the epidermis and the dermis. Recent studies have elucidated the biological roles of the basement membrane and its pathophysiological involvement in bullous diseases. To understand the functions of the basement membrane, it is essential to have clear and precise information regarding the ultrastructural molecular organization of the epidermal basement membrane. Immunoelectron microscopy is a powerful technique and the only method available for the clarification of the ultrastructural localization or orientation of molecules. This review summarizes the latest information regarding the molecular organization of the epidermal basement membrane as determined by immunoelectron microscopy as well as the blistering diseases that occur in the epidermal basement membrane zone.

Glycine substitution mutations by different amino acids in the same codon of COL7A1 lead to heterogeneous clinical phenotypes of dominant dystrophic epidermolysis bullosa

Abstract Dystrophic epidermolysis bullosa (DEB), caused by mutations in the gene encoding type VII collagen (COL7A1), is known to show heterogeneous clinical phenotypes. Certain correlations between the nature or position of COL7A1 mutations and the resultant DEB phenotypes have been suggested, although such relationships may be more complex than initially thought. The purpose of the present study was to clarify the molecular basis of two different subtypes of dominant DEB (DDEB), EB pruriginosa and classical type. Interestingly, we found that both cases were caused by a missense glycine substitution mutation by different amino acids in the same codon of COL7A1 (G2028R and G2028A). These results further support the notion that different glycine substitution mutations in the same codon can lead to heterogeneous clinical phenotypes of DDEB, EB pruriginosa and classical type.

Bidimensional analysis of desmoglein 1 distribution on the outermost corneocytes provides the structural and functional information of the stratum corneum

BACKGROUND The stratum corneum (SC) plays an important role in cutaneous barrier function. Recent clarification of the pathophysiology of several keratoses has suggested that adhesive molecules contribute not only to SC construction but also to SC barrier function. OBJECTIVE The purpose of this study is to clarify how the distribution of adhesion molecules on corneocytes contributes to the construction of the SC and the overall organization and function of the cutaneous barrier. METHODS To investigate the distribution of desmoglein 1 (Dsg1), which may be a main component of corneodesmosomes (CDSs) in the SC, we used a bidimensional observation method using tape-stripped corneocytes and several immunohistochemical techniques to demonstrate the distribution of Dsg1 and to deduce internal events in the SC. RESULTS Immunofluorescence labeling showed that Dsg1 distributed on corneocytes of the outermost SC with a characteristic pattern at the periphery, or over the entire surface, and differences in this distribution pattern correlated with the transepidermal water loss (TEWL). Furthermore, electron microscopic analysis showed that (1) Dsg1 was localized on CDSs involved in adhesion, and (2) CDSs on the periphery of corneocytes contributed to the generation of the characteristic basket-weave structure. CONCLUSION We explored the distribution pattern of Dsg1 in the SC via a non-invasive investigation tool. Our findings indicate the significance of adhesion molecules in the formation and function of the SC, and suggest that adhesion molecules are one of the important elements in barrier formation in addition to corneocytes, which act as bricks, and intercellular lipids, which act as mortar.

Differences in recurrent COL7A1 mutations in dystrophic epidermolysis bullosa: ethnic-specific and worldwide recurrent mutations

Dystrophic epidermolysis bullosa (DEB) is caused by mutations in the gene encoding type VII collagen (COL7A1). Although most COL7A1 mutations are unique to individual families, small numbers of mutations are recurrent. The recurrent mutations R578X, 7786delG, and R2814X seem to be exclusive to a specific ethnic group, the British population. The mutations 5818delC, 6573+1G→C, and E2857X are present only in individuals of Japanese ethnic origin. On the other hand, the mutations 425A→G and G2043R have been found in several different ethnic groups. The purpose of this study was to clarify whether these recurrent mutations are also found in patients of other ethnic groups with DEB, mainly Asian patients. We demonstrated the absence of the recurrent mutations R578X, 7786delG, and R2814X in 42 non-British patients with DEB and detected the mutations 425A→G in a French patient and G2043R in Japanese and Chinese patients with DEB. The mutations 5818delC, 6573+1G→C, and E2857X were detected in 11 Japanese patients (13 alleles) with DEB. Our results confirm that R578X, 7786delG, and R2814X mutations are specifically limited to British patients, and the mutations 5818delC, 6573+1G→C, and E2857X are frequent in Japanese patients. On the other hand, the mutations 425A→G and G2043R can be found in different ethnic groups. In conclusion, our results further support the notion that recurrent mutations can be classified into two types, ethnic-specific mutation and worldwide mutation.

Demonstration of desmosomal antigens by electron microscopy using cryofixed and cryosubstituted skin with silver-enhanced gold probe.

In a previous post-embedding immunogold electron microscopic (EM) studies, localization of various desmosomal antigens was possible at high but not at low magnification. We developed a method for simultaneous demonstration of epidermal desmosomal antigens at both low- and high-power EM magnifications by a method based on cryofixation and acetone cryosubstitution and the use of a 1-nm gold probe with silver enhancement. Ultra-thin sections of Lowicryl K11M were incubated with primary antibodies against desmoplakin, desmocollin, or desmoglein, followed by 1-nm gold-conjugated secondary antibody. Silver enhancement for 12 min provided the ideal labeling size for low-power visualization, whereas silver enhancement for 4-6 min was ideal for high-power EM observation. Each desmosome immunolabeled with the gold probe was clearly demonstrated, even at very low-power magnification. The level of background labeling could be determined easily and the area of interest for high-power observation selected accurately. The fine ultrastructural appearance of desmosomal molecules was precisely demonstrated on high-power observation. This system should be useful for the immunocytochemical study of a variety of desmosomal antigens as well as other molecules of interest.

Dystrophic form of inherited epidermolysis bullosa in a dog (Akita Inu).

We report a dog with dystrophic epidermolysis bullosa. This 4‐year‐old female Akita Inu, a species of Canis familiaris var. japanicus Temminck, had a 3‐year‐history of ulcers and scars over the pressure areas on the limbs, and dystrophic nails, since the age of 1 year, which corresponds lo early adulthood in humans. Electron microscopy of a blister revealed separation beneath the lamina densa, and a reduction in the number of anchoring fibrils. The NC‐1 domain of type VII collagen was positively stained with monoclonal anlibody LH7.2 at the basement membrane zone. These findings indicate that humans and dogs have a similar response to antibody LH7.2, which may aid the development of an animal model for this disease.

New non‐invasive method for evaluation of the stratum corneum structure in diseases with abnormal keratinization by immunofluorescence microscopy of desmoglein 1 distribution in tape‐stripped samples

The corneodesmosomes in the stratum corneum are critical for the maintenance of stratum corneum integrity. To evaluate the normal and diseased keratinization states in the epidermis, we studied the distribution of desmoglein 1 (DSG1), a major component of corneodesmosomes, in samples of the stratum corneum obtained by tape stripping, a non‐invasive method. Samples were collected from lesional skin of four patients with psoriasis and three with lichen planus, and from non‐lesional skin of three volunteers. Upper stratum corneum cells were obtained by tape stripping and skin biopsies were obtained from adjacent sites. Tape‐stripped samples were examined by immunofluorescence microscopy using anti‐DSG1 monoclonal antibody, in combination with histopathology of skin biopsies. In normal human stratum corneum, which shows basket‐woven orthokeratosis, DSG1‐containing fluorescent dots were distributed on the lateral cell–cell contact areas of plasma membrane, but not on the dorsal/ventral plasma membrane, and formed a well‐ordered hexagonal network structure. In psoriatic stratum corneum, fluorescent dots were distributed throughout the cell membrane at ventral aspects of corneocytes as well as at the lateral cell–cell contacts. In lichen planus, fluorescent dots were distributed homogeneously and/or heterogeneously on the ventral surface in some cells. Adjacent cells lacked DSG1 at the lateral cell–cell contacts, but were instead separated by distinctive black‐gap lines. These results suggest that the intercellular adhesion by DSG1 may depend on the lateral plasma membrane in normal human stratum corneum, on the dorsal/ventral plasma membrane in lichen planus, and on both lateral and dorsal/ventral plasma membranes in psoriatic stratum corneum. Tape stripping and DSG1 immunofluorescence visualizes adhesion features of corneocytes and has considerable potential for evaluation of abnormal keratinization and the process of healing in response to treatment.

A novel de novo splice-site mutation in the COL7A1 gene in dominant dystrophic epidermolysis bullosa (DDEB): specific exon skipping could be a prognostic factor for DDEB pruriginosa

We report a Japanese infant who had a novel de novo splice‐site mutation in the COL7A1 gene, which resulted in in‐frame exon 87 skipping. Very interestingly, most of the previously reported cases with the same exon skipping presented as dystrophic epidermolysis bullosa (DEB) pruriginosa. The proband in this study showed an extremely mild clinical phenotype, with no nail dystrophy, pruritus or prurigo‐like lesions. However, dominant (DDEB) pruriginosa often shows a typical mild DEB phenotype until the onset of pruritus, making it likely that as she gets older the proband will present with features consistent with DDEB pruriginosa. By knowing in advance the anticipated clinical course, it might be possible to reduce or even prevent development of nodular prurigo‐like lesions by sufficient control of pruritus. Our study should contribute to further refinement of the genotype–phenotype correlations in DEB, emphasizing the significance of mutation analysis for correct diagnosis and possibly for prediction of prognosis.

DETECTION OF ANTIGENS BY IMMUNOFLUORESCENCE ON ULTRATHIN CRYOSECTIONS OF SKIN

Cryoultramicrotomy was originally established to provide ultrathin cryosections as substrates for on-section immunolabeling in immunoelectron microscopy. Recently, we recognized that ultrathin cryosections of skin (0.2 μm thick) could serve as substrates for immunofluorescence (IF) with excellent resolution. To assess the advantages and the limitations of IF on ultrathin cryosections, we compared the labeling of IF on 0.2-μm ultrathin cryosections of skin with those of routine IF on 6-μm cryostat sections, confocal laser scanning microscopy (LSM), and immunogold electron microscopy using several markers of keratinocyte cell surface and basement membrane zone molecules. IF on ultrathin cryosections clearly demonstrated a lack of bullous pemphigoid antigens beneath the melanocytes, desmosomal antigens as discontinuous dot-like labeling, and nondesmosomal plasma membrane antigen as a ladder-like pattern. IF on ultrathin cryosections provided convincing images with higher resolution than confocal LSM, which corresponded well to those of immunogold electron microscopy. IF on ultrathin cryosections had superior resolution compared to routine IF or confocal LSM and should serve as a powerful tool in future studies for the analysis of skin antigens.

Does the position of the premature termination codon in COL7A1 correlate with the clinical severity in recessive dystrophic epidermolysis bullosa

Abstract:  Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited skin disease caused by mutations in the gene encoding type VII collagen (COL7A1). The mutations are highly variable and this greatly complicates the study of the genotype–phenotype relationships. To date, three recurrent mutations, specific to Japanese RDEB patients have been reported. By comparing the phenotypes of RDEB patients with different recurrent mutations, the upstream positions of the premature termination codons (PTCs) showed strong correlation with the RDEB clinical disease severity. However, such correlations have not been supported by patients with mutations that were different from these recurrent Japanese patients mutations. In this study, we report a case of RDEB with a very mild clinical phenotype, who was a compound heterozygote harbouring both a recurrent Japanese mutation and a novel deletion mutation resulting in a more upstream PTC. The patient and his mother were shown to have a recurrent donor splice site mutation within intron 81 (6573 + 1G > C), a recurrent Japanese mutation that activates a cryptic donor splicing site and results in a downstream PTC. The patient and his father shared a single‐nucleotide deletion within exon 64 (5504delA), which causes a downstream frame shift in five amino acids before creating a PTC. Occurrence of the PTCs in mRNA was confirmed by reverse transcription‐polymerase chain reaction (RT)‐PCR. The patients skin showed reduced immunofluorescence staining for COL7A1 and reduced number of abnormal or short anchoring fibrils by electron microscopy. Although the position of the mutation 5504delA PTC was located upstream of the previous mutations reported in combination with the 6573 + 1G > C mutation, the two mutations together give an apparently milder clinical phenotype. Therefore, genotype–phenotype relationships in RDEB cannot be explained purely by the position of PTC.