Susan M. Morley

A Homozygous Missense Mutation in TGM5 Abolishes Epidermal Transglutaminase 5 Activity and Causes Acral Peeling Skin Syndrome

Peeling skin syndrome is an autosomal recessive genodermatosis characterized by the shedding of the outer epidermis. In the acral form, the dorsa of the hands and feet are predominantly affected. Ultrastructural analysis has revealed tissue separation at the junction between the granular cells and the stratum corneum in the outer epidermis. Genomewide linkage analysis in a consanguineous Dutch kindred mapped the gene to 15q15.2 in the interval between markers D15S1040 and D15S1016. Two homozygous missense mutations, T109M and G113C, were found in TGM5, which encodes transglutaminase 5 (TG5), in all affected persons in two unrelated families. The mutation was present on the same haplotype in both kindreds, indicating a probable ancestral mutation. TG5 is strongly expressed in the epidermal granular cells, where it cross-links a variety of structural proteins in the terminal differentiation of the epidermis to form the cornified cell envelope. An established, in vitro, biochemical cross-linking assay revealed that, although T109M is not pathogenic, G113C completely abolishes TG5 activity. Three-dimensional modeling of TG5 showed that G113C lies close to the catalytic domain, and, furthermore, that this glycine residue is conserved in all known transglutaminases, which is consistent with pathogenicity. Other families with more-widespread peeling skin phenotypes lacked TGM5 mutations. This study identifies the first causative gene in this heterogeneous group of skin disorders and demonstrates that the protein cross-linking function performed by TG5 is vital for maintaining cell-cell adhesion between the outermost layers of the epidermis.

Keratin mutations of epidermolysis bullosa simplex alter the kinetics of stress response to osmotic shock

The intermediate filament cytoskeleton is thought to confer physical resilience on tissue cells, on the basis of extrapolations from the phenotype of cell fragility that results from mutations in skin keratins. There is a need for functional cell assays in which the impact of stress on intermediate filaments can be induced and analyzed. Using osmotic shock, we have induced cytoskeleton changes that suggest protective functions for actin and intermediate filament systems. Induction of the resulting stress response has been monitored in keratinocyte cells lines carrying K5 or K14 mutations, which are associated with varying severity of epidermolysis bullosa simplex. Cells with severe mutations were more sensitive to osmotic stress and took longer to recover from it. Their stress-activated response pathways were induced faster, as seen by early activation of JNK, ATF-2 and c-Jun. We demonstrate that the speed of a cells response to hypotonic stress, by activation of the SAPK/JNK pathway, is correlated with the clinical severity of the mutation carried. The response to hypo-osmotic shock constitutes a discriminating stress assay to distinguish between the effects of different keratin mutations and is a potentially valuable tool in developing therapeutic strategies for keratin-based skin fragility disorders.

Expression of individual lamins in basal cell carcinomas of the skin

In this study we used a unique collection of type specific anti-lamin antibodies to study lamin expression patterns in normal human skin and in skin derived from patients with basal cell carcinomas (BCCs). Lamin expression in serial sections from frozen tissue samples was investigated by single and double indirect immunofluorescence. In normal skin, lamin A was expressed in dermal fibroblasts and in suprabasal epithelial cells but was absent from all basal epithelial cells. Lamin C was expressed in dermal fibroblasts, suprabasal epithelial cells and a majority of basal epithelial cells. However, lamin C was not expressed in quiescent basal epithelial cells. Lamin B1was expressed in all epithelial cells but was not expressed in dermal fibroblasts. Finally, lamin B2was expressed in all epithelial cells but was not expressed in dermal fibroblasts. Finally, lamin B2was expressed in all cell types in normal skin. Lamin expression was also investigated in a collection of 16 BCCs taken from a variety of body sites. Based upon patterns of lamin expression the BCCs were classified into four groups: A-negative (10/16 tumours), C-negative (5/16 tumours), A/C-negative (1/16 tumours) and A/B2-negative (1/16 tumours). Lamin expression was also compared to cell proliferation index by staining serial sections with the proliferation marker Ki67. 9/10 of the lamin A negative tumours were highly proliferative, whereas 4/5 of the lamin C negative tumours were slow growing. Thus as a general rule absence of lamin A was correlated with rapid growth within the tumour, while absence of lamin C was correlated with slow growth within the tumour. Our data supports the hypothesis that lamin A has a negative influence on cell proliferation and its down regulation may be a requisite of tumour progression.

Generation and characterization of epidermolysis bullosa simplex cell lines: scratch assays show faster migration with disruptive keratin mutations.

Summary Background Epidermolysis bullosa simplex (EBS) is an inherited skin fragility disorder caused by mutations in keratin intermediate filament proteins. While discoveries of these mutations have increased understanding of the role of keratins and other intermediate filaments in epithelial tissues, progress towards the development of therapy for these disorders is much slower.

K15 Expression Implies Lateral Differentiation within Stratified Epithelial Basal Cells

Keratins are intermediate filament proteins whose expression in epithelial tissues is closely linked to their differentiated state. The greatest complexity of this expression is seen in the epidermis and associated structures. The critical basal (proliferative) cell layer expresses the major keratin pair, K5 and K14, but it also expresses an additional type I keratin, K15, about which far less is known. We have compared the expression of K15 with K14 in normal, pathological, and tissue culture contexts; distinct differences in their expression patterns have been observed that imply different regulation and function for these two genes. K15 appears to be preferentially expressed in stable or slowly turning over basal cells. In steady-state epidermis, K15 is present in higher amounts in basal cells of thin skin but in lower amounts in the rapidly turning over thick plantar skin. Although remaining high in basal cell carcinomas (noninvasive) it is suppressed in squamous cell carcinomas (which frequently metastasize). Wounding-stimulated epidermis loses K15 expression, whereas K14 is unchanged. In cultured keratinocytes, K15 levels are suppressed until the culture stratifies, whereas K14 is constitutively expressed throughout. Therefore, unlike K14, which appears to be a fundamental component of all keratinocytes, K15 expression appears to be more tightly coupled to a mature basal keratinocyte phenotype.

Phase IIa randomized, placebo‐controlled study of antimicrobial photodynamic therapy in bacterially colonized, chronic leg ulcers and diabetic foot ulcers: a new approach to antimicrobial therapy

Background  With increasing problems of antibiotic resistance, photodynamic therapy (PDT) is being developed as a novel antimicrobial treatment. Following light activation, cationic photosensitizer PPA904 [3,7‐bis(N,N‐dibutylamino) phenothiazin‐5‐ium bromide] kills a broad spectrum of bacteria in vitro and this has a variety of potential clinical applications.

Functional improvement of mutant keratin cells on addition of desmin: an alternative approach to gene therapy for dominant diseases.

A major challenge to the concept of gene therapy for dominant disorders is the silencing or repairing of the mutant allele. Supplementation therapy is an alternative approach that aims to bypass the defective gene by inducing the expression of another gene, with similar function but not susceptible to the disrupting effect of the mutant one. Epidermolysis bullosa simplex (EBS) is a genetic skin fragility disorder caused by mutations in the genes for keratins K5 or K14, the intermediate filaments present in the basal cells of the epidermis. Keratin diseases are nearly all dominant in their inheritance. In cultured keratinocytes, mutant keratin renders cells more sensitive to a variety of stress stimuli such as osmotic shock, heat shock or scratch wounding. Using a ‘severe’ disease cell culture model system, we demonstrate reversion towards wild-type responses to stress after transfection with human desmin, an intermediate filament protein normally expressed in muscle cells. Such a supplementation therapy approach could be widely applicable to patients with related individual mutations and would avoid some of the financial obstacles to gene therapy for rare diseases.

Novel and recurrent mutations in keratin 10 causing bullous congenital ichthyosiform erythroderma

Abstract: Bullous congenital ichthyosiform erythroderma (BCIE) is a dominantly inherited keratinizing disorder characterized by erythroderma and blistering in neonates and generalized epidermolytic hyperkeratosis (EH) in adulthood. Previously, it has been shown that BCIE can be caused by mutations in either of the genes encoding K1 or K10, the keratins predominantly expressed in suprabasal layers of the epidermis. Using direct sequencing of genomic PCR fragments, we have analyzed 4 British families with BCIE, all of whom were found to carry mutations in K10. In 1 family, the affected person was found to have an unusual dinucleotide transversion mutation, 2138CCÁ, causing two amino acid substitutions, D155E and R156S, also in the 1A domain of the K10 polypeptide. In 2 further kindreds, the previously reported hotspot mutations 2139C‐T and 2140G‐A were found. These mutations predict amino acid substitutions in the helix 1A domain of K10, designated R156C and R156H respectively. The proband in the fourth family was found to carry a novel mutation 4724T‐C, predicting the amino acid change L452P in the helix 2B domain of K10. All mutations were confirmed in the affected persons and were excluded from a population of 50 normal, unrelated individuals by restriction enzyme analysis. The location of these mutations in the highly conserved helix boundary motif sequences of K10 are consistent with previously reported dominant negative mutations in K10 and other keratins. Despite the unusual nature of two of these mutations, in particular the double missense mutation, the phenotypes of the affected individuals in these 4 families were entirely typical of BCIE.