Mild recessive dystrophic epidermolysis bullosa associated with two compound heterozygous COL7A1 mutations.

Dystrophic epidermolysis bullosa is a group of inherited skin blistering disorders caused by mutations in the COL7A1 gene coding for type VII collagen. More than 500 different COL7A1 mutations have been detected in dystrophic epidermolysis bullosa to date. Clarification of genotype-phenotype correlations is of particular importance for the development of novel therapeutic approaches. Here we report a female patient with mild dystrophic epidermolysis bullosa harbouring two compound heterozygous COL7A1 mutations, namely the intronic splice site mutation c.3832-2A > G and the glycine substitution p.G1347W. Our data extend the current knowledge on genotype-phenotype correlations in dystrophic epidermolysis bullosa.

Primary perforating granulomatous folliculitis--scarring deep type.

Perforating folliculitis is characterized by asymptomatic skin-coloured or erythematous scattered and aggregated follicular papules with a central keratotic plug. Histologically, a superficial type can be distinguished from the profound type where perforations and rupture of the follicular wall take place at different levels of the hair follicle. This goes along with a granulomatous reaction of the entire pilary complex with destruction of the follicle epithelium and sebaceous gland. Often cases are associated with systemic disorders such as renal diseases or diabetes mellitus. We describe two patients with the profunda type of perforating folliculitis with scarring that manifested in early adulthood without any underlying disorders.

The molecular basis of human keratin disorders.

Keratins are cytoskeletal proteins that provide structural support to epithelial cells and tissues. Perturbation causes cell and tissue fragility and accounts for a large number of genetic disorders in humans. In humans, 54 functional keratin genes exist and 21 different keratin genes including hair keratins and hair follicle-specific epithelial keratins have been associated with hereditary disorders. Moreover, keratins have been implicated in more complex traits such as liver disease and inflammatory bowel disease. Understanding the molecular basis of keratin disorders has been the basis for improved diagnosis with prognostic implications, genetic counseling and prenatal testing for severe disorders. Besides their mechanical role, keratins have newly identified functions in apoptosis, cell growth, tissue polarity, wound healing and tissue remodeling. Improved understanding of the regulatory functions of keratins may offer novel approaches to overcome current treatment limitations.

Keratins and their associated skin disorders.

Keratins are the largest group of intermediate filament proteins that are expressed in the cytoplasm of epithelial cells. They form a cytoskeletal scaffold that maintains cell and tissue integrity and provides vital mechanical support to epithelia. Mutations in 19 different keratin genes have so far been identified as the cause of at least 15 different genetic diseases. Identification of the molecular basis of the keratin disorders has contributed to definite diagnoses and has facilitated genetic counselling. Better understanding of the structure, function and regulatory mechanisms of keratins will be the basis for the development of novel therapeutic approaches to overcome the current treatment limitations.

Novel and recurrent mutations in keratin KRT5 and KRT14 genes in epidermolysis bullosa simplex: implications for disease phenotype and keratin filament assembly.

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominant genetic skin disorders caused by mutations of the keratin genes KRT5 and KRT14. It is characterised by lysis of basal keratinocytes leading to the development of intraepidermal blisters upon minor mechanical trauma. We investigated 27 EBS patients and families of mainly German origin by sequence analysis of the entire coding sequences of KRT5 and KRT14 and identified 12 novel and seven previously reported mutations within the KRT5 and KRT14 genes. The study discusses possible implications of the novel mutations on protein structure, keratin intermediate filament (KIF) formation and the corresponding phenotype, and summarises the spectrum of mutations reported so far in EBS. Detailed knowledge of the spectrum of EBS mutations and their genotype-phenotype correlation is essential for accurate genetic counselling and prenatal diagnosis.

Silicone granuloma of the face treated with minocycline.

Siliconoma represents a granulomatous foreign body reaction to silicone, which is often used for soft tissue augmentation. Although considered as biologically inert for a long time, silicone has been implicated in various undesirable local and systemic reactions, sometimes with a latency period of up to several decades. Treatment of siliconomas is difficult and granulomas involving the face are a therapeutic challenge. We present a 43-year-old woman with a severely disfiguring facial silicone granuloma who was successfully treated with minocycline.

Anti-B- cell-directed immunotherapy (rituximab) in the treatment of refractory pemphigus--an update.

Rituximab is a monoclonal antibody directed against the CD20 surface antigen present on B lymphocytes. Following its application, B cells are rapidly and specifically depleted. Rituximab has been approved for the treatment of relapsed and therapy-refractory non-Hodgkin lymphoma and has been incorporated into numerous chemotherapy regimes with promising results. Eradication of auto-reactive B cell clones is the rationale for its application in a variety of autoimmune disorders including the pemphigus group where B cells are thought to play a critical role in the pathogenesis. Preliminary reports in autoimmune disorders are encouraging. Adverse effects are generally well controlled and although severe infections have been reported following rituximab, the overall risk does not seem to be significantly increased. In the pemphigus group, rituximab has been successfully employed in refractory cases and a recent study suggests that a single course induces long-term remission in this patient group.

Epidermal expression of neuropilin 1 protects murine keratinocytes from UVB-induced apoptosis.

BACKGROUND: Neuropilin 1 (NRP1) is expressed on several cell types including neurons and endothelial cells, where it functions as an important regulator in development and during angiogenesis. As a cell surface receptor, NRP1 is able to bind to members of the VEGF family of growth factors and to secreted class 3 semaphorins. Neuropilin 1 is also highly expressed in keratinocytes, but the function of NRP1 in epidermal physiology and pathology is still unclear. METHODS AND RESULTS: To elucidate the role of NRP1 in skin in vivo we generated an epidermis-specific neuropilin 1 knock out mouse model by using the Cre-LoxP-System. Mice were viable and fertile and did not display any obvious skin or hair defects. After challenge with UVB irradiation, we found that deletion of epidermal NRP1 leads to increased rates of apoptosis both in vitro and in vivo. NRP1-deficient primary keratinocytes cultured in vitro showed significantly higher rates of apoptosis 24 hours after UVB. Likewise, there is a significant increase of active caspase 3 positive cells in the epidermis of Keratin 14-Cre-NRP1 (-/-) mice 24 hours after UVB irradiation. By Western Blot analysis we could show that NRP1 influences the cytosolic levels of Bcl-2, a pro-survival member of the Bcl-2 family. After UVB irradiation the amounts of Bcl-2 decrease in both protein extracts from murine epidermis and in NRP1-deficient keratinocytes in vitro, whereas wild type cells retain their Bcl-2 levels. Likewise, levels of phospho-Erk and Rac1 were lower in NRP1-knock out keratinocytes, whereas levels of pro-apoptotic p53 were higher. CONCLUSION: NRP1 expression in keratinocytes is dispensable for normal skin development. Upon UVB challenge, NRP1 contributes to the prevention of keratinocyte apoptosis. This pro-survival function of NRP1 is accompanied by the maintenance of high levels of the antiapoptotic regulator Bcl-2 and by lower levels of pro-apoptotic p53.

Laminin-5-deficient human keratinocytes: defective adhesion results in a saltatory and inefficient mode of migration.

Laminin-5 is a major adhesion protein of the skin basement membrane and crucially involved in integrin-mediated cell substrate attachment of keratinocytes, which is important for hemidesmosomal anchorage as well as for keratinocyte migration during epidermal wound healing. To investigate its role in keratinocyte migration, we analyzed laminin-5-deficient cells of patients with a lethal variant of junctional epidermolysis bullosa. Normal migrating keratinocytes adopted monopolar morphology with a distinct front lamella and employed a continuous mode of translocation. In contrast, laminin-5-deficient cells assumed a stretched bipolar shape with two lamella regions and migrated in a discontinuous, saltatory manner characterized by significantly decreased directional persistence and reduced migration velocity. The distinct morphology as well as the migratory phenotype apparently resulted from a defect in the formation of cell substrate adhesions that were completely missing in the cell body and less stable in the lamella regions. Accordingly in normal keratinocytes, a bipolar shape and a saltatory migration mode were inducible by blocking laminin-5-mediated substrate adhesion. Our findings clearly point to an essential role of laminin-5 in forming dynamic cell substrate adhesion during migration of epidermal keratinocytes and provide an explanation for the cellular mechanisms that underlie the lethal form of junctional epidermolysis bullosa.

A human keratin 10 knockout causes recessive epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis (EHK) is a blistering skin disease inherited as an autosomal-dominant trait. The disease is caused by genetic defects of the epidermal keratin K1 or K10, leading to an impaired tonofilament network of differentiating epidermal cells. Here, we describe for the first time a kindred with recessive inheritance of EHK. Sequence analysis revealed a homozygous nonsense mutation of the KRT10 gene in the affected family members, leading to a premature termination codon (p.Q434X), whereas the clinically unaffected consanguineous parents were both heterozygous carriers of the mutation. Semi-quantitative RT-PCR and western blot analysis demonstrated degradation of the KRT10 transcript, resulting in complete absence of keratin K10 protein in the epidermis and cultured keratinocytes of homozygous patients. This K10 null mutation leads to a severe phenotype, clinically resembling autosomal-dominant EHK, but differing in form and distribution of keratin aggregates on ultrastructural analysis. Strong induction of the wound-healing keratins K6, K16 and K17 was found in the suprabasal epidermis, which are not able to compensate for the lack of keratin 10. We demonstrate that a recessive mutation in KRT10 leading to a complete human K10 knockout can cause EHK. Identification of the heterogeneity of this disorder has a major impact for the accurate genetic counseling of patients and their families and also has implications for gene-therapy approaches.

Inducible mouse models for inherited skin diseases: implications for skin gene therapy.

Stem cells are crucial for the formation and maintenance of tissues and organs. To understand the role of stem cells in the pathogenesis of mosaic skin disorders, we generated inducible mouse models for two autosomal dominant keratin disorders, epidermolytic hyperkeratosis (EHK) and epidermolysis bullosa simplex (EBS), that enable activation of the respective mutation in epidermal stem cells in a spatially and temporally controlled manner using a ligand-inducible Cre recombinase. Whereas mosaic forms have been reported for EHK, which is caused by mutations in the suprabasal keratins K1 or K10, this has never been reported for EBS, which is due to mutations in the basal keratins K5 or K14. When we induced the phenotype in these models by topical application of the inducer, we found phenotypic areas in the EHK model that persisted for the life of the mouse. On the contrary, the induced blisters in the EBS model healed within a few weeks by migration of surrounding non-phenotypic stem cells into the wound bed. Our results indicate that lack of selective pressure against certain mutations in epidermal stem cells could explain why mosaic forms exist for EHK, but not for EBS. These findings have important implications for the development of new strategies for somatic gene therapy of dominant genodermatoses, and we are currently using these inducible mouse models to test gene therapy approaches.

Disruption of dihydronicotinamide riboside:quinone oxidoreductase 2 (NQO2) leads to myeloid hyperplasia of bone marrow and decreased sensitivity to menadione toxicity.

Dihydronicotinamide riboside (NRH):quinone oxidoreductase 2 (NQO2) is a flavoenzyme that catalyzes the reductive metabolism of quinones. To examine the in vivo role of NQO2, NQO2-null (NQO2-/-) mice were generated using targeted gene disruption. Mice lacking NQO2 gene expression showed no detectable developmental abnormalities and were indistinguishable from wild-type (NQO2+/+) mice. However, NQO2-null mice exhibited myeloid hyperplasia of the bone marrow and increased neutrophils, basophils, eosinophils, and platelets in the peripheral blood. Decreased apoptosis of bone marrow cells and circulating granulocytes contributed to myeloid hyperplasia and hyperactivity of bone marrow in NQO2-null mice. The hematological changes in NQO2-/- mice were specifically associated with loss of the NQO2 gene because histological analysis of various tissues including spleen, thymus, blood cultures, and urine analysis demonstrated no sign of infection. NQO2-null mice also demonstrated decreased toxicity when exposed to menadione or menadione with NRH. These results establish a role for NQO2 in protection against myelogenous hyperplasia and in metabolic activation of menadione, leading to hepatic toxicity. The NQO2-null mice are a model for NQO2 deficiency in humans and can be used to determine the role of this enzyme in sensitivities to toxicity and carcinogenesis.