Dimitra Kiritsi

Integrin α3 mutations with kidney, lung, and skin disease.

Integrin α(3) is a transmembrane integrin receptor subunit that mediates signals between the cells and their microenvironment. We identified three patients with homozygous mutations in the integrin α(3) gene that were associated with disrupted basement-membrane structures and compromised barrier functions in kidney, lung, and skin. The patients had a multiorgan disorder that included congenital nephrotic syndrome, interstitial lung disease, and epidermolysis bullosa. The renal and respiratory features predominated, and the lung involvement accounted for the lethal course of the disease. Although skin fragility was mild, it provided clues to the diagnosis.

Kindler syndrome: extension of FERMT1 mutational spectrum and natural history.

Mutations in the FERMT1 gene (also known as KIND1), encoding the focal adhesion protein kindlin‐1, underlie the Kindler syndrome (KS), an autosomal recessive skin disorder with an intriguing progressive phenotype comprising skin blistering, photosensitivity, progressive poikiloderma with extensive skin atrophy, and propensity to skin cancer. Herein we review the clinical and genetic data of 62 patients, and delineate the natural history of the disorder, for example, age at onset of symptoms, or risk of malignancy. Although most mutations are predicted to lead to premature termination of translation, and to loss of kindlin‐1 function, significant clinical variability is observed among patients. There is an association of FERMT1 missense and in‐frame deletion mutations with milder disease phenotypes, and later onset of complications. Nevertheless, the clinical variability is not fully explained by genotype–phenotype correlations. Environmental factors and yet unidentified modifiers may play a role. Better understanding of the molecular pathogenesis of KS should enable the development of prevention strategies for disease complications. Hum Mutat 32:1204–1212, 2011. ©2011 Wiley Periodicals, Inc.

Lack of plakoglobin leads to lethal congenital epidermolysis bullosa: a novel clinico-genetic entity

Epidermal integrity is essential for skin functions. It is maintained by adhesive structures between keratinocytes, mainly the desmosomes and adherens junctions, which provide resistance against mechanical stress and regulate the formation of the skin barrier. As a constituent of both types of intercellular junctions, plakoglobin has multiple interaction partners and mutations in its gene [junction plakoglobin (JUP)] have been associated with mild cutaneous disease, palmoplantar keratoderma and arrhythmogenic heart disease. Here we report a novel lethal phenotype caused by a homozygous nonsense JUP mutation, c.1615C>T, p.Q539X, which is very different from any human or murine JUP phenotype described so far. The patient suffered from severe congenital skin fragility with generalized epidermolysis and massive transcutaneous fluid loss, but apparently no cardiac dysfunction. In contrast to previously reported JUP mutations where truncated proteins were still present, in this case there was complete loss of plakoglobin in the patients skin, as demonstrated by immunofluorescence and immunoblot analysis. As a consequence, only very few abnormal desmosomes were formed and no adhesion structures between keratinocytes were recognizable. The expression and distribution of desmosomal components was severely affected, suggesting an essential role for plakoglobin in desmosomal assembly. Adherens junction proteins were localized to keratinocyte plasma membrane, but did not provide proper cell-cell adhesion. This lethal congenital epidermolysis bullosa highlights the fundamental role of plakoglobin in epidermal cohesion.

Forty-two novel COL7A1 mutations and the role of a frequent single nucleotide polymorphism in the MMP1 promoter in modulation of disease severity in a large European dystrophic epidermolysis bullosa cohort.

Background  Dystrophic epidermolysis bullosa (DEB) is a severe genetic skin blistering disorder caused by mutations in the gene COL7A1, encoding collagen VII. Recently, the MMP1 promoter single nucleotide polymorphism (SNP) rs1799750, designated as 1G 2G, was shown to be involved in modulation of disease severity in patients with recessive DEB (RDEB), and was proposed as a genetic modifier.

Losartan ameliorates dystrophic epidermolysis bullosa and uncovers new disease mechanisms

Genetic loss of collagen VII causes recessive dystrophic epidermolysis bullosa (RDEB)—a severe skin fragility disorder associated with lifelong blistering and disabling progressive soft tissue fibrosis. Causative therapies for this complex disorder face major hurdles, and clinical implementation remains elusive. Here, we report an alternative evidence‐based approach to ameliorate fibrosis and relieve symptoms in RDEB. Based on the findings that TGF‐β activity is elevated in injured RDEB skin, we targeted TGF‐β activity with losartan in a preclinical setting. Long‐term treatment of RDEB mice efficiently reduced TGF‐β signaling in chronically injured forepaws and halted fibrosis and subsequent fusion of the digits. In addition, proteomics analysis of losartan‐ vs. vehicle‐treated RDEB skin uncovered changes in multiple proteins related to tissue inflammation. In line with this, losartan reduced inflammation and diminished TNF‐α and IL‐6 expression in injured forepaws. Collectively, the data argue that RDEB fibrosis is a consequence of a cascade encompassing tissue damage, TGF‐β‐mediated inflammation, and matrix remodeling. Inhibition of TGF‐β activity limits these unwanted outcomes and thereby substantially ameliorates long‐term symptoms.

Laminin 332 in junctional epidermolysis bullosa

Laminin 332 is an essential component of the dermal-epidermal junction, a highly specialized basement membrane zone that attaches the epidermis to the dermis and thereby provides skin integrity and resistance to external mechanical forces. Mutations in the LAMA3, LAMB3 and LAMC2 genes that encode the three constituent polypeptide chains, α3, β3 and γ2, abrogate or perturb the functions of laminin 332. The phenotypic consequences are diminished dermal-epidermal adhesion and, as clinical symptoms, skin fragility and mechanically induced blistering. The disorder is designated as junctional epidermolysis bullosa (JEB). This article delineates the signs and symptoms of the different forms of JEB, the mutational spectrum, genotype-phenotype correlations as well as perspectives for future molecular therapies.

Molecular mechanisms of phenotypic variability in junctional epidermolysis bullosa

Background Junctional epidermolysis bullosa (JEB), a group of hereditary skin fragility disorders, is associated with a wide variety of phenotypes, although all forms are characterised by trauma induced skin blistering and tissue separation at the dermal–epidermal junction zone. A subgroup, coined JEB-other, is associated with mutations in the COL17A1 gene encoding collagen XVII or, more rarely, with mutations in the laminin 332 genes LAMA3, LAMB3, or LAMC2. The objective of this study is comprehensive genotype–phenotype analysis in JEB-other patients with COL17A1 mutations and elucidation of disease mechanisms underlying different skin phenotypes. Methods and results COL17A1 mutations and their clinical and cellular consequences were systematically analysed in 43 patients with JEB-other. Cell culture, RT-PCR, and protein biochemistry were applied to assess the effects of splice site mutations—that is, the nature and amounts of transcripts and polypeptides synthesised and their association with the phenotypic outcome. 34 distinct COL17A1 mutations were disclosed, 12 of them novel. mRNA and protein analyses demonstrated that patients with only about 12–14% of the physiological collagen XVII levels had mild cutaneous involvement and a long life span. Conclusions In contrast to complete null phenotypes, presence of minor amounts of collagen XVII protein in JEB skin is associated with mild phenotypic manifestations. The data have significant implications for design of molecular therapies for JEB, since they suggest that already a low extent of collagen XVII restoration will improve skin stability and alleviate symptoms.

Loss of desmoglein 1 associated with palmoplantar keratoderma, dermatitis and multiple allergies

Monoallelic desmoglein 1 mutations have been known for many years to cause striate palmoplantar keratoderma, but only recently, biallelic loss‐of‐function mutations were associated with a new disorder, designated as SAM syndrome (comprising severe dermatitis, multiple allergies and metabolic wasting) in two consanguineous families. We report on a new case from a third independent family with the homozygous nonsense mutation, c.2659C>T, p.R887* in exon 15 of DSG1 (desmoglein 1 gene). This mutation led to mRNA decay and loss of expression of desmoglein 1. The clinical phenotype consisted of severe palmoplantar keratoderma, dermatitis and multiple allergies. In contrast to the previous cases, malabsorption, hypoalbuminaemia, developmental delay, hypotrichosis or severe recurrent infections were not observed.

Acral Peeling Skin Syndrome with TGM5 Gene Mutations May Resemble Epidermolysis Bullosa Simplex in Young Individuals

TO THE EDITOR The acral peeling skin syndrome (APSS) is a rare autosomal recessive condition characterized by superficial painless peeling of the skin predominantly on the dorsal aspects of hands and feet (Shwayder et al., 1997; Cassidy et al., 2005). The condition is usually aggravated by heat, humidity, and exposure to water. Microscopically, the cleavage level is located in the upper epidermis, between the stratum granulosum and the stratum corneum (Garcia et al., 2005). Only 15 patients with APSS have been reported since 1997 (Shwayder et al., 1997; Brusasco et al., 1998; Hashimoto et al., 2000; Cassidy et al., 2005; Garcia et al., 2005; Kharfi et al., 2009; Oumakhir et al., 2009; Wakade et al., 2009) (Table 1). In several of them, in addition to superficial peeling, acral blisters were also described (Wakade et al., 2009). The genetic basis of the disease was determined in only three families, in whom two different missense mutations in the TGM5 gene encoding transglutaminase 5 (TGase 5) were disclosed (Table 1) (Cassidy et al., 2005; Kharfi et al., 2009). It remains unclear whether the other patients have mutations in the same gene, or whether APSS is clinically and genetically heterogeneous (Cassidy et al., 2005). In this study, we investigated nine unrelated patients, eight children and one adult, clinically suspected to have epidermolysis bullosa simplex (EBS) because of acral skin blistering. The patients and/or diagnostic samples were referred to the Epidermolysis bullosa Center of the University Medical Center Freiburg (Volz et al., 2007) for molecular diagnostics of EBS. EDTA-blood and skin samples were obtained after informed consent of the patients and, if available, of family members. EDTAblood samples of 50 clinically unaffected Central European individuals were used as controls. The study was conducted according to the Declaration of Helsinki Principles. Immunofluorescence staining of skin cryosections was performed using a panel of antibodies to components of the epidermal basement membrane zone (Kern et al., 2006), as well as antibodies to loricrin (Abcam, Cambridge, UK), filaggrin (clone 15C10; Novocastra, Newcastle, UK), involucrin (clone SY5; Sigma, Taufkirchen, Germany), cytokeratin 10 (clone DE-K10; Dako, Glostrup, Denmark), TGase 1 (clone B.C1; Biomedical Technologies, Madrid Spain), TGase 3 (Jackson Immunoresearch Laboratories, West Grove, PA), and TGase 5 (Novus Biologicals, Littleton, CO). Genomic DNA was extracted from EDTA-blood using the QiAmp DNA mini kit (Qiagen, Hilden, Germany). Amplification of all KRT5 (NC_000012.11, National Center for Biotechnology Information (NCBI)), KRT14 (NC_000017.10, NCBI), and TGM5 (NC_000015.9, NCBI) exons and exon–intron boundaries, and sequencing were performed as described (Schuilenga-Hut et al., 2003; Wood et al., 2003; Cassidy et al., 2005). Mutations were confirmed by resequencing. The mutation c.763T4C was verified in 100 control chromosomes by Abbreviations: APSS, acral peeling skin syndrome; EBS, epidermolysis bullosa simplex; TGase, transglutaminase

Dynamic Interactions of Epidermal Collagen XVII with the Extracellular Matrix Laminin 332 as a Major Binding Partner

Transmembrane collagen XVII, a major component of the hemidesmosomes, is crucial for stable adhesion of the epidermis and dermis in the skin, and its dysfunction results in blistering diseases. The ectodomain of collagen XVII (Ecto-ColXVII) is constitutively shed from the cell surface, but its binding partner(s) in the extracellular matrix (ECM) and the physiologic roles of the ligand interactions remain elusive. Herein, we used a new cleavage site-specific antibody to address the dynamics of collagen XVII shedding and the interactions of Ecto-ColXVII with the ECM. Ecto-ColXVII was present in the migration tracks of primary human keratinocytes and co-localized with laminin 332. The presence of this laminin, but also of collagen IV and Matrigel, in the ECM enhanced shedding and incorporation of Ecto-ColXVII into the matrix. Laminin 332 is a major, but not exclusive, interaction partner in vivo because Ecto-ColXVII deposited in the ECM of laminin 332-deficient keratinocytes was drastically reduced, but Ecto-ColXVII was present in laminin 332-negative human skin. Expression of collagen XVII deletion mutants in HEK 293 cells identified the C-terminal ectodomain stretch Ser(978)-Pro(1497) as necessary for ECM binding. Taken together, migrating keratinocytes shed the Ecto-ColXVII, and this dynamically binds via its C-terminal domain to distinct partners in the ECM.