Management of advanced foot deformities in dystrophic epidermolysis bullosa

Treatment for the characteristic hand deformities of dystrophic epidermolysis bullosa has been well described in the literature. We present our first experience delineating the surgical treatment for advanced foot deformities in this disease. This case emphasizes the need for release of contractures, the use of split-thickness skin grafts, and the need for rigid fixation to maintain surgical correction until healing is complete.     

Study of elastase-type activity in blister fluids of recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is characterized clinically by blister formation due to minor trauma and ultrastructurally by a progressive disappearance of anchoring fibrils at the dermoepidermal junction and of the oxytalan-type fibers which belong to the elastic fiber system. In this study, we determined the elastase-type activity in blister fluid obtained from 8 patients suffering from RDEB as compared to the suction fluid of experimental blisters in a healthy person and to the blister fluid of a patient suffering from epidermolysis bullosa simplex. One patient with dominant dystrophic epidermolysis of the albopapuloid type was also studied. Seven of the eight children with RDEB showed highly elevated values. The eighth child, treated with etretinate, as well as the patient suffering from dominant epidermolysis bullosa had moderately increased values. The determination of elastase-type activity in the blister fluid could therefore be useful to establish the differential diagnosis of recessive dystrophic epidermolysis bullosa.     

EGFR inhibition for metastasized cutaneous squamous cell carcinoma in dystrophic epidermolysis bullosa

The ACTA2 gene encodes for smooth muscle specific α-actin, a critical component of the contractile apparatus of the vascular smooth muscle cell. Pathogenic variants in the ACTA2 gene are the most frequently encountered genetic cause of non-syndromic hereditary thoracic aortic disease (HTAD). Although thoracic aortic aneurysm and/or dissection is the main clinical manifestation, a variety of occlusive vascular disease and extravascular manifestations occur in ACTA2-related vasculopathy. Current data suggest possible mutation-specific manifestations of vascular and extra-aortic traits.

Despite its relatively high prevalence, comprehensive recommendations on the care of patients and families with pathogenic variants in ACTA2 have not yet been established. We aimed to develop a consensus document to provide medical guidance for health care professionals involved in the diagnosis and treatment of patients and relatives with pathogenic variants in ACTA2.

The HTAD Working Group of the European Reference Network for Rare Vascular Diseases (VASCERN) convened to review current literature and discuss expert opinions on clinical management of ACTA2 related vasculopathy. This consensus statement summarizes our recommendations on diagnosis, monitoring, treatment, pregnancy, genetic counselling and testing in patients with ACTA2-related vasculopathy. However, there is a clear need for additional prospective multicenter studies to further define proper guidelines.

     

Characterization of molecular mechanisms underlying mutations in dystrophic epidermolysis bullosa using site-directed mutagenesis

Type VII collagen (C7) is a major component of anchoring fibrils, structures that mediate epidermal-dermal adherence. Mutations in gene COL7A1 encoding for C7 cause dystrophic epidermolysis bullosa (DEB), a genetic mechano-bullous disease. The biological consequences of specific COL7A1 mutations and the molecular mechanisms leading to DEB clinical phenotypes are unknown. In an attempt to establish genotype-phenotype relationships, we generated four individual substitution mutations that have been associated with recessive DEB, G2049E, R2063W, G2569R, and G2575R, and purified the recombinant mutant proteins. All mutant proteins were synthesized and secreted as a 290-kDa mutant C7 alpha chain at levels similar to wild type C7. The G2569R and G2575R glycine substitution mutations resulted in mutant C7 with increased sensitivity to protease degradation and decreased ability to form trimers. Limited proteolytic digestion of mutant G2049E and R2063W proteins yielded aberrant fragments and a triple helix with reduced stability. These two mutations next to the 39-amino acid helical interruption hinge region caused local destabilization of the triple-helix that exposed an additional highly sensitive proteolytic site within the region of the mutation. Our functional studies demonstrated that C7 is a potent pro-motility matrix for skin human keratinocyte migration and that this activity resides within the triple helical domain. Furthermore, G2049E and R2063W mutations reduced the ability of C7 to support fibroblast adhesion and keratinocyte migration. We conclude that known recessive DEB C7 mutations perturb critical functions of the C7 molecule and likely contribute to the clinical phenotypes of DEB patients.     

Injection of recombinant human type VII collagen restores collagen function in dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is a family of inherited mechano-bullous disorders that are caused by mutations in the type VII collagen gene and for which ex vivo gene therapy has been considered. To develop a simpler approach for treating DEB, we evaluated the feasibility of protein-based therapy by intradermally injecting human recombinant type VII collagen into mouse skin and a DEB human skin equivalent transplanted onto mice. The injected collagen localized to the basement membrane zone of both types of tissues, was organized into human anchoring fibril structures and reversed the features of DEB disease in the DEB skin equivalent.     

The recombinant expression of full-length type VII collagen and characterization of molecular mechanisms underlying dystrophic epidermolysis bullosa.

Type VII collagen is a major component of anchoring fibrils, attachment structures that mediate dermal-epidermal adherence in human skin. Dystrophic epidermolysis bullosa (DEB) is an inherited mechano-bullous disorder caused by mutations in the type VII collagen gene and perturbations in anchoring fibrils. In this study, we produced recombinant human type VII collagen in stably transfected human 293 cell clones and purified large quantities of the recombinant protein from culture media. The recombinant type VII collagen was secreted as a correctly folded, disulfide-bonded, helical trimer resistant to protease degradation. Purified type VII collagen bound to fibronectin, laminin-5, type I collagen, and type IV collagen and also supported human dermal fibroblast adhesion. In an attempt to establish genotype-phenotype relationships, we generated two individual substitution mutations that have been associated with recessive DEB, R2008G and G2749R, and purified the recombinant mutant proteins. The G2749R mutation resulted in mutant type VII collagen with increased sensitivity to protease degradation and decreased ability to form trimers. The R2008G mutation caused the intracellular accumulation of type VII collagen. We conclude that structural and functional studies of in vitro generated type VII collagen mutant proteins will aid in correlating genetic mutations with the clinical phenotypes of DEB patients.     

Recurrent nonsense mutations within the type VII collagen gene in patients with severe recessive dystrophic epidermolysis bullosa.

The generalized mutilating form of recessive dystrophic epidermolysis bullosa (i.e., the Hallopeau-Siemens type; HS-RDEB) is a life-threatening disease characterized by extreme mucocutaneous fragility associated with absent or markedly altered anchoring fibrils (AF). Recently, we reported linkage between HS-RDEB and the type VII collagen gene (COL7A1), which encodes the major component of AF. In this study, we investigated 52 unrelated HS-RDEB patients and 2 patients with RDEB inversa for the presence, at CpG dinucleotides, of mutations changing CGA arginine codons to premature stop codons TGA within the COL7A1 gene. Eight exons containing 10 CGA codons located in the amino-terminal domain of the COL7A1 gene were studied. Mutation analysis was performed using denaturing gradient gel electrophoresis of PCR-amplified genomic fragments. Direct sequencing of PCR-amplified products with altered electrophoretic mobility led to the characterization of three premature stop codons, each in a single COL7A1 allele, in four patients. Two patients (one affected with HS-RDEB and the other with RDEB inversa) have the same C-to-T transition at arginine codon 109. Two other HS-RDEB patients have a C-to-T transition at arginine 1213 and 1216, respectively. These nonsense mutations predict the truncation of approximately 56%-92% of the polypeptide, including the collagenous and the noncollagenous NC-2 domains. On the basis of linkage analysis, which showed no evidence for locus heterogeneity in RDEB, it is expected that these patients are compound heterozygotes and have additional mutations on the other COL7A1 allele, leading to impaired AF formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidermolysis bullosa dystrophica dominans (Pasini)—A primary structural defect of the anchoring fibrils

Summary In epidermolysis bullosa dystrophica dominans Pasini a structural defect of the anchoring fibrils, a structural protein of the epidermo-dermal junction, has been demonstrated by electron microscopy to be constantly present not only in involved skin but also in intact skin of nonpredilection sites of blister formation. These findings render support to the concept that in dominant disorders an abnormality in a non-enzymic structural protein is more likely than in an enzymic protein.Supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich Klinische Genetik, SFB 35/B 5.     

Denaturing HPLC-based approach for detection of COL7A1 gene mutations causing dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is a rare clinically heterogeneous genodermatosis due to genetic defects in type VII collagen gene (COL7A1). Identification of COL7A1 mutations is a challenge since this gene comprises 118 exons and more than 300 mutations scattered over the gene have been reported. Here, we describe for the first time the use of denaturing high performance liquid chromatography (DHPLC) for COL7A1 mutation detection. To validate the method, exon-specific DHPLC conditions were applied to screen DNA samples from patients carrying known COL7A1 mutations. Abnormal DHPLC profiles were obtained for all known mutations. Subsequent DHPLC analysis of 17 DEB families of unknown genotype allowed the identification of 21 distinct mutations, 9 of which were novel. The DHPLC mutation detection rate was significantly higher compared with our mutation scanning rate with conventional techniques (97% vs 86%), indicating DHPLC as the method of choice for COL7A1 molecular characterization in DEB patients.     

Mutation analysis and molecular genetics of epidermolysis bullosa.

Cutaneous basement membrane zone (BMZ) consists of a number of attachment structures that are critical for stable association of the epidermis to the underlying dermis. These include hemidesmosomes, anchoring filaments and anchoring fibrils which form an interconnecting network extending from the intracellular milieu of basal keratinocytes across the dermal-epidermal basement membrane to the underlying dermis. Aberrations in this network structure, e.g. due to genetic lesions in the corresponding genes, can result in fragility of the skin at the level of the cutaneous BMZ. The prototype of such diseases is epidermolysis bullosa (EB), a heterogeneous group of genodermatoses characterized by fragility and blistering of the skin, often associated with extracutaneous manifestations, and inherited either in an autosomal dominant or autosomal recessive manner. Based on constellations of the phenotypic manifestations, severity of the disease, and the level of tissue separation within the cutaneous BMZ, EB has been divided into clinically distinct subcategories, including the simplex, hemidesmosomal, junctional and dystrophic variants. Elucidation of BMZ gene/protein systems and development of mutation detection strategies have allowed identification of mutations in 10 different BMZ genes which can explain the clinical heterogeneity of EB. These include mutations in the type VII collagen gene (COL7A1) in the dystrophic (severely scarring) forms of EB; mutations in the laminin 5 genes (LAMA3, LAMB3 and LAMC2) in a lethal (Herlitz) variant of junctional EB; aberrations in the type XVII collagen gene (COL17A1) in non-lethal forms of junctional EB; mutations in the alpha6 and beta4 integrin genes in a distinct hemidesmosomal variant of EB with congenital pyloric atresia; and mutations in the plectin gene (PLEC1) in a form of EB associated with late-onset muscular dystrophy. Identification of mutations in these gene/protein systems attests to their critical importance in the overall stability of the cutaneous BMZ. Furthermore, elucidation of mutations in different variants of EB has direct clinical applications in terms of refined classification, improved genetic counseling, and development of DNA-based prenatal testing in families with EB.     

ABCB5+ mesenchymal stromal cells facilitate complete and durable wound closure in recessive dystrophic epidermolysis bullosa

Background and aimsRecessive dystrophic epidermolysis bullosa (RDEB) is a hereditary, rare, devastating and life-threatening skin fragility disorder with a high unmet medical need. In a recent international, single-arm clinical trial, treatment of 16 patients (aged 6–36 years) with three intravenous infusions of 2 × 10⁶ immunomodulatory ABCB5⁺ dermal mesenchymal stromal cells (MSCs)/kg on days 0, 17 and 35 reduced disease activity, itch and pain. A post-hoc analysis was undertaken to assess the potential effects of treatment with ABCB5⁺ MSCs on the overall skin wound healing in patients suffering from RDEB.MethodsDocumentary photographs of the affected body regions taken on days 0, 17, 35 and at 12 weeks were evaluated regarding proportion, temporal course and durability of wound closure as well as development of new wounds.ResultsOf 168 baseline wounds in 14 patients, 109 (64.9%) wounds had closed at week 12, of which 63.3% (69 wounds) had closed already by day 35 or day 17. Conversely, 74.2% of the baseline wounds that had closed by day 17 or day 35 remained closed until week 12. First-closure ratio within 12 weeks was 75.6%. The median rate of newly developing wounds decreased significantly (P = 0.001) by 79.3%.ConclusionsComparison of the findings with published data from placebo arms and vehicle-treated wounds in controlled clinical trials suggests potential capability of ABCB5⁺ MSCs to facilitate wound closure, prolongate wound recurrence and decelerate formation of new wounds in RDEB. Beyond suggesting therapeutic efficacy for ABCB5⁺ MSCs, the analysis might stimulate researchers who develop therapies for RDEB and other skin fragility disorders to not only assess closure of preselected target wounds but pay attention to the patients’ dynamic and diverse overall wound presentation as well as to the durability of achieved wound closure and the development of new wounds.Trial registrationClinicaltrials.gov NCT03529877; EudraCT 2018-001009-98.