Dataset Information

Characterization of mutant type VII collagens underlying the inversa subtype of recessive dystrophic epidermolysis bullosa.

ABSTRACT:

Background

Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional type VII collagen (C7) leading to skin fragility, bullae, and erosive wounds. RDEB-Inversa (RDEB-I), a subset of RDEB, is characterized by lesions localized to body areas with higher skin temperatures such as flexures and skin folds.

Objective

We aimed to determine if C7 derived from RDEB-I mutations had structural and functional aberrancies that were temperature sensitive and could be reversed by lowering the temperature.

Methods

In this study, we generated 12 substitution mutations associated with RDEB-I via site-directed mutagenesis and purified recombinant C7 protein. These C7 mutants were evaluated for structural parameters (trimer formation and protease sensitivity) and the ability to promote keratinocyte migration at 37 °C (the temperature of skin folds) and 30 °C (the maximum skin temperature of arms and legs). Fibroblasts derived from RDEB-I patients were evaluated for C7 secretion and cellular migration at both temperatures.

Results

C7s from RDEB-I mutations exhibited decreased thermal stability, increased sensitivity to protease digestion, diminished formation of collagen trimers, and reduced ability to promote keratinocyte migration compared with wild-type C7. In addition, fibroblasts derived from RDEB-I patients demonstrated intracellular accumulation of C7 and abnormal cell migration at 37 °C. All of these aberrancies were corrected by reducing the temperature to 30 °C. C7s generated from severe-RDEB mutations (non-Inversa) did not display temperature-dependent perturbations.

Conclusion

These data demonstrate that RDEB-I mutations generate C7 aberrancies that are temperature dependent. This may explain why RDEB-I patients develop clinical lesions in areas where their skin is considerably warmer.

SUBMITTER: Woodley DT

PROVIDER: S-EPMC8639788 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Publications

Characterization of mutant type VII collagens underlying the inversa subtype of recessive dystrophic epidermolysis bullosa.

Woodley David T DT Cogan Jon J Mosallaei Daniel D Yim Kaitlyn K Chen Mei M

Journal of dermatological science 20210923 2

<h4>Background</h4>Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional type VII collagen (C7) leading to skin fragility, bullae, and erosive wounds. RDEB-Inversa (RDEB-I), a subset of RDEB, is characterized by lesions localized to body areas with higher skin temperatures such as flexures and skin folds.<h4>Objective</h4>We aimed to determine if C7 derived from RDEB-I mutations had structural and functional aberrancies that were temperature sensitive and could be rever ...[more]

PMID: 34674926

Similar Datasets

Project description:BackgroundRecessive dystrophic epidermolysis bullosa (RDEB) is an incurable disease caused by mutations in the gene encoding type VII collagen, the major component of anchoring fibrils (AF). We previously demonstrated that gentamicin produced functional type VII collagen in RDEB cells harboring nonsense mutations. Herein, we determined whether topical or intradermal gentamicin administration induces type VII collagen and AFs in RDEB patients.MethodsA double-blind, placebo-controlled pilot trial assessed safety and efficacy of topical and intradermal gentamicin in 5 RDEB patients with nonsense mutations. The topical arm tested 0.1% gentamicin ointment or placebo application 3 times daily at 2 open erosion sites for 2 weeks. The intradermal arm tested daily intradermal injection of gentamicin solution (8 mg) or placebo into 2 intact skin sites for 2 days in 4 of 5 patients. Primary outcomes were induction of type VII collagen and AFs at the test sites and safety assessment. A secondary outcome assessed wound closure of topically treated erosions.ResultsBoth topical and intradermal gentamicin administration induced type VII collagen and AFs at the dermal-epidermal junction of treatment sites. Newly created type VII collagen varied from 20% to 165% of that expressed in normal human skin and persisted for 3 months. Topical gentamicin corrected dermal-epidermal separation, improved wound closure, and reduced blister formation. There were no untoward side effects from gentamicin treatments. Type VII collagen induction did not generate anti-type VII collagen autoantibodies in patients' blood or skin.ConclusionTopical and intradermal gentamicin suppresses nonsense mutations and induces type VII collagen and AFs in RDEB patients. Gentamicin therapy may provide a readily available treatment for RDEB patients with nonsense mutations.Trial registrationClinicalTrials.gov NCT02698735.FundingEpidermolysis Bullosa Research Partnership, Epidermolysis Bullosa Medical Research Foundation, NIH, and VA Merit Award.

Project description:BackgroundRevertant mosaicism has been described previously in recessive dystrophic epidermolysis bullosa (RDEB), manifesting as regions of skin with normal mechanical and biological characteristics. Here we report the discovery of revertant dermal fibroblasts, unique in that all other documented cases of revertant mosaicism occur in epidermal keratinocytes.ObjectivesTo determine the cause of revertant mosaicism found in a patient with RDEB from isolated epidermal keratinocytes and dermal fibroblasts in blister and mosaic skin regions.MethodsSkin biopsies were taken from blister and mosaic skin regions of a patient with RDEB. Allele identification was confirmed and the type VII collagen (C7) content and COL7A1 expression profile of isolated keratinocytes and fibroblasts was determined.ResultsKeratinocytes isolated from the mosaic area had a slight increase in C7, although overall expression of COL7A1 was unchanged between blister and mosaic fibroblasts. Differential allele expression was identified in blister and mosaic fibroblasts using targeted RNA sequencing (TREx), where the allele harbouring a point mutation was preferentially expressed over that containing a frameshift mutation. A crossing over event was identified in mosaic fibroblasts that was not present in blister fibroblasts, yielding a functional COL7A1 allele in a subset of cells.ConclusionsIn documenting a novel case of revertant mosaicism in RDEB, we have identified dermal fibroblasts as having the capacity to correct blistering functionally. We have also pioneered the use of TREx in quantifying allele-specific expression. Using fibroblasts instead of keratinocytes for RDEB therapies offers advantages in the local and systemic therapy of RDEB. What's already known about this topic? Revertant mosaicism has been previously documented in patients with recessive dystrophic epidermolysis bullosa (RDEB), however, it has only been found in epidermal keratinocytes. What does this study add? We have demonstrated that COL7A1 gene reversion in dermal fibroblasts occurs and is able to form functional skin in a patient with RDEB. Additionally, we have pioneered a new application for targeted RNA sequencing in quantifying allele-specific expression in fibroblasts and keratinocytes. What is the translational message? This opens up possibilities for using fibroblasts as local and systemic therapy for patients with RDEB.

Project description:BackgroundRecessive dystrophic epidermolysis bullosa is an incurable, often fatal mucocutaneous blistering disease caused by mutations in COL7A1, the gene encoding type VII collagen (C7). On the basis of preclinical data showing biochemical correction and prolonged survival in col7 −/− mice, we hypothesized that allogeneic marrow contains stem cells capable of ameliorating the manifestations of recessive dystrophic epidermolysis bullosa in humans.MethodsBetween October 2007 and August 2009, we treated seven children who had recessive dystrophic epidermolysis bullosa with immunomyeloablative chemotherapy and allogeneic stem-cell transplantation. We assessed C7 expression by means of immunofluorescence staining and used transmission electron microscopy to visualize anchoring fibrils. We measured chimerism by means of competitive polymerase-chain-reaction assay, and documented blister formation and wound healing with the use of digital photography.ResultsOne patient died of cardiomyopathy before transplantation. Of the remaining six patients, one had severe regimen-related cutaneous toxicity, with all having improved wound healing and a reduction in blister formation between 30 and 130 days after transplantation. We observed increased C7 deposition at the dermal-epidermal junction in five of the six recipients, albeit without normalization of anchoring fibrils. Five recipients were alive 130 to 799 days after transplantation; one died at 183 days as a consequence of graft rejection and infection. The six recipients had substantial proportions of donor cells in the skin, and none had detectable anti-C7 antibodies.ConclusionsIncreased C7 deposition and a sustained presence of donor cells were found in the skin of children with recessive dystrophic epidermolysis bullosa after allogeneic bone marrow transplantation. Further studies are needed to assess the long-term risks and benefits of such therapy in patients with this disorder. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT00478244.)

Dataset Information

Characterization of mutant type VII collagens underlying the inversa subtype of recessive dystrophic epidermolysis bullosa.

Background

Objective

Methods

Results

Conclusion

Publications

Characterization of mutant type VII collagens underlying the inversa subtype of recessive dystrophic epidermolysis bullosa.

Similar Datasets

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