Revertant mosaic fibroblasts in recessive dystrophic epidermolysis bullosa

Abstract

Summary Background Revertant 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. Objectives To 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. Methods Skin 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. Results Keratinocytes 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. Conclusions In 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.

QR-313, an Antisense Oligonucleotide, Shows Therapeutic Efficacy for Treatment of Dominant and Recessive Dystrophic Epidermolysis Bullosa: A Preclinical Study

“…Even so, the level of collagen VII after QR-313 treatment would not be expected to reach that of unaffected heterozygous carriers. Nevertheless, reports indicate that a relatively modest increase in functional collagen VII has significant clinical benefit (Fritsch et al 2008;Kern et al 2009;Schwieger-Briel et al 2015;Twaroski et al 2019).…”

“…In addition, the critical concentration of collagen VII needed to nucleate anchoring fibril formation may not have been reached. It has to be emphasized though that collagen VII may still support epidermal-to-dermal cohesion without assembling into well-formed anchoring fibrils (Bornert et al 2016;Schwieger-Briel et al 2015;Twaroski et al 2019).…”

Molecular Therapeutics in Development for Epidermolysis Bullosa: Update 2020

“…The mechanisms of the mutation reversion are multiple, including mitotic recombinations, back mutations and second site mutations [48][49][50]. Revertant mosaicism in patients with RDEB has been primarily documented in keratinocytes, but evidence of revertant mosaic fibroblasts has also recently been reported [51]. Attempts to establish long-term cultures of keratinocytes from areas of skin with revertant mosaicism have been mostly unsuccessful, primarily due to depletion of stem cells in the affected skin.…”

Meeting Report: The First Global Congress on Epidermolysis Bullosa, EB2020 London: Toward Treatment and Cure

“…In the case of EB, iPSCs can also be generated from skin areas manifesting revertant mosaicism, a form of natural gene therapy, where the mutations have spontaneously reversed manifesting as patches of normal-appearing skin. It should be noted that revertant mosaicism in skin diseases has been primarily documented in keratinocytes, but revertant mosaic fibroblasts have also been identified in the skin of patients with RDEB (Twaroski et al, 2019).…”