Twaroski K, Eide C, Riddle M J, Xia L, Lees C J, Chen W, Mathews W, Keene D R, McGrath J A, Tolar J
Stem Cell Institute and.
Department of Pediatrics, Division of Blood and Marrow Transplantation, Medical School, University of Minnesota, Minneapolis, MN, U.S.A.
Br J Dermatol. 2019 Dec;181(6):1247-1253. doi: 10.1111/bjd.17943. Epub 2019 Jul 8.
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.
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.
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.
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.
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.
先前已在隐性营养不良性大疱性表皮松解症（RDEB）中描述过回复性镶嵌现象，表现为具有正常机械和生物学特性的皮肤区域。在此，我们报告了回复性真皮成纤维细胞的发现，其独特之处在于，所有其他已记录的回复性镶嵌病例均发生在表皮角质形成细胞中。
确定在一名RDEB患者的水疱和镶嵌皮肤区域分离出的表皮角质形成细胞和真皮成纤维细胞中发现的回复性镶嵌现象的原因。
从一名RDEB患者的水疱和镶嵌皮肤区域采集皮肤活检样本。确认等位基因鉴定，并测定分离出的角质形成细胞和成纤维细胞的VII型胶原蛋白（C7）含量及COL7A1表达谱。
从镶嵌区域分离出的角质形成细胞中C7略有增加，尽管水疱和成纤维细胞之间COL7A1的总体表达没有变化。使用靶向RNA测序（TREx）在水疱和成纤维细胞中鉴定出差异等位基因表达，其中携带点突变的等位基因比含有移码突变的等位基因优先表达。在成纤维细胞中鉴定出一个水疱成纤维细胞中不存在的交叉事件，在一部分细胞中产生了功能性COL7A1等位基因。
在记录RDEB中一例新的回复性镶嵌病例时，我们已确定真皮成纤维细胞具有在功能上纠正水疱形成的能力。我们还率先使用TREx来量化等位基因特异性表达。在RDEB治疗中使用成纤维细胞而非角质形成细胞在局部和全身治疗方面具有优势。关于该主题已知的信息有哪些？先前已在隐性营养不良性大疱性表皮松解症（RDEB）患者中记录过回复性镶嵌现象，然而，仅在表皮角质形成细胞中发现过。本研究增加了什么内容？我们已证明真皮成纤维细胞中发生COL7A1基因回复，并且能够在一名RDEB患者中形成功能性皮肤。此外，我们率先将靶向RNA测序应用于量化成纤维细胞和角质形成细胞中的等位基因特异性表达。转化信息是什么？这为将成纤维细胞用作RDEB患者的局部和全身治疗开辟了可能性。
