Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and most often severe genetic disease characterized by recurrent blistering and erosions of the skin and mucous membranes after minor trauma, leading to major local and systemic complications. The disease is caused by loss-of-function variants in COL7A1 encoding type VII collagen (C7), the main component of anchoring fibrils, which form attachment structures stabilizing the cutaneous basement membrane zone. Alterations in C7 protein structure and/or expression lead to abnormal, rare or absent anchoring fibrils resulting in loss of dermal-epidermal adherence and skin blistering. To date, more than 1,200 distinct COL7A1 deleterious variants have been reported and 19% are splice variants. Here, we describe two RDEB patients for whom we identified two pathogenic deep intronic pathogenic variants in COL7A1 . One of these variants (c.7795-97C > G) promotes the inclusion of a pseudoexon between exons 104 and 105 in the COL7A1 transcript, while the other causes partial or complete retention of intron 51. We used antisense oligonucleotide (ASO) mediated exon skipping to correct these aberrant splicing events in vitro. This led to increased normal mRNA splicing above 94% and restoration of C7 protein expression at a level (up to 56%) that should be sufficient to reverse the phenotype. This first report of exon skipping applied to counteract deep intronic variants in COL7A1 represents a promising therapeutic strategy for personalized medicine directed at patients with intronic variants at a distance of consensus splice sites.

中文翻译：

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剪接调制策略应用于 COL7A1 中的深层内含子变异，导致隐性营养不良性大疱性表皮松解症


隐性营养不良性大疱性表皮松解症（RDEB）是一种罕见且最常见的严重遗传性疾病，其特征是轻微创伤后皮肤和粘膜反复出现水疱和糜烂，导致严重的局部和全身并发症。该疾病是由编码 VI​​I 型胶原 (C7) 的 COL7A1 功能丧失变异引起的，C7 是锚定原纤维的主要成分，形成稳定皮肤基底膜区域的附着结构。 C7 蛋白结构和/或表达的改变导致锚定原纤维异常、罕见或缺失，从而导致真皮-表皮粘附力丧失和皮肤起泡。迄今为止，已报道了超过 1,200 个不同的 COL7A1 有害变体，其中 19% 是剪接变体。在这里，我们描述了两名 RDEB 患者，我们在 COL7A1 中鉴定了两个致病性深部内含子致病变异。这些变体之一 (c.7795-97C >G) 促进 COL7A1 转录物中外显子 104 和 105 之间包含假外显子，而另一种则导致内含子 51 部分或完全保留。我们使用反义寡核苷酸 (ASO) 介导的外显子跳跃可以在体外纠正这些异常剪接事件。这导致正常 mRNA 剪接增加超过 94%，并且 C7 蛋白表达恢复到足以逆转表型的水平（高达 56%）。这是第一份关于外显子跳跃应用于抵消 COL7A1 中深层内含子变异的报告，代表了一种有前途的个性化医疗治疗策略，针对在共有剪接位点距离处具有内含子变异的患者。