Generation of a recessive dystrophic epidermolysis bullosa mouse model with patient-derived compound heterozygous mutations.
Generation of a recessive dystrophic epidermolysis bullosa mouse model with patient-derived compound heterozygous mutations.
复制标题
产生具有患者来源的复合杂合突变的隐性营养不良性大疱性表皮松解症小鼠模型。
DOI：
10.1038/s41374-022-00735-5
复制
发表时间：
2022
期刊：
影响因子：
5
通讯作者：
Tamai K
中科院分区：
文献类型：
--
作者：
Takaki S;Shimbo T;Ikegami K;Kitayama T;Yamamoto Y;Yamazaki S;Mori S;Tamai K
隐性营养不良性大疱性表皮病（RDEB）是一种由COL 7A 1基因突变引起的难治性遗传性皮肤病。大多数RDEB患者携带复合杂合突变-每个染色体上有两个不同的突变-在COL 7A 1突变模式中没有任何明显的热点。这种情况使得建立具有准确模拟患者基因组背景的突变的可靠RDEB小鼠模型具有挑战性。在这里，我们使用基于CRISPR的基因组编辑技术i-GONAD建立了一种以复合杂合方式携带患者型突变的RDEB小鼠模型。我们选择了两个突变，c.5818delC和E2857 X，这两个突变在日本RDEB患者队列中经常被发现。这些突变被引入到小鼠基因组中与患者中鉴定的位置相对应的位置。5818 delC突变的纯合子小鼠出现了严重的RDEB样表型，并在出生后立即死亡，而E2857 X纯合子小鼠与野生型小鼠相比寿命并没有缩短。成年E2857 X纯合子小鼠表现出毛发异常、并指（趾）畸形和指甲营养不良;这些发现表明E2857 X确实在小鼠中具有致病性。具有c.5818delC/E2857 X复合杂合突变的小鼠呈现介于c.5818delC和E2857 X纯合小鼠之间的中间表型。单细胞RNA测序进一步阐明，在c.5818delC/E2857 X复合杂合子小鼠的滤泡内角质形成细胞表现出异常的细胞周期调控。除了已建立的小鼠系之外，提出的产生复合杂合小鼠的策略将促进对RDEB发病机制的研究，以开发治疗这种毁灭性疾病的方法。
Recessive dystrophic epidermolysis bullosa (RDEB) is an intractable genetic disease of the skin caused by mutations in theCOL7A1gene. The majority of patients with RDEB harbor compound heterozygous mutations—two distinct mutations on each chromosome—without any apparent hotspots in theCOL7A1mutation pattern. This situation has made it challenging to establish a reliable RDEB mouse model with mutations that accurately mimic the genomic background of patients. Here, we established an RDEB mouse model harboring patient-type mutations in a compound heterozygous manner, using the CRISPR-based genome-editing technology i-GONAD. We selected two mutations, c.5818delC and E2857X, that have frequently been identified in cohorts of Japanese patients with RDEB. These mutations were introduced into the mouse genome at locations corresponding to those identified in patients. Mice homozygous for the 5818delC mutation developed severe RDEB-like phenotypes and died immediately after birth, whereas E2857X homozygous mice did not have a shortened lifespan compared to wild-type mice. Adult E2857X homozygous mice showed hair abnormalities, syndactyly, and nail dystrophy; these findings indicate that E2857X is indeed pathogenic in mice. Mice with the c.5818delC/E2857X compound heterozygous mutation presented an intermediate phenotype between the c.5818delC and E2857X homozygous mice. Single-cell RNA sequencing further clarified that the intrafollicular keratinocytes in c.5818delC/E2857X compound heterozygous mice exhibited abnormalities in cell cycle regulation. The proposed strategy to produce compound heterozygous mice, in addition to the established mouse line, will facilitate research on RDEB pathogenesis to develop a cure for this devastating disease.