目的 分析3个单纯型大疱性表皮松解症（EBS）家系的致病基因变异特征，探讨基因型与表型的关联性，为EBS的分子诊断和遗传咨询提供依据。方法 收集2019年至2023 年就诊的3个EBS家系为研究对象，行全外显子组测序筛选候选致病突变，经Sanger测序验证；利用AlphaFold 3及PDBePISA对KRT5新突变进行蛋白结构建模与功能预测；制定个体化干预方案并随访。结果 3个家系患者均符合EBS局限型（EBS-loc）诊断标准，且在皮损范围、严重程度及病程演变上存在差异。遗传学检测发现家系1携带KRT5基因新错义突变 (NM_000424.4)：c.1291A>G（p.Lys431Glu），为常染色体显性遗传；家系2携带KRT14基因已知错义突变（NM_000526.5）：c.374G>A（p.Arg125His），与表型共分离；家系3携带KRT5基因新发错义突变（NM_000424.4）：c.428T>A（p.Val143Asp）。蛋白结构预测显示 p.Lys431Glu 突变会削弱KRT5-KRT14异源二聚体的结合亲和力与结构稳定性。结论 本研究发现KRT5（NM_000424.4）：c.1291A>G（p.Lys431Glu）为未报道新突变，丰富了EBS突变谱，蛋白结构预测揭示了该突变致病机制。个体化综合管理可显著改善EBS患者预后，基因检测对明确诊断、遗传咨询及个体化管理提供了重要依据。

Objective To analyze the pathogenic gene mutation characteristics in three families with epidermolysis bullosa simplex (EBS) and explore the association between genotype and phenotype, providing evidence for molecular diagnosis and genetic counseling of EBS. Methods Three EBS families admitted between 2019 and 2023 were enrolled. Whole-exome sequencing (WES) was performed to screen candidate pathogenic mutations, followed by Sanger sequencing validation. For the novel KRT5 mutation, protein structure modeling and functional prediction were conducted using AlphaFold 3 and PDBePISA. Individualized intervention plans were formulated and followed up. Results All patients in the three families met the diagnostic criteria for localized EBS (EBS-loc), and there were differences in the extent, severity, and disease course. Genetic testing revealed that family 1 carried a novel missense mutation in the KRT5 gene (NM_000424.4): c.1291A>G (p.Lys431Glu), which was an autosomal dominant inheritance; family 2 carried a known missense mutation in the KRT14 gene (NM_000526.5): c.374G>A (p.Arg125His), which was co-segregated with the phenotype; family 3 carried a novel missense mutation in the KRT5 gene (NM_000424.4): c.428T>A (p.Val143Asp). Protein structure prediction showed that the p.Lys431Glu mutation would weaken the binding affinity and structural stability of the KRT5-KRT14 heterodimer. Conclusion This study identified a novel KRT5 (NM_000424.4): c.1291A>G (p.Lys431Glu) mutation that has not been reported before, enriching the EBS mutation spectrum. Protein structure prediction revealed the pathogenic mechanism of this mutation. Individualized comprehensive management can significantly improve the prognosis of EBS patients, and genetic testing provides an important basis for clear diagnosis, genetic counseling, and individualized management.