Abstract：
OBJECTIVE: To investigate the genetic causes of 22 patients with clinically high suspicion of X-linked hypohidrotic ectodermal dysplasia from 20 unrelated Chinese families, expand the spectrum of ectodysplasin-A mutations, and provide more evidence for variants of uncertain significance.
METHODS: Whole-exome sequencing was performed and potentially pathogenic variants were verified by Sanger sequencing. Western blotting, real-time PCR and immunofluorescence analyses were performed to investigate the preliminary functions of the candidate variants.
RESULTS: Nineteen ectodysplasin-A variants were identified, six of which were not previously reported. Among these variants, we identified a patient who carried two mutations in ectodysplasin-A and exhibited more severe phenotypes. Additionally, mutant protein expression levels decreased, whereas mRNA transcription levels increased. Cellular sublocalisation of the variants located in the tumour necrosis factor homologous domain showed that the proteins accumulated in the nucleus, whereas wild-type proteins remained in the cell membrane. A rare indel variant and two classical splicing variants that lead to exon 7 skipping were detected.
CONCLUSIONS: This study provides definitive diagnoses for 20 families with suspected X-linked hypohidrotic ectodermal dysplasia and additional information on clinical heterogeneity and genotype-phenotype relationships.
METHODS: Whole-exome sequencing was performed and potentially pathogenic variants were verified by Sanger sequencing. Western blotting, real-time PCR and immunofluorescence analyses were performed to investigate the preliminary functions of the candidate variants.
RESULTS: Nineteen ectodysplasin-A variants were identified, six of which were not previously reported. Among these variants, we identified a patient who carried two mutations in ectodysplasin-A and exhibited more severe phenotypes. Additionally, mutant protein expression levels decreased, whereas mRNA transcription levels increased. Cellular sublocalisation of the variants located in the tumour necrosis factor homologous domain showed that the proteins accumulated in the nucleus, whereas wild-type proteins remained in the cell membrane. A rare indel variant and two classical splicing variants that lead to exon 7 skipping were detected.
CONCLUSIONS: This study provides definitive diagnoses for 20 families with suspected X-linked hypohidrotic ectodermal dysplasia and additional information on clinical heterogeneity and genotype-phenotype relationships.
摘要：
目的：调查来自20个无关中国家庭的22例临床高度怀疑X连锁低汗性外胚层发育不良患者的遗传原因。扩大外生体异常蛋白-A突变谱，并为不确定意义的变体提供更多证据。
方法：进行全外显子组测序，并通过Sanger测序验证潜在致病变异。西方印迹,进行实时PCR和免疫荧光分析以研究候选变体的初步功能。
结果：鉴定了19种外泌体异常蛋白-A变体，其中6个以前没有报道过。在这些变体中，我们确定了1例携带2个外生体异常蛋白-A突变的患者,并且表现出更严重的表型.此外,突变蛋白表达水平下降，而mRNA转录水平增加。位于肿瘤坏死因子同源域中的变体的细胞亚定位表明，蛋白质在细胞核中积累，而野生型蛋白保留在细胞膜上。检测到一个罕见的indel变体和两个导致外显子7跳跃的经典剪接变体。
结论：本研究为20个疑似X连锁多汗性外胚层发育不良的家庭提供了明确的诊断，并提供了有关临床异质性和基因型-表型关系的其他信息。
方法：进行全外显子组测序，并通过Sanger测序验证潜在致病变异。西方印迹,进行实时PCR和免疫荧光分析以研究候选变体的初步功能。
结果：鉴定了19种外泌体异常蛋白-A变体，其中6个以前没有报道过。在这些变体中，我们确定了1例携带2个外生体异常蛋白-A突变的患者,并且表现出更严重的表型.此外,突变蛋白表达水平下降，而mRNA转录水平增加。位于肿瘤坏死因子同源域中的变体的细胞亚定位表明，蛋白质在细胞核中积累，而野生型蛋白保留在细胞膜上。检测到一个罕见的indel变体和两个导致外显子7跳跃的经典剪接变体。
结论：本研究为20个疑似X连锁多汗性外胚层发育不良的家庭提供了明确的诊断，并提供了有关临床异质性和基因型-表型关系的其他信息。
