PDF(Pubmed)
Abstract：
BACKGROUND: Premature ovarian insufficiency (POI) is a severe disorder leading to female infertility. Genetic mutations are important factors causing POI. TP63-truncating mutation has been reported to cause POI by increasing germ cell apoptosis, however what factors mediate this apoptosis remains unclear.
METHODS: Ninety-three patients with POI were recruited from Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Whole-exome sequencing (WES) was performed for each patient. Sanger sequencing was used to confirm potential causative genetic variants. A minigene assay was performed to determine splicing effects of TP63 variants. A TP63-truncating plasmid was constructed. Real-time quantitative PCR, western blot analyses, dual luciferase reporter assays, immunofluorescence staining, and cell apoptosis assays were used to study the underlying mechanism of a TP63-truncating mutation causing POI.
RESULTS: By WES of 93 sporadic patients with POI, we found a 14-bp deletion covering the splice site in the TP63 gene. A minigene assay demonstrated that the 14-bp deletion variant led to exon 13 skipping during TP63 mRNA splicing, resulting in the generation of a truncated TP63 protein (TP63-mut). Overexpression of TP63-mut accelerated cell apoptosis. Mechanistically, the TP63-mut protein could bind to the promoter region of CLCA2 and activate the transcription of CLCA2 several times compared to that of the TP63 wild-type protein. Silencing CLCA2 using a specific small interfering RNA (siRNA) or inhibiting the Ataxia Telangiectasia Mutated (ATM) pathway using the KU55933 inhibitor attenuated cell apoptosis caused by TP63-mut protein expression.
CONCLUSIONS: Our findings revealed a crucial role for CLCA2 in mediating apoptosis in POI pathogenesis, and suggested that CLCA2 is a potential therapeutic target for POI.
METHODS: Ninety-three patients with POI were recruited from Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Whole-exome sequencing (WES) was performed for each patient. Sanger sequencing was used to confirm potential causative genetic variants. A minigene assay was performed to determine splicing effects of TP63 variants. A TP63-truncating plasmid was constructed. Real-time quantitative PCR, western blot analyses, dual luciferase reporter assays, immunofluorescence staining, and cell apoptosis assays were used to study the underlying mechanism of a TP63-truncating mutation causing POI.
RESULTS: By WES of 93 sporadic patients with POI, we found a 14-bp deletion covering the splice site in the TP63 gene. A minigene assay demonstrated that the 14-bp deletion variant led to exon 13 skipping during TP63 mRNA splicing, resulting in the generation of a truncated TP63 protein (TP63-mut). Overexpression of TP63-mut accelerated cell apoptosis. Mechanistically, the TP63-mut protein could bind to the promoter region of CLCA2 and activate the transcription of CLCA2 several times compared to that of the TP63 wild-type protein. Silencing CLCA2 using a specific small interfering RNA (siRNA) or inhibiting the Ataxia Telangiectasia Mutated (ATM) pathway using the KU55933 inhibitor attenuated cell apoptosis caused by TP63-mut protein expression.
CONCLUSIONS: Our findings revealed a crucial role for CLCA2 in mediating apoptosis in POI pathogenesis, and suggested that CLCA2 is a potential therapeutic target for POI.
摘要：
背景：过早卵巢功能不全（POI）是一种导致女性不孕的严重疾病。基因突变是引起POI的重要因素。据报道，TP63截短突变可通过增加生殖细胞凋亡而导致POI，然而，是什么因素介导这种细胞凋亡仍不清楚。
方法：选取北京妇产医院93例POI患者，首都医科大学。对每位患者进行全外显子组测序(WES)。Sanger测序用于确认潜在的致病遗传变异。进行小基因测定以确定TP63变体的剪接效应。构建了TP63截短质粒。实时定量PCR,蛋白质印迹分析，双荧光素酶报告分析，免疫荧光染色，和细胞凋亡测定用于研究TP63截短突变导致POI的潜在机制。
结果：通过93例散发性POI患者的WES，我们在TP63基因的剪接位点上发现了一个14bp的缺失.小基因分析表明，在TP63mRNA剪接过程中，14bp缺失变体导致外显子13跳跃，导致产生截短的TP63蛋白(TP63-mut)。过表达TP63-mut加速细胞凋亡。机械上,与TP63野生型蛋白相比，TP63-mut蛋白可以结合CLCA2的启动子区并激活CLCA2的转录数倍。使用特定的小干扰RNA（siRNA）沉默CLCA2或使用KU55933抑制剂抑制共济失调毛细血管扩张突变（ATM）途径减弱了由TP63-mut蛋白表达引起的细胞凋亡。
结论：我们的发现揭示了CLCA2在POI发病机制中介导细胞凋亡的关键作用，并提示CLCA2是POI的潜在治疗靶点。
方法：选取北京妇产医院93例POI患者，首都医科大学。对每位患者进行全外显子组测序(WES)。Sanger测序用于确认潜在的致病遗传变异。进行小基因测定以确定TP63变体的剪接效应。构建了TP63截短质粒。实时定量PCR,蛋白质印迹分析，双荧光素酶报告分析，免疫荧光染色，和细胞凋亡测定用于研究TP63截短突变导致POI的潜在机制。
结果：通过93例散发性POI患者的WES，我们在TP63基因的剪接位点上发现了一个14bp的缺失.小基因分析表明，在TP63mRNA剪接过程中，14bp缺失变体导致外显子13跳跃，导致产生截短的TP63蛋白(TP63-mut)。过表达TP63-mut加速细胞凋亡。机械上,与TP63野生型蛋白相比，TP63-mut蛋白可以结合CLCA2的启动子区并激活CLCA2的转录数倍。使用特定的小干扰RNA（siRNA）沉默CLCA2或使用KU55933抑制剂抑制共济失调毛细血管扩张突变（ATM）途径减弱了由TP63-mut蛋白表达引起的细胞凋亡。
结论：我们的发现揭示了CLCA2在POI发病机制中介导细胞凋亡的关键作用，并提示CLCA2是POI的潜在治疗靶点。
