Central Laboratory, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing Maternal and Child Health Care Hospital, Beijing, 100006, China.
Department of Gynecology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing Maternal and Child Health Care Hospital, Beijing, 100026, China.
J Ovarian Res. 2024 Mar 25;17(1):67. doi: 10.1186/s13048-024-01396-2.
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.
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.
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.
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.
卵巢早衰（POI）是导致女性不孕的严重疾病。遗传突变是导致 POI 的重要因素。TP63 截断突变已被报道通过增加生殖细胞凋亡导致 POI，然而介导这种凋亡的因素尚不清楚。
从首都医科大学北京妇产医院招募了 93 名 POI 患者。对每位患者进行全外显子组测序（WES）。使用 Sanger 测序证实潜在的致病遗传变异。进行微基因检测以确定 TP63 变异的剪接效应。构建了一个 TP63 截断质粒。使用实时定量 PCR、western blot 分析、双荧光素酶报告基因检测、免疫荧光染色和细胞凋亡检测来研究 TP63 截断突变导致 POI 的潜在机制。
通过对 93 名散发性 POI 患者的 WES 分析，我们发现了一个 14 个碱基对的缺失，覆盖了 TP63 基因的剪接位点。微基因检测表明，14 个碱基对缺失变体导致 TP63 mRNA 剪接时外显子 13 跳过，产生截断的 TP63 蛋白（TP63-mut）。TP63-mut 的过表达加速了细胞凋亡。机制上，TP63-mut 蛋白可以结合 CLCA2 的启动子区域，并激活 CLCA2 的转录，其转录活性比 TP63 野生型蛋白高几倍。使用特异性小干扰 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）通路，可减弱由 TP63-mut 蛋白表达引起的细胞凋亡。
我们的研究结果揭示了 CLCA2 在介导 POI 发病机制中的细胞凋亡中的关键作用，并表明 CLCA2 是 POI 的潜在治疗靶点。
