家族性高血钾高血压致病基因CUL3突变c.1221A>G的发病机制和剪接调控寡核苷酸的治疗干预研究

As we all know that CUL3/KLHL3-WNK-SPAK/OSR1 signaling cascade is important for regulating blood pressure, and mutations in the CUL3、KLHL3 and WNK genes have been identified as the main cause of familial hyperkalemic hypertension (FHH). So far, a total of 17 mutations of CUL3 gene have been described including the novel c.1221A>G recently identified by us. Of note, all CUL3 gene mutations are involved in splicing of exon 9, which finally lead to skipping of exon 9（ΔE9）. Exploration of the pathogenic mechanism of ΔE9 could bring insight to the development of new antihypertensive drugs, however, the precise mechanism of ΔE9 remains elusive, particularly with a lack of study on splicing modulation. Our previous research found that c.1221A>G probably lead to a destruction of an exonic splicing enhancer, which was accompanied with a generation of a splicing silencer. The exon 9 of CUL3 has a weak 3’ acceptor site, meanwhile it has some characteristics of cassette exon (30.4% GC content). Furthermore, strong splicing inhibition motifs located in the intronic sequence adjacent to exon 9 have been found by us. Thus, in the context of a weak exon 9, some specific variants of CUL3 gene could easily led to abnormal splicing of this exon. This project aims to explore the splicing modulation net of CUL3 exon 9 by in vitro/vivo splicing assay and humanized CUL3 knock-in mice. Based on above expected findings, we will make further efforts to investigate the pathogenic mechanism of c.1221A>G to design the targeted splice-switching oligonucleotides and observe their treatment effect on different biological levels.

CUL3/KLHL3-WNK-SPAK/OSR1是机体调节血压的重要通路，CUL3、KLHL3或WNK的突变与家族性高血钾高血压(FHH)有关。CUL3有关突变（共17种），包括我们新发现的c.1221A>G，均无例外地导致9号外显子（E9）的剪接丢失（ΔE9）。揭示ΔE9的发病机制有助开发新的降压药，但机制仍不明确，尤其缺乏剪接调控水平的探讨。我们前期发现c.1221A>G可能导致了外显子剪接增强子破坏并伴随沉默子产生。E9存在较弱的3’剪接位点，具备盒式外显子（GC含量30.4%）的某些特征，且E9邻近的内含子序列存在强力的剪接抑制基序，这些特点决定了ΔE9的易感性。本项目拟从体外、体内剪接实验和人源化基因敲入小鼠模型研究E9的剪接调控网络，在此基础上探讨c.1221A>G的发病机制，设计靶向剪接调控寡核苷酸并在不同水平验证其治疗干预作用。

2型假性醛固酮减少症（又称家族性高血钾高血压）相关的CUL3基因突变均导致不同程度的9号外显子跳跃，但导致这一异常剪切突变的分子致病机制仍然不清楚。本项目的目的是通过探讨2个同义突变c.1221A > G (p. Glu407Glu)和 c.1236G > A (p. Leu412Leu)在RNA剪接调控中的参与机制，借以寻找通过靶向特有的调控序列达到纠正异常剪接，进而达到治疗的目的。本团队通过一系列的RNA pulldown、银染、Western Blotting、RNA干扰、体外表达以及定点突变（单点、非连续双点或连续多点）等技术，首次探索了CUL3基因突变导致9号外显子剪切跳过的内在机制，确定了同义突变c.1221A > G和 c.1236G > A参与疾病的机制主要与产生新的外显子剪切抑制子，以及增强了与异质核核糖核蛋白（hnRNPs）的相互作用有关。而同义突变c.1221A > G和 c.1236G > A参与疾病的机制次要因素则是破坏了外显子剪切增强子有关。另外，我们证实如果额外引入另一个同义突变c.1224A > G则明显纠正c.1221A > G和 c.1236G > A导致的剪切异常，为治疗CUL3基因突变导致的2型假性醛固酮减少症提供了理论基础。