CTNNB1 pathogenic variants analysed in the study.

Germline variants in the CTNNB1 gene, encoding β-catenin protein, cause severe neurodevelopmental alterations manifested early in the infancy, and define the CTNNB1 syndrome. Patients with CTNNB1 syndrome display heterogeneous clinical manifestations, and most of them carry CTNNB1 pathogenic nonsense or frameshift variants that generate premature termination codons (PTC). We have previously described the neuropsychological manifestations of a group of CTNNB1 syndrome patients harboring novel β-catenin variants. Here, we have analysed the molecular and functional characterization of these β-catenin variants, performed genotype-phenotype analyses, and tested for β-catenin functional reconstitution. We describe a complex variety of N-terminal and C-terminal truncated β-catenin proteoforms generated by PTC. Protein stability of truncated proteoforms was variable, as indicated by their expression levels and biophysical analysis, and high protein stability correlated with better patient performance in visuospatial tests. Transcriptional activity was abrogated in most of the β-catenin variants, although some specific truncations, as well as a three-residues in-frame deletion variant, retained partial transcriptional activity. Reconstitution of full-length β-catenin expression and function was achieved in specific β-catenin PTC variants by induction of translational readthrough with aminoglycosides and protein synthesis stimulators. Inhibition of β-catenin degradation by MG-132 proteasome inhibitor also resulted in partial rescue of β-catenin transcriptional activity. Our results suggest the existence of intricate patterns of truncated β-catenin proteoforms in CTNNB1 syndrome patients, which may correlate with clinical manifestations, and provide insights to increase the function of β-catenin in patients carrying CTNNB1 pathogenic variants.

编码β-连环蛋白（β-catenin）的CTNNB1基因生殖系变异，可引发婴幼儿早期即显现的严重神经发育异常，进而定义了CTNNB1综合征。该综合征患者的临床表现具有显著异质性，多数患者携带可产生提前终止密码子（PTC）的CTNNB1致病性无义变异或移码变异。我们此前曾报道过一组携带新型β-连环蛋白变异的CTNNB1综合征患者的神经心理学表现。本研究针对上述β-连环蛋白变异开展了分子与功能特征分析，完成了基因型-表型关联研究，并验证了β-连环蛋白的功能重构效果。本研究揭示了一类由提前终止密码子所产生的、包含多种复杂类型的N端与C端截短型β-连环蛋白蛋白变体。截短型蛋白变体的稳定性存在显著差异，这一点可通过其表达水平与生物物理分析得以印证；且蛋白稳定性越高，患者在视觉空间测试中的表现越佳。大多数β-连环蛋白变异会使其转录活性完全丧失，但部分特定截短变异以及一处三残基框内缺失变异仍可保留部分转录活性。通过氨基糖苷类药物与蛋白质合成刺激剂诱导翻译通读，可在特定携带提前终止密码子的β-连环蛋白变异样本中，实现全长β-连环蛋白的表达与功能重构。通过MG-132蛋白酶体抑制剂抑制β-连环蛋白的降解，同样可部分恢复其转录活性。本研究结果表明，CTNNB1综合征患者体内存在复杂多样的截短型β-连环蛋白蛋白异构体模式，该模式或与临床表型密切相关；本研究同时为提升携带CTNNB1致病性变异患者的β-连环蛋白功能提供了全新思路。