K蛋白介导SHH远距离调控子（ZRS）新突变导致先天性I型轴前多指的机制研究

Preaxial polydactyly (PPD) is a common congenital hand malformation and is classified into four types as PPD-I, II, III, IV. However, no genes have been identified as the isolated PPD-I. In our previous studies, we recruited a rare four-generation Chinese Han PPD-I pedigree. By genome-wide genotyping and linkage analysis, we identified that a novel point mutation within the zone of polarizing activity regulatory sequence (ZRS) region in LMBR1 gene was responsible for the isolated PPD-I. It has been reported that ZRS mutations can cause ectopic expression of its target gene Sonic hedgehog (SHH ), leading to ectopic digits, but the molecular mechanisms is unclear. We hypothesize that the ZRS novel mutation might change some transcription factors (TFs) affinities to its binding sites and activate SHH abnormal expression in the limb bud. Pre-experiment showed that the novel point mutants enhanced the binding potential to the protein K. In order to further elucidate the pathogenesis of PPD-I, we will use Caco-2 cells to perform the DNA-pull down analysis, protein mass spectrometry analysis, the dual luciferase reporter gene assay, and zebrafish model genome-edited by the CRISPR/Cas9 system in this project. Not only a novel causative gene has been found for PPD-I, but also the specific pathogenesis will be further investigated. This will certainly provide theoretical foundation for disease prevention and gene therapy of PPD-I.

轴前多指/趾（Preaxial polydactyly，PPD)是常见先天性肢体畸形，分四种亚型，I型轴前多指(PPD-I)的致病基因及机制不明。本课题针对一个汉族四代PPD-I家系，利用全基因组基因分型和连锁分析，定位出LMBR1基因中ZRS区（Sonic hedgehog，SHH远距离调控子)新发突变是PPD-I的致病突变。研究表明，ZRS突变导致SHH异常表达产生其它多指亚型、机制不明。我们提出转录调控可能涉及ZRS突变导致SHH异常表达的科研假设，预实验证明该点突变造成ZRS区与转录因子K蛋白亲和力显著增加，本项目拟利用表达SHH相关转录因子的Caco-2细胞模型、CRISPR/Cas9系统建立的斑马鱼模型，应用DNA-pull down、质谱分析、双荧光素酶报告基因检测等技术，研究K蛋白如何介导ZRS突变调控SHH异常表达导致PPD-I发生的分子机制，阐明PPD-I的致病机理。

轴前多指/趾（Preaxial polydactyly，PPD)是常见先天性肢体畸形，分四种亚型，I型轴前多指(PPD-I)的致病基因及机制不明。本课题针对一个汉族四代PPD-I家系，利用全基因组基因分型和连锁分析，定位出LMBR1基因中ZRS区（Sonic hedgehog，SHH远距离调控子)新发突变g.101779T>A 是PPD-I的致病突变。研究表明，ZRS突变导致SHH异常表达产生其它多指亚型、机制不明。我们的研究表明该ZRS点突变造成ZRS区与核不均一核糖核蛋白K亲和力显著增加，进一步促进SHH异常表达。ZRS点突变小鼠成功模拟了PPD-I表型，并且E11.5胎鼠肢芽前轴出现SHH的异位和过量表达。本项目利用表达SHH相关转录因子的Caco-2细胞模型、CRISPR/Cas9系统建立的小鼠模型，应用DNA-pull down、质谱分析、双荧光素酶报告基因检测等技术，揭示了Hnrnp K介导ZRS突变调控SHH异常表达导致PPD-I发生的分子机制，阐明PPD-I的致病机理。