Analyses of a novel L130F missense mutation in FOXC1
Analyses of a novel L130F missense mutation in FOXC1
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DOI：
10.1001/archopht.125.1.128
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发表时间：
2007-01-01
影响因子：
--
通讯作者：
Walter, Michael A.
中科院分区：
文献类型：
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作者：
Ito, Yoko A.;Footz, Tim K.;Walter, Michael A.
目的：了解在2例Axenfeld-Rieger综合征患者中发现的新型L130F突变如何破坏叉头盒C1蛋白（FOXC1）的功能。方法：对Axenfeld-Rieger综合征患者的DNA进行测序，发现了一种新的错义突变，导致FOXC1基因的L130F替换。利用定点诱变将L130F突变引入FOXC1互补DNA。免疫印迹法检测L130F蛋白表达水平。我们确定了突变蛋白定位到细胞核、结合DNA和反激活报告基因结构的能力。结果：FOXC1 L130F突变蛋白的表达水平与野生型FOXC1相似。然而，与野生型蛋白相比，L130F蛋白以明显降低的分子量迁移，这表明突变蛋白和野生型蛋白可能被不同的磷酸化。L130F蛋白定位细胞核、结合DNA和反激活报告基因的能力也明显受损。结论：L130F致病突变进一步证明叉头结构域螺旋3对FOXC1蛋白正确定位到细胞核，结合DNA，激活基因表达具有重要作用。临床相关性：由于L130F突变导致FOXC1无法发挥功能，这为FOXC1基因的破坏如何导致人类Axenfeld-Rieger综合征提供了进一步的见解。
Objective: To understand how the novel L130F mutation, found in 2 patients with Axenfeld-Rieger syndrome, disrupts function of the forkhead box C1 protein (FOXC1).Methods: Sequencing DNA from patients with Axenfeld-Rieger syndrome identified a novel missense mutation that results in an L130F substitution in the FOXC1 gene. Site-directed mutagenesis was used to introduce the L130F mutation into the FOXC1 complementary DNA. The level of L130F protein expression was determined by means of immunoblotting. We determined the mutant protein's ability to localize to the nucleus, bind DNA, and transactivate a reporter construct.Results: The FOXC1 L130F mutant protein is expressed at levels similar to those of wild-type FOXC1. The L130F protein, however, migrated at an apparent reduced molecular weight compared with the wild-type protein, suggesting that the mutant and wild-type proteins may be differentially phosphorylated. The L130F protein also had a significantly impaired capacity to localize to the nucleus, bind DNA, and transactivate reporter genes.Conclusions: The disease-causing L130F mutation further demonstrates that helix 3 of the forkhead domain is important for the FOXC1 protein to properly localize to the nucleus, bind DNA, and activate gene expression.Clinical Relevance: The inability of FOXC1 to function owing to the L130F mutation provides further insight into how disruptions in the FOXC1 gene lead to human Axenfeld-Rieger syndrome.