Molecular analysis of a human PAX6 homeobox mutant
Molecular analysis of a human PAX6 homeobox mutant
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DOI：
10.1038/sj.ejhg.5201579
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发表时间：
2006-06-01
影响因子：
5.2
通讯作者：
Damante, Giuseppe
中科院分区：
文献类型：
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作者：
D'Elia, Angela Valentina;Puppin, Cinzia;Damante, Giuseppe
Pax6 控制眼睛、胰腺和大脑的形态发生。在人类中，杂合 PAX6 突变会导致无虹膜和各种其他先天性眼睛异常。最常见的 PAX6 错义突变位于配对结构域 (PD) 中，而在同源结构域 (HD) 中发现的错义突变极少。在本报告中，我们描述了人类 PAX6 R242T 错义突变的分子分析，该突变位于 HD 的第二螺旋。在一名左眼部分无虹膜的男孩中发现了这种情况，表现为假性缺损。凝胶延迟测定表明突变型HD 与野生型HD 一样能结合DNA。此外，突变不会改变 PD 的 DNA 结合特性。细胞转染测定表明全长突变蛋白的稳态水平高于野生型蛋白。在共转染测定中，突变蛋白比野生型蛋白更高程度地激活PAX6响应启动子。体外有限的蛋白水解测定表明，突变的存在降低了对胰蛋白酶消化的敏感性。因此，我们认为 R242T 人类表型可能是由于 PAX6 蛋白的异常增加所致，这与报道的眼睛表型对增加 PAX6 剂量的敏感性一致。
Pax6 controls eye, pancreas and brain morphogenesis. In humans, heterozygous PAX6 mutations cause aniridia and various other congenital eye abnormalities. Most frequent PAX6 missense mutations are located in the paired domain (PD), while very few missense mutations have been identified in the homeodomain (HD). In the present report, we describe a molecular analysis of the human PAX6 R242T missense mutation, which is located in the second helix of the HD. It was identified in a male child with partial aniridia in the left eye, presenting as a pseudo- coloboma. Gel-retardation assays revealed that the mutant HD binds DNA as well as the wild- type HD. In addition, the mutation does not modify the DNA-binding properties of the PD. Cell transfection assays indicated that the steady-state levels of the full length mutant protein are higher than those of the wild- type one. In cotransfection assays a PAX6 responsive promoter is activated to a higher extent by the mutant protein than by the wild- type protein. In vitro limited proteolysis assays indicated that the presence of the mutation reduces the sensitivity to trypsin digestion. Thus, we suggest that the R242T human phenotype could be due to abnormal increase of PAX6 protein, in keeping with the reported sensitivity of the eye phenotype to increased PAX6 dosage.