Song Zixun, Wang Lianqing, Liu Yaping, Xiao Wei
Department of Ophthalmology, Shengjing Hospital of China Medical University, Shenyang, Liaoning, 110004, China.
Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, P. R. China.
PLoS One. 2015 Mar 24;10(3):e0119296. doi: 10.1371/journal.pone.0119296. eCollection 2015.
To detect the causative mutation for congenital posterior polar cataracts in a five-generation Chinese family and further explore the potential pathogenesis of this disease.
Coding exons, with flanking sequences of five candidate genes, were screened using direct DNA sequencing. The identified mutations were confirmed by restriction fragment length polymorphism (RFLP) analysis. A full-length wild-type or an Y219* mutant aquaporin0 (AQP0) fused with an N-terminal FLAG tag, was transfected into HEK293T cells. For co-localization studies, FLAG-WT-AQP0 and Myc-Y219*-AQP0 constructs were co-transfected. Quantitative real-time RT-PCR, western blotting and immunofluorescence studies were performed to determine protein expression levels and sub-cellular localization, respectively.
We identified a novel nonsense mutation in MIP (c.657 C>G; p.Y219*) (major intrinsic protein gene) that segregates with congenital posterior polar cataract in a Chinese family. This mutation altered a highly conserved tyrosine to a stop codon (Y219*) within AQP0.When FLAG-WT-AQP0 and FLAG-Y219*-AQP0 expression constructs were singly transfected into HEK 293T cells, mRNA expression showed no significant difference between the wild-type and the mutant, while Y219*-AQP0 protein expression was significantly lower than that of wild-type AQP0. Wild-type AQP0 predominantly localized to the plasma membrane, while the mutated protein was abundant within the cytoplasm of HEK293T cells. However, when FLAG-WT-AQP0 andMyc-MU-AQP0were co-expressed, both proteins showed high fluorescence in the cytoplasm.
The novel nonsense mutation in the MIP gene (c.657 C>G) identified in a Chinese family may cause posterior polar cataracts. The dominant negative effect of the mutated protein on the wild-type protein interfered with the trafficking of wild-type protein to the cell membrane and both the mutant and wild-type protein were trapped in the cytoplasm. Consequently, both wild-type and mutant protein lost their function as a water channel on the cell membrane, and may result in a cataract phenotype. Our data also expands the spectrum of known MIP mutations.
检测一个五代中国家系中先天性后极性白内障的致病突变，并进一步探索该疾病的潜在发病机制。
使用直接DNA测序法筛选五个候选基因的编码外显子及其侧翼序列。通过限制性片段长度多态性（RFLP）分析确认所鉴定的突变。将全长野生型或与N端FLAG标签融合的Y219突变型水通道蛋白0（AQP0）转染到HEK293T细胞中。为了进行共定位研究，将FLAG-WT-AQP0和Myc-Y219-AQP0构建体共转染。分别进行定量实时RT-PCR、蛋白质印迹和免疫荧光研究以确定蛋白质表达水平和亚细胞定位。
我们在中国家系中鉴定出MIP（c.657 C>G；p.Y219*）（主要内在蛋白基因）中的一个新的无义突变，该突变与先天性后极性白内障共分离。此突变将AQP0内一个高度保守的酪氨酸改变为终止密码子（Y219*）。当将FLAG-WT-AQP0和FLAG-Y219*-AQP0表达构建体单独转染到HEK 293T细胞中时，野生型和突变型之间的mRNA表达无显著差异，而Y219*-AQP0蛋白表达明显低于野生型AQP0。野生型AQP0主要定位于质膜，而突变蛋白在HEK293T细胞的细胞质中大量存在。然而，当FLAG-WT-AQP0和Myc-MU-AQP0共表达时，两种蛋白在细胞质中均显示出高荧光。
在中国家系中鉴定出的MIP基因新的无义突变（c.657 C>G）可能导致后极性白内障。突变蛋白对野生型蛋白的显性负效应干扰了野生型蛋白向细胞膜的运输，突变型和野生型蛋白都被困在细胞质中。因此，野生型和突变型蛋白都失去了其在细胞膜上作为水通道的功能，并可能导致白内障表型。我们的数据也扩展了已知的MIP突变谱。
