Zhang Li-Yun, Yam Gary Hin-Fai, Fan Dorothy Shu-Ping, Tam Pancy Oi-Sin, Lam Dennis Shun-Chiu, Pang Chi-Pui
Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong, China.
Mol Vis. 2007 Nov 7;13:2096-104.
To investigate a novel deletion variant of gammaD-crystallin (CRYGD) identified in a Chinese family with nuclear congenital cataract.
A Chinese family with five affected members diagnosed with nuclear cataract and four unaffected members were recruited for the mutational screening of 15 known candidate genes for autosomal dominant congenital cataract. Two-point linkage analysis with single nucleotide polymorphism markers and microsatellite markers flanking these genes together with direct sequencing was applied to identify the disease-causing mutation. Recombinant NH(2)-terminal FLAG-tagged wildtype or mutant gammaD-crystallin was expressed in COS-7 cells. The expression pattern, protein solubility and intracellular distribution were analyzed by western blotting and confocal double immunofluorescence.
Linkage analysis located the candidate region in the gammaC-crystallin and gammaD-crystallin gene cluster. Direct sequencing identified a c.494delG in CRYGD, which cosegregated with the disease in all affected members. Neither the unaffected family members nor the 103 unrelated controls carried this deletion mutation, which causes a frameshift and an early termination of polypeptide to become G165fs. A significantly reduced solubility was observed for this mutant. Unlike wildtype gammaD-crystallin, which existed in both the nucleus and cytoplasm, G165fs was colocalized with lamin A/C on the nuclear envelope.
We have identified a novel mutation, c.494delG, in CRYGD, which was associated with nuclear cataract. This is the first deletion mutation of CRYGD found to cause autosomal dominant congenital cataract. The mutant protein with loss of solubility and localization to the nuclear envelope is hypothesized to impair nuclear transfiguration and degradation in lens fiber cell differentiation, leading to opacity formation during lens development.
研究在中国一个患有核性先天性白内障的家系中鉴定出的γD-晶状体蛋白（CRYGD）的一种新型缺失变异体。
招募了一个有5名被诊断患有核性白内障的患病成员和4名未患病成员的中国家系，对15个已知的常染色体显性先天性白内障候选基因进行突变筛查。应用与这些基因侧翼的单核苷酸多态性标记和微卫星标记进行两点连锁分析以及直接测序来鉴定致病突变。重组的带有NH(2)-末端FLAG标签的野生型或突变型γD-晶状体蛋白在COS-7细胞中表达。通过蛋白质印迹和共聚焦双免疫荧光分析表达模式、蛋白质溶解性和细胞内分布。
连锁分析将候选区域定位在γC-晶状体蛋白和γD-晶状体蛋白基因簇中。直接测序在CRYGD中鉴定出一个c.494delG，其在所有患病成员中与疾病共分离。未患病的家庭成员和103名无关对照均未携带这种缺失突变，该突变导致移码并使多肽提前终止为G165fs。观察到该突变体的溶解性显著降低。与存在于细胞核和细胞质中的野生型γD-晶状体蛋白不同，G165fs与核纤层蛋白A/C在核膜上共定位。
我们在CRYGD中鉴定出一种新型突变c.494delG，其与核性白内障相关。这是首次发现的CRYGD缺失突变导致常染色体显性先天性白内障。推测溶解性丧失且定位于核膜的突变蛋白会损害晶状体纤维细胞分化过程中的核变形和降解，导致晶状体发育过程中形成混浊。
