Department of Ophthalmology and Visual Science, Eye and ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
NHC Key Laboratory of Myopia, Chinese Academy of Medical Sciences, Shanghai Key Laboratory of Visual Impairment and Restoration (Fudan University), Shanghai, China.
Mol Vis. 2024 Jun 26;30:260-267. eCollection 2024.
Congenital cataract is an important cause of visual impairment in childhood. Our previous study reported that the c.110G>C (p.R36P) mutation in the γD-crystallin gene (CRYGD) was associated with congenital cataract in a Chinese family. This study aimed to investigate the potential underlying mechanism through which the p.R36P mutation leads to congenital cataract.
Plasmids encoding wide-type human γD-crystallin and the mutant R36P γD-crystallin were transfected into HEK293T and SRA01/04 cells. Protein expression levels, including total, soluble, and insoluble fractions, were quantified by Western blotting. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to assess the mRNA expression of other crystallin genes. Cell viability and apoptosis were evaluated using the CCK-8 assay and flow cytometry, respectively.
The total protein, especially the soluble fraction, was significantly reduced in the R36P mutant, while the insoluble part remained unaffected. The decrease of soluble R36P γD-crystallin could not be rescued by the proteinase inhibitor MG132. The mRNA expression of the R36P mutation was lower, but other crystallin RNAs were unchanged. Cell viability was slightly decreased (11%, p<0.05), and cell apoptosis was not significantly increased (12%, p=0.31).
The significant decrease in soluble R36P γD-crystallin may represent a novel mechanism underlying congenital cataract caused by CRYGD gene mutation.
先天性白内障是儿童视力损害的一个重要原因。我们之前的研究报道，γD-晶体蛋白（CRYGD）基因中的 c.110G>C（p.R36P）突变与一个中国家族的先天性白内障有关。本研究旨在探讨该 p.R36P 突变导致先天性白内障的潜在机制。
将编码野生型人 γD-晶体蛋白和突变型 R36P γD-晶体蛋白的质粒转染到 HEK293T 和 SRA01/04 细胞中。通过 Western 印迹定量分析总蛋白、可溶性和不溶性分数的蛋白表达水平。使用定量逆转录聚合酶链反应（RT-PCR）评估其他晶体蛋白基因的 mRNA 表达。分别用 CCK-8 测定法和流式细胞术评估细胞活力和细胞凋亡。
与野生型相比，R36P 突变体的总蛋白，特别是可溶性部分显著减少，而不溶性部分不受影响。溶菌酶抑制剂 MG132 不能挽救可溶性 R36P γD-晶体蛋白的减少。R36P 突变的 mRNA 表达较低，但其他晶体素 RNA 不变。细胞活力略有下降（11%，p<0.05），细胞凋亡没有明显增加（12%，p=0.31）。
可溶性 R36P γD-晶体蛋白的显著减少可能代表 CRYGD 基因突变导致先天性白内障的一种新机制。
