Eye Hospital, First Affiliated Hospital, Harbin Medical University, 23 Youzheng Street, Harbin, 150001, China.
Beijing Institute of Ophthalmology, Beijing Tongren Hospital, Beijing Ophthalmology & Visual Science Key Laboratory, Capital Medical University, Beijing, 100000, China.
Graefes Arch Clin Exp Ophthalmol. 2020 Dec;258(12):2737-2751. doi: 10.1007/s00417-020-04846-x. Epub 2020 Aug 13.
The present study investigated a pathogenic mutation and its mechanism on membranous cataract in a congenital membranous cataract family.
An autosomal dominant four-generation Chinese congenital membranous cataract family was recruited and whole-exome sequencing was performed to screen for sequence variants. Candidate variants were validated using polymerase chain reaction and Sanger sequencing. Wild-type and mutant low-density lipoprotein receptor-related protein 5-like (LRP5L) plasmids were constructed and transfected into human lens epithelial cells (HLE B-3) and human anterior lens capsules. The cell lysates, nuclear and cytoplasmic proteins, and basement membrane components of HLE B-3 cells were harvested. LRP5L and laminin γ1 were knocked down in HLE B-3 cells using specific small-interfering RNA. The protein expression levels of LRP5L, laminin γ1, and c-MAF were detected using immunoblotting and immunofluorescence.
We identified a novel suspected pathogenic mutation in LRP5L (c.107C > G, p.P36R) in the congenital membranous cataract family. This mutation was absent in 300 normal controls and 300 age-related cataract patients. Bioinformatics analysis with PolyPhen-2 and SIFT suggested that LRP5L-P36R was pathogenic. LRP5L upregulated laminin γ1 expression in the cytoplasmic proteins of HLE B-3 cells and human anterior lens capsules, and LRP5L-P36R inhibited the effects of LRP5L. LRP5L upregulated c-MAF expression in the nucleus and cytoplasm of HLE B-3 cells, and LRP5L-P36R inhibited c-MAF expression via inhibition of laminin γ1.
Our study identified a novel gene, LRP5L, associated with congenital membranous cataract, and its mutant LRP5L-P36R contributed to membranous cataract development via inhibition of laminin γ1 and c-MAF.
本研究旨在探讨一个常染色体显性遗传的四代中国人先天性膜性白内障家系的致病突变及其机制。
招募一个常染色体显性遗传的四代中国人先天性膜性白内障家系，进行全外显子组测序以筛选序列变异。使用聚合酶链反应和 Sanger 测序验证候选变异。构建野生型和突变低密度脂蛋白受体相关蛋白 5 样（LRP5L）质粒，并转染人晶状体上皮细胞（HLE B-3）和人前晶状体囊。提取 HLE B-3 细胞的细胞裂解物、核和细胞质蛋白以及基底膜成分。使用特异性小干扰 RNA 敲低 HLE B-3 细胞中的 LRP5L 和层粘连蛋白 γ1。使用免疫印迹和免疫荧光检测 LRP5L、层粘连蛋白 γ1 和 c-MAF 的蛋白表达水平。
我们在先天性膜性白内障家系中发现了 LRP5L 中的一个新的疑似致病突变（c.107C>G，p.P36R）。该突变在 300 名正常对照和 300 名年龄相关性白内障患者中均不存在。PolyPhen-2 和 SIFT 的生物信息学分析表明 LRP5L-P36R 是致病性的。LRP5L 在 HLE B-3 细胞的细胞质蛋白和人前晶状体囊上调层粘连蛋白 γ1 的表达，而 LRP5L-P36R 抑制了 LRP5L 的作用。LRP5L 在 HLE B-3 细胞的核和细胞质中上调 c-MAF 的表达，而 LRP5L-P36R 通过抑制层粘连蛋白 γ1 抑制 c-MAF 的表达。
本研究鉴定了一个与先天性膜性白内障相关的新基因 LRP5L，其突变 LRP5L-P36R 通过抑制层粘连蛋白 γ1 和 c-MAF 导致膜性白内障的发生。
