Department of Ophthalmology, Nanjing Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, Jiangsu, China.
Institute of Laboratory Medicine, Nanjing Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, Jiangsu, China.
Invest Ophthalmol Vis Sci. 2023 Jun 1;64(7):18. doi: 10.1167/iovs.64.7.18.
To describe a novel spontaneous cataract inbred strain isolated from large-scale breeding SD rats, identify the responsible gene mutation, and understand how this mutation affects lens function.
Exome sequencing of 12 cataract-associated genes was performed in the affected and healthy relatives. Sequences of rat wild-type or mutant gap junction protein alpha 8 gene (Gja8) were transfected into cells. The expression level of protein was assayed by Western blot analysis. Subcellular localization of connexin 50 (Cx50) was analyzed in confocal fluorescent images. Wound-healing, 5-ethynyl-2'-deoxyuridine incorporation, and attachment assay were performed to characterize the cell migration, proliferation and adhesion.
The abnormality was found to be inheritable in an autosomal semi-dominant pattern through different mating patterns. We found a G to T transversion at codon 655 in Gja8, leading to a substitution of valine by phenylalanine (p.V219F). Gja8V219F/+ heterozygotes expressed nuclear cataract while Gja8V219F/V219F homozygotes manifested microphthalmia in addition to cataract. Histology revealed fiber disorders and loss of organelle-free zone in the mutant lens. Cx50V219F altered its location in HeLa cells and inhibited the proliferation, migration and adhesion abilities of HLEB3 cells. The mutation also reduced the expression of focal adhesion kinase and its phosphorylation.
The c.655G>T mutation (p.V219F) is a novel mutation in Gja8, inducing semi-dominant nuclear cataracts in a new spontaneous cataract rat model. The p.V219F mutation altered Cx50 distribution, inhibited lens epithelial cell proliferation, migration, and adhesion, and disrupted fiber cell differentiation. As a consequence, the nuclear cataract and small lens formed.
描述从大规模饲养的 SD 大鼠中分离出的一种新型自发白内障近交系，鉴定其相关基因突变，并了解该突变如何影响晶状体功能。
对 12 种白内障相关基因进行外显子组测序，在受影响和健康的亲属中进行。将野生型或突变的间隙连接蛋白α 8 基因（Gja8）的大鼠序列转染到细胞中。通过 Western blot 分析测定蛋白表达水平。通过共聚焦荧光图像分析连接蛋白 50（Cx50）的亚细胞定位。进行划痕愈合、5-乙炔基-2'-脱氧尿苷掺入和附着试验，以表征细胞迁移、增殖和黏附。
发现该异常通过不同的交配模式以常染色体半显性模式遗传。我们在 Gja8 密码子 655 处发现了 G 到 T 的颠换，导致缬氨酸被苯丙氨酸取代（p.V219F）。Gja8V219F/+杂合子表达核性白内障，而 Gja8V219F/V219F 纯合子除白内障外还表现为小眼球。组织学显示突变晶状体纤维紊乱和细胞器游离区缺失。Cx50V219F 改变了其在 HeLa 细胞中的位置，并抑制了 HLEB3 细胞的增殖、迁移和黏附能力。该突变还降低了粘着斑激酶及其磷酸化的表达。
c.655G>T 突变（p.V219F）是 Gja8 的一种新突变，在一种新的自发性白内障大鼠模型中诱导半显性核性白内障。p.V219F 突变改变了 Cx50 的分布，抑制了晶状体上皮细胞的增殖、迁移和黏附，并破坏了纤维细胞的分化。因此，形成了核性白内障和小晶状体。
