Abstract：
In this study, we report on two different GJA8 variants related to congenital eye anomalies in two unrelated families, respectively. GJA8 (or Cx50) encoding a transmembrane protein to form lens connexons has been known as a common causative gene in congenital cataracts and its variants have recently been reported related to a wide phenotypic spectrum of eye defects. We identified two GJA8 variants, c.134G>T (p.Try45Leu, W45L) detected in a cataract family by Sanger sequencing and c.281G>A (p.Gly94Glu, G94E) found in a family with severe eye malformations including microphthalmia by whole-exome sequencing. These two variants were absent in healthy population and predicted deleterious by bioinformatic analysis. Furthermore, we compared the expression in cell lines between these mutants and the wildtype to explore their potential mechanism. Cell counting kit-8 assay showed that overexpression of either W45L or G94E decreased cell viability compared with wild-type Cx50 and the control. A lower protein level in W45L found by western blotting and fewer punctate fluorescent signals showed by fluorescence microscopy suggested that W45L may have less protein expression. A higher G94E protein level and abundant dotted distribution indicated that G94E may cause aberrant protein degradation and accumulation. Such results from in vitro assays confirmed the impact of these two variants and gave us a hint about their different pathogenic roles in different phenotypes. In conclusion, our study is the first to have the functional analysis of two GJA8 variants c.134G>T and c.281G>A in Chinese pedigrees and explore the impact of these variants, which can help in prenatal diagnosis and genetic counseling as well in basic studies on GJA8.
摘要：
在这项研究中,我们报道了两个不相关的家族中与先天性眼部异常相关的两种不同的GJA8变异，分别。已知编码跨膜蛋白以形成晶状体连接子的GJA8(或Cx50)是先天性白内障中的常见致病基因,并且最近报道其变体与眼睛缺陷的广泛表型谱有关。我们确定了两种GJA8变体，c.134G>T（p。Try45Leu,W45L）通过Sanger测序在白内障家族中检测到，c.281G>A（p。Gly94Glu,G94E）通过全外显子组测序在一个患有严重眼睛畸形的家庭中发现，包括小眼症。这两种变体在健康人群中不存在，并且通过生物信息学分析预测有害。此外，我们比较了这些突变体和野生型在细胞系中的表达，以探索它们的潜在机制。细胞计数试剂盒-8测定显示,与野生型Cx50和对照相比,W45L或G94E的过表达降低了细胞活力。通过蛋白质印迹发现的W45L中的较低蛋白质水平和通过荧光显微镜显示的较少点状荧光信号表明W45L可能具有较少的蛋白质表达。较高的G94E蛋白水平和丰富的点状分布表明G94E可能导致异常的蛋白降解和积累。来自体外测定的这些结果证实了这两种变体的影响，并为我们提供了关于它们在不同表型中的不同致病作用的提示。总之,我们的研究首次对中国家系中两个GJA8变异c.134G>T和c.281G>A进行功能分析，并探讨这些变异的影响，这可以帮助产前诊断和遗传咨询以及GJA8的基础研究。
