Chung Doug D, Frausto Ricardo F, Lin Benjamin R, Hanser Evelyn M, Cohen Zack, Aldave Anthony J
Stein Eye Institute, David Geffen School of Medicine at University of California-Los Angeles, Los Angeles, California, United States.
Invest Ophthalmol Vis Sci. 2017 Jun 1;58(7):3202-3214. doi: 10.1167/iovs.17-21423.
To investigate the molecular basis of posterior polymorphous corneal dystrophy (PPCD) by examining the PPCD transcriptome and the effect of decreased ZEB1 expression on corneal endothelial cell (CEnC) gene expression.
Next-generation RNA sequencing (RNA-seq) analyses of corneal endothelium from two PPCD-affected individuals (one with PPCD3 and one of unknown genetic cause) compared with two age-matched controls, and primary human CEnC (pHCEnC) transfected with siRNA-mediated ZEB1 knockdown. The expression of selected differentially expressed genes was validated by quantitative polymerase chain reaction (qPCR) and/or assessed by in situ hybridization in the corneal endothelium of four independent cases of PPCD (one with PPCD3 and three of unknown genetic cause).
Expression of 16% and 46% of the 104 protein-coding genes specific to ex vivo corneal endothelium was lost in the endothelium of two individuals with PPCD. Thirty-two genes associated with ZEB1 and 3 genes (BMP4, CCND1, ZEB1) associated with OVOL2 were differentially expressed in the same direction in both individuals with PPCD. Immunohistochemistry staining and RNA-seq analyses demonstrated variable expression of type IV collagens in PPCD corneas. Decreasing ZEB1 expression in pHCEnC altered expression of 711 protein-coding genes, many of which are associated with canonical pathways regulating various cellular processes.
Identification of the altered transcriptome in PPCD and in a cell-based model of PPCD provided insight into the molecular alterations characterizing PPCD. Further study of the differentially expressed genes associated with ZEB1 and OVOL2 is expected to identify candidate genes for individuals with PPCD and without a ZEB1 or OVOL2 mutation.
通过检测后部多形性角膜营养不良（PPCD）的转录组以及ZEB1表达降低对角膜内皮细胞（CEnC）基因表达的影响，来研究PPCD的分子基础。
对两名受PPCD影响的个体（一名患有PPCD3，另一名遗传病因不明）的角膜内皮进行下一代RNA测序（RNA-seq）分析，并与两名年龄匹配的对照进行比较，同时对用小干扰RNA（siRNA）介导的ZEB1敲低处理的原代人CEnC（pHCEnC）进行分析。通过定量聚合酶链反应（qPCR）验证所选差异表达基因的表达，并/或在4例独立的PPCD病例（1例患有PPCD3，3例遗传病因不明）的角膜内皮中通过原位杂交进行评估。
在两名PPCD患者的内皮中，离体角膜内皮特有的104个蛋白质编码基因中分别有16%和46%的表达缺失。在两名PPCD患者中，与ZEB1相关的32个基因和与OVOL2相关的3个基因（BMP4、CCND1、ZEB1）在相同方向上差异表达。免疫组织化学染色和RNA-seq分析显示PPCD角膜中IV型胶原表达存在差异。降低pHCEnC中ZEB1的表达会改变711个蛋白质编码基因的表达，其中许多基因与调节各种细胞过程的经典通路相关。
PPCD及其细胞模型中改变的转录组的鉴定为PPCD的分子改变提供了见解。对与ZEB1和OVOL2相关的差异表达基因的进一步研究有望为没有ZEB1或OVOL2突变的PPCD患者鉴定候选基因。
