Ye Yang, Wu Menghan, Qiao Yue, Xie Tingting, Yu Yinhui, Yao Ke
Department of Eye Center, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China.
Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, China.
Ophthalmic Genet. 2019 Oct;40(5):428-435. doi: 10.1080/13816810.2019.1675179. Epub 2019 Oct 16.
: Congenital cataract is a significant cause of visual impairment and blindness. The present study examined the disease-causing mutations in three Chinese families with autosomal dominant congenital cataract (ADCC) to provide the preliminary evidence of the mechanisms underlying congenital cataract formation.: Three pedigrees affected with ADCC were recruited. All participants underwent detailed ophthalmic examinations. Leucocyte DNA was extracted from venous blood for direct sequencing of candidate genes. In silico bioinformatics analysis was conducted to verify the functional impacts of the mutant proteins. Distribution patterns of connexin proteins were assessed through fluorescence microscopy using an enhanced green fluorescent protein (EGFP)-labeled expression vector in stably transfected Hek293 cells.: We identified three Chinese pedigrees with ADCC. Family 1 and family 2 presented with pulverized cataract and family 3 with an unknown phenotype. Direct sequencing of family 1 and family 2 revealed a missense mutation of c.64G>A encoding for G22S of connexin46 (Cx46), while a similar c.64G>A encoding for G22S of connexin50 (Cx50) was found in family 3; both mutations co-segregated well within all affected individuals in their families and were absent from 100 unrelated controls. Bioinformatics analysis revealed with high confidence that both mutations were deleterious. Confocal microscopy revealed the accumulation of both mutant connexins in the cytoplasm with punctate staining and a failure of gap junction formation between adjacent cells.: Two novel G22S mutations of Cx46 and Cx50 were identified, and preliminary functional analysis revealed a potential deleterious effect of these mutations due to the malfunction of connexins. ADCC: autosomal dominant congenital cataract; Cx26: connexin26; Cx32: connexin32; Cx46: connexin46; Cx46WT: wild-type connexin46; Cx50: Connexin50; Cx50WT: wild-type connexin50; DAPI: 4',6-diamidino-2-phenylindole; EGFP: enhanced green fluorescent protein; FBS: fetal bovine serum; :gap junction alpha-; PCR: polymerase chain reaction; PolyPhen: polymorphism phenotyping; PSIC: position-specific independent count; RPMI: Roswell Park Memorial Institute; TM1: first transmembrane.
先天性白内障是导致视力损害和失明的重要原因。本研究检测了三个常染色体显性遗传性先天性白内障（ADCC）中国家系中的致病突变，以提供先天性白内障形成机制的初步证据。
招募了三个患有ADCC的家系。所有参与者均接受了详细的眼科检查。从静脉血中提取白细胞DNA，用于对候选基因进行直接测序。进行了计算机生物信息学分析，以验证突变蛋白的功能影响。通过荧光显微镜，在稳定转染的Hek293细胞中使用增强型绿色荧光蛋白（EGFP）标记的表达载体，评估连接蛋白的分布模式。
我们鉴定出三个患有ADCC的中国家系。家系1和家系2表现为粉末状白内障，家系3表现为未知表型。对家系1和家系2的直接测序显示，编码连接蛋白46（Cx46）的G22S的c.64G>A错义突变，而在家族3中发现了编码连接蛋白50（Cx50）的G22S的类似c.64G>A突变；这两种突变在其家系中的所有受影响个体中均表现出良好的共分离，且在100名无关对照中未出现。生物信息学分析高度确信这两种突变均有害。共聚焦显微镜显示，两种突变连接蛋白在细胞质中积累，呈点状染色，相邻细胞之间未能形成间隙连接。
鉴定出了Cx46和Cx50的两种新型G22S突变，初步功能分析显示，由于连接蛋白功能异常，这些突变可能具有有害作用。ADCC：常染色体显性遗传性先天性白内障；Cx26：连接蛋白26；Cx32：连接蛋白32；Cx46：连接蛋白46；Cx46WT：野生型连接蛋白46；Cx50：连接蛋白50；Cx50WT：野生型连接蛋白50；DAPI：4',6-二脒基-2-苯基吲哚；EGFP：增强型绿色荧光蛋白；FBS：胎牛血清；间隙连接α；PCR：聚合酶链反应；PolyPhen：多态性表型分析；PSIC：位置特异性独立计数；RPMI：罗斯威尔公园纪念研究所；TM1：第一跨膜区
