Identification and preliminary functional analysis of two novel congenital cataract associated mutations of Cx46 and Cx50
Identification and preliminary functional analysis of two novel congenital cataract associated mutations of Cx46 and Cx50
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Cx46和Cx50两种新型先天性白内障相关突变的鉴定和初步功能分析
DOI：
10.1080/13816810.2019.1675179
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发表时间：
2019-10-13
影响因子：
1.2
通讯作者：
Yao，Ke
中科院分区：
文献类型：
--
作者：
Ye，Yang;Wu，Menghan;Yao，Ke
摘要背景：先天性白内障是导致视力损害和致盲的重要原因。本研究检测了三个常染色体显性遗传性先天性白内障(ADCC)家系的致病基因突变，为先天性白内障的发病机制提供初步证据。方法：收集3个ADCC家系受试者。所有参与者都接受了详细的眼科检查。从静脉血中提取白细胞DNA，用于候选基因的直接测序。在计算机生物信息学分析中，验证了突变蛋白的功能影响。使用增强型绿色荧光蛋白(EGFP)标记的表达载体，通过荧光显微镜观察连接蛋白在稳定表达的Hek293细胞中的分布模式。结果：我们鉴定了3个中国人ADCC家系。家系1和家系2表现为粉末状白内障，家系3表现型不明。对家系1和家系2的直接测序发现了连接蛋白46(Cx46)G22S编码基因c.64G>；A的错义突变，而在家系3中发现了一个类似的连接蛋白50(CX50)G22S编码的c.64G>；A错义突变；这两个突变在他们的家族中所有患病个体中都很好地共分离，而在100名无关对照中都不存在。生物信息学分析显示，这两个突变都是有害的。共聚焦显微镜显示两种突变型连接蛋白均在细胞质中聚集，呈点状染色，相邻细胞之间的缝隙连接形成失败。结论：发现了Cx46和CX50两个新的G22S突变，初步的功能分析显示这些突变可能是连接蛋白功能障碍所致。缩写：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：4ʹ，6-二氨基-2-苯基吲哚；绿色荧光蛋白：增强型绿色荧光蛋白；胎牛血清；GJA-：缝隙连接α-；聚合酶链式反应：聚合酶链式反应；Polyphen：多态表型；PSIC：位置特异性计数；RPMI：罗斯韦尔公园纪念研究所；TM1：第一跨膜。
ABSTRACT Background: 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. Methods: 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. Results: 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. Conclusions: 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. Abbreviations: ADCC: autosomal dominant congenital cataract; Cx26: connexin26; Cx32: connexin32; Cx46: connexin46; Cx46WT: wild-type connexin46; Cx50: Connexin50; Cx50WT: wild-type connexin50; DAPI: 4ʹ,6-diamidino-2-phenylindole; EGFP: enhanced green fluorescent protein; FBS: fetal bovine serum; GJA-:gap junction alpha-; PCR: polymerase chain reaction; PolyPhen: polymorphism phenotyping; PSIC: position-specific independent count; RPMI: Roswell Park Memorial Institute; TM1: first transmembrane.