在一个有先天性白内障的中国家庭中鉴定和描述 CRYBA1 中的 p.G91 缺失。

The identification and characterization of the p.G91 deletion in CRYBA1 in a Chinese family with congenital cataracts.

机构信息

Eye Institute, Eye & ENT Hospital of Fudan University, 83 Fenyang Road, Shanghai, 200031, China.

NHC Key Laboratory of Myopia (Fudan University), 83 Fenyang Road, Shanghai, 200031, China.

出版信息

BMC Med Genet. 2019 Sep 5;20(1):153. doi: 10.1186/s12881-019-0882-z.

DOI:10.1186/s12881-019-0882-z

PMID:31488069

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6727356/

Abstract

BACKGROUND

Mutations in more than 52 genes have been identified in isolated congenital cataracts, the majority of which are located in crystalline and connexin (gap junction) genes. An in-frame one amino acid deletion in the beta-crystalline gene CRYBA1 has been reported in several different Chinese, Caucasian and Iranian families of congenital cataracts. Further functional studies are needed to confirm the variant pathogenicity.

METHODS

The purpose of this study is to identify the genetic causes that contribute to congenital cataracts with esotropia and nystagmus in a Chinese family. Whole-exome sequencing was performed on samples from all five family members. The two brothers of the father and their daughters were then enrolled in the study, and 40 suspected variants were sequenced among the 9 subjects using Sanger sequencing. The mRNA and protein levels of CRYBA1 in the lens epithelium from cataract patients and normal controls were compared using quantitative polymerase chain reaction (qPCR) and Western blot analyses. The wild-type and mutated forms (p.G91del) of CRYBA1 cDNA were transfected into two types of cell lines, and the expression level of exogenous CRYBA1 was measured by Western blot analysis. The exogenous CRYBA1 proteins were visualized by immunofluorescence staining.

RESULTS

In this two-generation family, all three descendants inherited congenital cataracts with esotropia and nystagmus from the father, while the mother's lens was normal. After two rounds of sequencing, CRYBA1 (c. 269-271 del, p.G91del) was identified as the mutation responsible for the autosomal dominant congenital cataract in the Chinese family. CRYBA1 showed lower expression in cataract lenses than in control lenses. The deleted form (p.G91del) of CRYBA1 showed lower expression and was more aggregate to the cell membrane than the wild-type CRYBA1.

CONCLUSIONS

We performed molecular experiments to confirm that the p.G91del mutation in CRYBA1 results in abnormal expression and distribution of CRYBA1 protein, and this study could serve as an example of the pathogenicity of an in-frame small deletion in an inherited eye disorder.

摘要

背景

在孤立性先天性白内障中已经鉴定出超过 52 个基因的突变，其中大多数位于晶状体和连接蛋白（缝隙连接）基因中。已经在几个不同的中国、高加索人和伊朗先天性白内障家族中报道了β-晶状体基因 CRYBA1 中的一个框内一个氨基酸缺失。需要进一步的功能研究来确认该变体的致病性。

方法

本研究的目的是鉴定导致一个中国家庭患有伴有内斜视和眼球震颤的先天性白内障的遗传原因。对所有五名家庭成员的样本进行全外显子组测序。然后招募父亲的两个兄弟及其女儿进行研究，在 9 名受试者中使用 Sanger 测序对 40 个疑似变体进行测序。使用定量聚合酶链反应（qPCR）和 Western blot 分析比较白内障患者和正常对照的晶状体上皮中的 CRYBA1 mRNA 和蛋白水平。将 CRYBA1 的野生型和突变型（p.G91del）cDNA 转染到两种细胞系中，并通过 Western blot 分析测量外源性 CRYBA1 的表达水平。通过免疫荧光染色观察外源性 CRYBA1 蛋白。

结果

在这个两代同堂的家庭中，所有三个后代均从父亲那里遗传了伴有内斜视和眼球震颤的先天性白内障，而母亲的晶状体则正常。经过两轮测序，确定 CRYBA1（c.269-271del，p.G91del）是导致这个中国家族常染色体显性先天性白内障的突变。CRYBA1 在白内障晶状体中的表达低于正常晶状体。与野生型 CRYBA1 相比，缺失形式（p.G91del）的 CRYBA1 表达水平更低，并且更容易聚集到细胞膜上。