一汉族常染色体显性遗传视网膜色素变性家系视紫红质基因检测分析

Detection and analysis of the Rhodopsin gene in a consanguineous Chinese Han autosomal dominant retinitis pigmentosa family

背景视紫红质（RHO）基因是常染色体显性遗传视网膜色素变性（adRP）最常见的致病基因，错误折叠的RHO突变型蛋白积聚在内质网从而引起内质网应激（ERS）是其引起视网膜色素变性（RP）的主要发病机制之一。目的检测一汉族adRP家系的致病突变基因，探讨其遗传学基础。方法对参与研究的所有家系成员进行详细的眼科检查。利用新一代测序技术（NGS）对该家系进行189个遗传性视网膜疾病相关基因的目标区域捕获测序，数据经分析滤过，候选突变进行Sanger测序验证和致病性评估。构建视紫红质一增强型绿色荧光蛋白质粒（RHO—pEGFP），包括RHO-pEGFP-N1野生型和RHO-pEGFP-N1突变型质粒，转染人视网膜色素上皮（RPE）细胞系（ARPEl9）和人肾胚293细胞系（HEK293）培养24h，用细胞免疫荧光法观察野生型和突变型RHO—GFP融合蛋白在细胞内的定位；采用荧光定量PCR（Q—PCR）及Westernblot法检测ERS关键因子X盒结合蛋白-1（XBPl）在细胞中的表达。结果该汉族家系共5代，遗传模式符合常染色体显性遗传，家系中患病者眼底特征为RP。NGS数据经分析过滤后获得惟一可能致病突变RHOP．P53R，Sanger测序验证该错义突变在该家系中呈共分离，现存的11例患者均呈杂合子。野生型RHO—GFP融合蛋白呈绿色荧光，分布于呈红色荧光的内质网及细胞膜，而突变型RHO—GFP融合蛋白仅积聚在内质网。与转染RHO-pEGFP—N1野生型的细胞相比，转染RHOp5-pEGFP—N1突变型的HEK293细胞中XBPl表达上调了（1．28±O．09）倍。表达突变型蛋白的HEK293细胞内XBPlmRNA中被特异性地剪切了26个碱基的内含子，转染突变型蛋白的HEK293细胞中XBPl蛋白水平表达明显高于转染野生型、转染空pEGFP—N1质粒细胞及未转染细胞。结论RHO p．P53R是该家系的致病突变基因，该突变型RHO蛋白不能有效地从内质网向细胞膜转运，蓄积在内质网，引起蛋白折叠错误，发生ERS。

Background Rhodopsin （RHO） gene is the most common disease gene for autosomal dominant retinitis pigmentosa （adRP） ,one of the main pathogenesis is that misfolded mutant RHO proteins accumulate in the endoplasmic retieulum and cause endoplasmic reticulum stress （ERS）. Objective This study aimed to determine the genetic basis for a consanguineous Chinese Han adRP family. Methods This study procedure complied with Helsinki Declaration. All participants in the family were investigated under the informed consent. Regular ocular examination was performed on the patients in this family. Next-generation sequencing （NGS） was carried out to screen the mutations in 189 genes associated with hereditary retinal diseases （HRDs）. After being analyzed and filtered, variations detected by NGS were validated by Sanger sequencing and evaluating of pathogenicity. The wild-type RHOWT and mutant RHO Rwere cloned into the vector pEGFP-N1. Then the two plasmids were transfected into adult retinal pigmentosa epithelium cell line（ARPE19） and human embryo kidney 293 line （HEK293） to observe the location of rhodopsin-GFP fusion protein in cells,and the expression of ERS related protein XBPI in the ceils was detected by quantitative-PCR and Western blot. Results This family included 5 generations with the typical adRP characteristics. Genetic analysis identified a heterozygous variation, p. P53R in RHO gene, which was fully cosegregated in the family. Wild-type RHOWr-GFP fusion proteins showed the green fluorescence on the endoplasmic reticulum and cytomembrane,but the misfolded mutant RHO-GFP fusion protein gathered only in endoplasmic reticulum. Compared to wild-type RHO,thc XBP1 was activated and increased by （ 1.28_＋0. 09） fold. Tbe introns of 26 bases in XBPI mRNA were removed in the HEK293 cells with mutant RHO-GFP fusion protein, and the expression of XBP1 was stronger in the HEK293 cells with mutant RHO-GFP than that in HEK293 cells with wild type RHO-GFP and cells with blank pEGFP-N1 plasmid. Conclusions Heterozygous variant RHO p. P53R is very likely the pathogenical mutation in the adRP family. The RHOpSsR mutant rhodopsin protein （;an not be delivery effectively from the endoplasmic reticulum to the cell membrane, and these proteins accumulate in the endoplasmic reticulum, which causes ERS.