51个视网膜色素变性家系遗传学特征分析

Genetic characteristics of 51 retinitis pigmentosa families

目的分析视网膜色素变性(RP)家系的致病基因。方法采用家系调查研究方法,收集2019年6月至2022年12月就诊于武汉大学人民医院的中国51个RP家系的临床资料,包括患者病史、家族史及眼科检查临床资料,眼科检查临床资料包括最佳矫正视力、裂隙灯显微镜、彩色眼底照相、眼底自发荧光、黄斑区光学相干断层扫描、视野及视网膜电图。采集患者及家属外周血,提取DNA,进行全外显子测序,对发现的变异位点进行生物信息学分析、Sanger验证。采用SIFT、Polyphen等在线软件预测错义变异致病性,采用Mutation Taster在线软件评估错义变异位点的保守性,采用varSEAK、spliceAI对剪切变异进行预测,并用Clustalw软件对新发现的变异位点进行多物种蛋白质氨基酸序列比对。结果51个家系中,2例先证者伴有听力障碍,被诊断为Usher综合征,2例先证者眼底成像除典型RP特征外还出现黄白色结晶样物质沉着,其余家系先证者眼底成像表现为典型RP。51个家系中,29个家系在15个致病基因中检测出38个单核苷酸变异(SNVs)和3个拷贝数变异,包括PRPF 6、PRPF 31、RHO、CYP 4V2、USH 2 A、EYS、MERTK、PCDH 15、ABCA 4、BBS 2、PROM 1、SPATA 7、RPE 65、RPGR、OFD 1基因,38个SNVs中有6个未曾报道过的新变异,分别是USH 2 A基因c.12523T>C(p.Trp4175Arg)、c.1723T>C(p.Cys575Arg)、c.1875C>G(p.Phe625Leu),CYP 4 V 2基因c.1441C>T(p.Leu481Phe),MERTK基因c.2487-8A>G和PCDH 15基因c.5183del(p.Arg1728LysfsTer116)。SIFT、Polyphen预测软件对USH 2 A基因p.Trp4175Arg、p.Cys575Arg、p.Phe625Leu和CYP 4 V 2基因p.Leu 481Phe这4个错义变异位点造成的氨基酸改变预测均为致病或有害,保守性分析显示其在多个物种中保守。spliceAI、varSEAK预测软件均提示,MERTK基因c.2487-8A>G可能会导致剪切异常,影响蛋白质功能。PCDH 15基因c.5183del(p.Arg1728LysfsTer116)为移码变异,会改变下游氨基酸序列并使翻译提前终止。CYP 4V2、USH2 A、RPGR基因为RP患者中变异频率较高的基因,占所有检出致病基因家系的50%以上,其中CYP 4V2基因致病的先证者起病较晚,但视功能下降和视网膜退变更严重。结论本研究发现了6个未报道过的变异可能是RP的致病变异;CYP 4V2、USH 2 A和RPGR基因是中国RP患者主要的致病基因;CYP 4V2基因致病的患者起病较晚,但疾病进展较快。

Objective To analyze the disease-causing genes of families affected by retinitis pigmentosa(RP).Methods A pedigree investigation study was performed.The clinical data of 51 Chinese families with RP treated at the Renmin Hospital of Wuhan University from June 2019 to December 2022 were collected,including patient history,family history and clinical data of ophthalmic examination.Ophthalmic examination including best corrected visual acuity,slit lamp microscopy,color fundus photography,fundus autofluorescence,macular optical coherence tomography,visual field and electroretinogram.Peripheral blood samples from patients and their family members were collected for DNA extraction and whole exome sequencing.The mutation sites found were analyzed by bioinformatics and verified by Sanger sequencing.The pathogenicity of the missense mutations was predicted using SIFT,Polyphen and other online software.Conservation of the missense mutation site was evaluated using Mutation Taster.The shear mutation was predicted using varSEAK and spliceAI.The amino acid sequences of the newly discovered mutation sites were compared using Clustalw software.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Renmin Hospital of Wuhan University(No.WDRY2019-K032).Results Among the 51 families,two proband patients had hearing impairment and were diagnosed as Usher syndrome.In addition to typical RP features,the two proband patients also showed yellow-white crystalline substance deposits in fundus imaging,while the other proband patients showed typical RP.A total of 38 single nucleotide variants(SNVs)and 3 copy number variants were detected in 15 pathogenic genes in 29 of 51 families,including PRPF 6,PRPF 31,RHO,CYP 4V2,USH 2 A,EYS,MERTK,PCDH 15,ABCA 4,BBS 2,PROM 1,SPATA 7,RPE 65,RPGR and OFD 1 genes.There were 6 of the 38 SNVs that were novel variants that had not been reported,which were USH 2 A gene c.12523T>C(p.Trp4175Arg),c.1723T>C(p.Cys575Arg),c.1875C>G(p.Phe625Leu),CYP 4V2 gene c.1441C>T(p.Leu481Phe),MERTK gene c.2487-8A>G and PCDH 15 gene c.5183del(p.Arg1728LysfsTer116).SIFT and Polyphen prediction software predicted that amino acid changes caused by the 4 missense variants,USH 2 A gene p.Trp4175Arg,p.Cys575Arg,p.Phe625Leu and CYP 4V2 gene p.Leu481Phe,are all pathogenic or harmful.Conservation analysis showed that they are conserved in multiple species.The prediction software spliceAI and varSEAK suggested that MERTK gene c.2487-8A>G may lead to abnormal shear and affect protein function.PCDH 15 gene c.5183del(p.Arg1728LysfsTer116)is a frameshift variant that alters the downstream amino acid sequence and terminates translation early.CYP 4V2,USH2 A,and RPGR were frequently mutated genes in RP patients,accounting for more than 50%of the families with pathogenic genes detected.The proband with CYP 4V2 variants had late onset,but severe visual impairment and retinal degeneration.Conclusions Six previously unreported variants may be novel pathogenic variants of RP.CYP 4V2,USH2 A,and RPGR may be the most common pathogenic genes in Chinese RP patients.Patients with CYP 4V2 variants have late onset,but faster disease progression.