全外显子测序技术在遗传性眼病诊断中的应用研究  

Application of whole exome sequencing technology in the diagnosis of hereditary eye diseases

作　　者：张丽 刘聪 陈松长 徐晨明 Zhang Li;Liu Cong;Chen Songchang;Xu Chenming(Genetics Center of Obstetrics and Gynecology,Obstetrics&Gynecology Hospital of Fudan University,Shanghai 200011,China)

机构地区：[1]复旦大学附属妇产科医院妇产科遗传中心,上海200011

出　　处：《中华医学杂志》2024年第26期2438-2444,共7页National Medical Journal of China

基　　金：国家重点研发计划(2023YFC2705600、2021YFC2701002);国家自然科学基金(81971344、82171677);上海市卫生健康委员会卫生行业临床研究专项(202140110)

摘　　要：目的评估全外显子组测序(WES)在遗传性眼病诊断中的应用。方法本研究收集2020年12月至2023年12月以先天性眼部疾病为主诉在复旦大学附属妇产科医院进行生殖遗传咨询的患者为研究对象,共24例。所有病例均已排除了已知感染或暴露于已知的致畸药物、核型和染色体微阵列分析(CMA)异常。采集先证者及其家系成员外周血样本,提取基因组DNA并进行WES检测,其中单人WES 3例,家系WES 21例,分析筛选出潜在致病位点并进行致病性分类及Sanger测序验证。针对RPGRIP1:c.1611+26G>A内含子变异构建minigene载体行逆转录实时荧光定量聚合酶链反应(RT-qPCR)检测mRNA剪接影响。结果24个家系中20(83.3%)个获得了明确病因的阳性结果,共涉及21个基因,鉴定出30个不同变异,其中19个为新变异。家系3产前诊断分析结果显示胎儿PRPF8基因携带c.6970G>T杂合无义突变;家系24中非经典剪接位点minigene载体RT-PCR结果提示突变型质粒转录产物12号内含子部分保留104 bp,即p.Glu538Valfs*12。结论WES在遗传性眼病诊断中的高检出率进一步支持了其应用优势,可作为明确遗传性眼病病因的重要分子检测工具。Objective To evaluate the application of whole exome sequencing(WES)in the diagnosis of hereditary eye diseases.Methods A total of 24 patients who came to the Obstetrics and Gynecology Hospital of Fudan University for reproductive genetic counseling from December 2020 to December 2023 with the main complaint of congenital eye disorders were included in this study.All cases had no known infections or exposure to known teratogenic drugs,karyotype and chromosome microarray analysis(CMA)abnormalities.Genomic DNA was extracted from the peripheral blood of the probands and their family members and tested for WES.Among them,three individual WES and 21 Trio WES were performed.Potential pathogenic sites were screened and analyzed by Sanger sequencing.For RPGRIP1:c.1611+26G>A site,minigene vector was constructed and RT-qPCR was performed to detect the effect of mRNA splicing.Results A total of 24 families were collected in this study,of which 20 yielded positive results,achieving a diagnosis rate of 83.3%(20/24).The results involved 21 genes and identified 30 distinct variants,19 of which were new variants reported.Prenatal diagnostic analysis of family 3 revealed that the fetus carried a c.6970G>T heterozygous nonsense mutation in the PRPF8 gene.The results of RT-PCR with the minigene vector at the non-classical splice site in family 24 indicated that the transcription product of the mutant plasmid was partially retained 104 bp in intron 12,resulting in a p.Glu538Valfs*12 alteration of the protein.Conclusions The high detection rate of WES in the diagnosis of hereditary eye diseases further supports the advantages of its application as an important molecular detection tool for determining the etiology of hereditary eye diseases.

关 键 词：眼疾病 遗传性 分子诊断技术 全外显子测序 剪接位点变异 minigene验证 

分 类 号：R714.55[医药卫生—妇产科学]