应用目标基因捕获结合二代测序技术检测无遗传性眼病家族史的遗传性视网膜疾病患者的致病基因

Rapid identification of pathogenic mutations in sporadic hereditary retinal dystrophies using targeted next-generation sequencing

背景遗传性视网膜疾病（HRDs）是以原发性视网膜病变为主要病理改变、视力和色觉等视敏度严重损害为主要临床表现的致盲眼病，其与遗传因素密切相关，是『晦床上难治性盲的首要原因。目的应用目标基因捕获结合二代测序技术检测HRDs的致病基因。方法选择2014年1月至2015年1月在宁夏眼科医院就诊的无遗传性眼病家族史的20例HRDs患者作为研究对象。收集所有患者及其家庭成员临床资料，完善眼科检查，抽取患者和家庭正常成员外周静脉血，提取DNA。针对232个HRDs已知致病基因的目标序列设计并定制目标序列捕获芯片，借助高通量二代测序技术检测患者的遗传变异，数据经分析滤过，候选突变进行Sanger测序验证和致病性评估，明确致病基因和突变，并对表型和基因型间的关系进行分析。结果20例HRDs患者中有11例患者检测到致病性突变位点，共涉及9个基因，阳性率为55％。其中有8例患者检测到复合杂合突变，3例患者为纯合子突变，均为新发现的突变位点。通过对家系成员的基因筛查分析，6例HRDs患者为常染色体隐性遗传，5例遗传类型不确定。结合临床表型以及基因检测结果分析，11例HRDs患者的诊断分别为锥杆细胞营养不良5例，致病基因分别为ABCA4、RPE65、USH2A、RIMSl和RHO；Leber先天性黑嚎3例，致病基因分别为CRBl（2例）和LCA5；先天性静止性夜盲1例，致病基因为PRP乃；Best卵黄样黄斑营养不良1例，致病基因为BESTl基因；Stargardt病1例，致病基因为ABCA4基因。结论目标基因捕获结合二代测序技术可以对视网膜疾病患者进行快速、有效的基因诊断，结合临床特征分析有助于提高隐性遗传及遗传方式不明的HRDs的临床诊断水平。

Background Hereditary retinal diseases （HRDs） are a group of retinal degenerative diseases with significant genetic and clinical heterogeneities. Traditional techniques are challenging for detection of pathogenic mutations. Objective This study was to identify the diseasing-causal genes in 20 Chinese families with a variety of HRDs. Methods Family histories and ophthalmic examinations were obtained from all participants in 20 sporadic families. Targeted sequence capture array technique with next-generation sequencing （NGS） was performed to detect pathogenic mutations in 232 identified genes associated with HRDs. Variants detected by NGS were filtered by bioinformatic analysis HRDs. Genotype-phenotype correlation was also assessed. Results We identified 11 patients with pathogenic mutations,including 8 compound heterozygous mutations and 3 homozygous mutations,which were not yet reported. These findings showed genetic diagnoses in 11 of 20 patients, with the positive rate of 55%. Among them,6 patients were autosomal recessive inheritance and 5 were unspecific. Identification of different mutations and divergent phenotypes revealed 5 patients were affected with cone-rod dystrophy, 3 patients with Leber congenital amaurosis, 1 patient with congenital stationary night blindness, 1 patient with Best vitelliform macular dystrophy and 1 patient with Stargardt disease. Conclusions Targeted NGS is an effective approach for the genetic diagnoses of HRDs. These findings provide insights into understanding the genotype-phenotype correlations in HRDs.