Department of Ophthalmology, Xinhua Hospital, Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Mol Vis. 2021 Jan 6;27:1-16. eCollection 2021.
Retinoblastoma (RB) is a pediatric ocular malignancy due to biallelic inactivation of the gene. Genetic testing is critically important for treatment decisions for this disease. Targeted next-generation sequencing (NGS) has been demonstrated to be an effective strategy for discovering all types of mutations in the gene. The aim of this study is the application of targeted NGS in a cohort of Chinese patients with retinoblastoma to identify germline mutations in the gene.
Blood samples were collected from 149 unrelated probands with retinoblastoma (62 bilaterally and 87 unilaterally) and their parent(s). Genomic DNA was analyzed with custom panel-based targeted NGS, and the panel was designed to include exons 1-27 of the gene with flanking intronic sequences. Single nucleotide variations (SNVs) and small insertions/deletions (InDels) identified were confirmed with Sanger sequencing. If the Sanger sequencing of a low-frequency variant (LFV) detected with targeted NGS was negative, PCR-based deep NGS was conducted for added confirmation. Copy number variations (CNVs) detected with targeted NGS were confirmed with multiplex ligation-dependent probe amplification (MLPA).
Overall, 74 germline mutations were detected in 48.3% of the probands (72/149, 56 bilateral and 16 unilateral cases). The total detection rate in the bilateral cases was 90.3% (56/62). These mutations included 64 SNVs and InDels (25 nonsense, 20 splicing, ten frameshift, eight missense, and one synonymous variants) and ten CNVs. All CNVs were confirmed with MLPA. Twenty-four (32.4%, 24/74) variants detected were novel, including nine splicing, six frameshift, five missense, and four nonsense variants. Eight LFVs (10.8%, 8/74) were found with targeted NGS; six of which were identified with Sanger sequencing, and two were identified with PCR-based deep NGS (13.16% and 3.000% mutant rates, respectively).
This study expanded the spectrum of germline mutations in using targeted NGS technology, which is a cost-saving and efficient method for genetic sequencing of retinoblastoma and may improve the molecular diagnosis of retinoblastoma.
视网膜母细胞瘤（RB）是一种小儿眼部恶性肿瘤，由 基因的双等位基因失活引起。基因检测对于这种疾病的治疗决策至关重要。靶向下一代测序（NGS）已被证明是一种有效的策略，可用于发现 基因中的所有类型的突变。本研究的目的是将靶向 NGS 应用于一组中国视网膜母细胞瘤患者中，以鉴定 基因中的种系突变。
收集 149 名（62 名双侧和 87 名单侧）未患病的视网膜母细胞瘤患者及其父母的血液样本。使用基于定制面板的靶向 NGS 分析基因组 DNA，该面板设计用于包含 基因的外显子 1-27 及其侧翼内含子序列。用 Sanger 测序法对鉴定出的单核苷酸变异（SNVs）和小插入/缺失（InDels）进行确认。如果靶向 NGS 检测到的低频变异（LFV）的 Sanger 测序结果为阴性，则进行基于 PCR 的深度 NGS 进行补充确认。用多重连接依赖性探针扩增（MLPA）法对靶向 NGS 检测到的拷贝数变异（CNVs）进行确认。
总体而言，在 48.3%的患者（72/149，56 例双侧和 16 例单侧病例）中检测到 74 种种系突变。双侧病例的总检出率为 90.3%（56/62）。这些突变包括 64 种 SNVs 和 InDels（25 种无义突变、20 种剪接突变、10 种移码突变、8 种错义突变和 1 种同义突变）和 10 种 CNVs。所有的 CNVs 均经 MLPA 法确认。24 种（32.4%，24/74）检测到的变异是新的，包括 9 种剪接突变、6 种移码突变、5 种错义突变和 4 种无义突变。用靶向 NGS 发现了 8 种 LFVs（10.8%，8/74），其中 6 种用 Sanger 测序法确认，2 种用基于 PCR 的深度 NGS 法确认（突变率分别为 13.16%和 3.000%）。
本研究利用靶向 NGS 技术扩展了 基因的种系突变谱，这是一种节省成本且高效的视网膜母细胞瘤遗传测序方法，可提高视网膜母细胞瘤的分子诊断水平。
