Department of Ophthalmology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
Research Center, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
Transl Vis Sci Technol. 2022 Sep 1;11(9):30. doi: 10.1167/tvst.11.9.30.
The study aimed to generate a stepwise method to reduce the workload of full-scale RB1 sequencing for germline mutation screening in retinoblastoma (RB) patients. The implication of germline mutation in tumor focality was also determined in this study.
A stepwise method was created on the basis of "hotspot" exons analyzed using data on germline RB1 mutation in the RB1-Leiden Open Variation Database and then tested for mutation screening in the blood DNA of 42 patients with RB. The method was compared with the clinical next-generation sequencing (NGS) panel in terms of sequencing outcomes. The germline RB1 mutation was examined in association with multifocality in RB.
Germline RB1 mutation was identified in 61% of all bilateral cases in the first step of the 3 stepwise method and in 78% and 89% for the two and three steps combined, respectively. NGS detected a mosaic variant of RB1 that was not detected by the first two steps and increased the sensitivity from 78% to 83%. Analysis of the relationship between mutation status and tumor focality indicated that multifocality in RB was dependent on germline RB1 mutation, confirming a higher tendency to have a germline RB1 mutation in patients with multifocal RB.
A 3 stepwise method reduces the workload needed for sequencing of the RB1 for bilateral cases. NGS outweighs conventional sequencing in terms of the identification of germline mosaic variants. Multifocal tumors in RB may be used to presume germline mutation.
The presence of "hotspot" exons of germline RB1 mutation in bilateral cases facilitates a mutation screening. However, when genetic testing is not available, multifocality in RB regardless of tumor laterality is predictive of germline RB1 mutation.
本研究旨在生成一种逐步方法，以减少对视网膜母细胞瘤（RB）患者种系突变筛查的全规模 RB1 测序工作量。本研究还确定了种系突变对肿瘤局灶性的影响。
基于 RB1-Leiden 开放变异数据库中的种系 RB1 突变数据分析“热点”外显子，创建了一种逐步方法，然后在 42 名 RB 患者的血液 DNA 中进行突变筛查。该方法在测序结果方面与临床下一代测序（NGS）面板进行了比较。
在三步骤逐步方法的第一步中，61%的双侧病例中发现了种系 RB1 突变，两步和三步结合的分别为 78%和 89%。NGS 检测到 RB1 的镶嵌变体，而前两步未检测到，从而将敏感性从 78%提高到 83%。对突变状态与肿瘤局灶性的关系进行分析表明，RB 的多灶性取决于种系 RB1 突变，证实多灶性 RB 患者更倾向于存在种系 RB1 突变。
三步骤逐步方法减少了双侧病例中 RB1 测序所需的工作量。与传统测序相比，NGS 更能识别种系镶嵌变体。RB 中的多灶性肿瘤可用于推测种系突变。
张昊玥
