Oculoplasty and Oncology Services, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi.
Department of Anatomy, All India Institute of Medical Sciences, New Delhi.
Indian J Ophthalmol. 2024 Jul 1;72(7):1012-1016. doi: 10.4103/IJO.IJO_234_23. Epub 2024 Mar 8.
Retinoblastoma (RB) is the most common intraocular tumor in pediatric age group. The role of genetics has been explored in predicting survival prognosis, but its role in predicting globe salvage remains largely unexplored. We hereby aim to isolate cell-free DNA (cfDNA) from aqueous humor (AH) in RB eyes and validate its use for genetic studies.
AH was obtained from 26 eyes undergoing enucleation (arm A) or intravitreal chemotherapy (arm B). Isolation of cfDNA was done using QIAamp ® Circulating Nucleic Acid kit, and the cfDNA was utilized for targeted sequencing of RB1 gene.
We could isolate cfDNA in all eyes (72% unilateral and 28% bilateral) with a distribution peak between 140 and 160 bp and a mean concentration of 27.75 ng/µl for arm A and 14 ng/µl for arm B. Targeted sequencing done on four samples showed RB1 gene mutations, namely, inframe deletion (c. 78-80del, p.Pro29del), start-loss mutation (c.1A>T, p.Met1?), nonsense mutations (c.2236G>T, p.Glu746Ter), (c.1659T>A, p.Cys553Ter), and (c.2065C>T, p.Gln689Ter), and novel missense mutations (c.672C>A, p.Asp224Glu) and c.692C>T (p.Pro231Leu). Genetic profile of cfDNA extracted from AH and genomic DNA from the tumor tissue was comparable.
Our study supports the previous reports that AH may be used as a source of tumor-derived cfDNA. This is the first report from South Asia on isolation and genetic analysis of cfDNA from AH of RB eyes and, therefore, a big step forward in paving the role of tumor genetics in RB. Further studies are required to elucidate concordance between the tumor and AH genetic profile.
视网膜母细胞瘤（RB）是儿童期最常见的眼内肿瘤。遗传学在预测生存预后方面的作用已经得到了探索，但在预测眼球保存方面的作用仍在很大程度上尚未得到探索。我们旨在从 RB 眼中的房水中分离无细胞 DNA（cfDNA），并验证其在遗传研究中的用途。
从 26 只接受眼球摘除术（A 组）或玻璃体内化疗（B 组）的眼中获得房水。使用 QIAamp ® 循环核酸试剂盒分离 cfDNA，并利用 cfDNA 对 RB1 基因进行靶向测序。
我们可以从所有眼睛（72%单侧和 28%双侧）中分离出 cfDNA，分布峰值在 140 和 160 bp 之间，A 组的平均浓度为 27.75 ng/µl，B 组为 14 ng/µl。对四个样本进行的靶向测序显示 RB1 基因突变，即框内缺失（c.78-80del，p.Pro29del）、起始缺失突变（c.1A>T，p.Met1？）、无义突变（c.2236G>T，p.Glu746Ter）、（c.1659T>A，p.Cys553Ter）和（c.2065C>T，p.Gln689Ter），以及新的错义突变（c.672C>A，p.Asp224Glu）和 c.692C>T（p.Pro231Leu）。从 AH 中提取的 cfDNA 和肿瘤组织中提取的基因组 DNA 的遗传谱是可比的。
我们的研究支持了之前的报告，即 AH 可能被用作肿瘤衍生 cfDNA 的来源。这是南亚关于从 RB 眼中的 AH 中分离和遗传分析 cfDNA 的第一篇报告，因此在为 RB 中的肿瘤遗传学铺平道路方面迈出了一大步。需要进一步的研究来阐明肿瘤和 AH 遗传谱之间的一致性。
