Kiet Nguyen Cong, Khuong Le Thai, Minh Do Duc, Quan Nguyen Huynh Minh, Xinh Phan Thi, Trang Nguyen Ngoc Chau, Luan Nguyen Thanh, Khai Nguyen Minh, Vu Hoang Anh
Department of Ophthalmology, Faculty of Medicine, University of Medicine and Pharmacy at Ho Chi Minh City, Ho Chi Minh City, Vietnam.
Center for Molecular Biomedicine, University of Medicine and Pharmacy at Ho Chi Minh City, Ho Chi Minh City, Vietnam.
Mol Vis. 2019 Apr 4;25:215-221. eCollection 2019.
Retinoblastoma (RB) is a rare childhood malignant disorder caused by the biallelic inactivation of the gene. Early diagnosis and identification of carriers of heritable mutations in can improve disease outcome and management. In this study, we present the spectrum of mutations in the gene in Vietnamese patients with RB.
Tumor RNA from 50 probands with RB, including 12 bilateral and 38 unilateral cases, was extracted. cDNA, after reverse transcription, was sequenced to identify the RNA mutation of the gene. At the genomic DNA level, mutational analysis of all exons, exon-intron boundaries, and the promoter region was conducted using PCR and direct sequencing. Multiplex ligation-dependent probe amplification (MLPA) analysis was performed for patients for whom the first two results were negative. For patients for whom either the sequencing or MLPA results were positive for a tumor mutation, patients' and their parents' blood DNA was analyzed to determine the germline mutation.
Forty-one different kinds of tumor mutations were identified in 41 probands (82.0%), including 11 of 12 bilateral cases (91.7%) and 30 of 38 unilateral cases (78.9%). The majority of the detected mutations were nonsense (15 different kinds), followed by frameshift (11 kinds), and splice site mutations (nine kinds). Each splice site mutation was confirmed to create a deletion of the corresponding exon with RNA sequencing. The single promoter mutation c.-197G>A was reported previously; however, both missense mutations identified in exon 6 (c.601G>C: p.A201P) and exon 22 (c.2264T>C: p.F755S) were novel. Gross deletions were detected with MLPA in three probands. The detection rate of germline mutations in bilateral and unilateral cases with mutations were 81.8% and 30.0%, respectively. Only one father out of the 20 parents tested was positive for a germline mutation.
Mutations in the gene in Vietnamese patients were heterogeneous and highly prevalent with pathogenic truncated mutations. With advancement in therapeutics, early detection of RB is important for eye salvation.
视网膜母细胞瘤（RB）是一种由该基因双等位基因失活引起的罕见儿童恶性疾病。早期诊断和识别该基因可遗传突变的携带者可改善疾病的治疗效果和管理。在本研究中，我们展示了越南RB患者中该基因的突变谱。
提取了50例RB先证者的肿瘤RNA，其中包括12例双侧病例和38例单侧病例。逆转录后的cDNA进行测序以鉴定该基因的RNA突变。在基因组DNA水平，使用PCR和直接测序对所有外显子、外显子 - 内含子边界和启动子区域进行突变分析。对于前两项结果为阴性的患者进行多重连接依赖探针扩增（MLPA）分析。对于测序或MLPA结果显示肿瘤突变呈阳性的患者，分析患者及其父母的血液DNA以确定种系突变。
在41例先证者（82.0%）中鉴定出41种不同类型的肿瘤突变，包括12例双侧病例中的11例（91.7%）和38例单侧病例中的30例（78.9%）。检测到的大多数突变是无义突变（15种不同类型），其次是移码突变（11种）和剪接位点突变（9种）。通过RNA测序证实每个剪接位点突变都会导致相应外显子的缺失。单一启动子突变c.-197G>A先前已有报道；然而，在外显子6（c.601G>C：p.A201P）和外显子22（c.2264T>C：p.F755S）中鉴定出的两个错义突变均为新发现的。在3例先证者中通过MLPA检测到大片段缺失。双侧和单侧有突变病例的种系突变检测率分别为81.8%和30.0%。在检测的20名父母中只有1名父亲的种系突变呈阳性。
越南患者中该基因的突变具有异质性且高度普遍，存在致病性截短突变。随着治疗方法的进步，RB的早期检测对挽救视力很重要。
