Tomar Swati, Sethi Raman, Sundar Gangadhara, Quah Thuan Chong, Quah Boon Long, Lai Poh San
Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Department of Ophthalmology, National University Hospital, Singapore, Singapore.
PLoS One. 2017 Jun 2;12(6):e0178776. doi: 10.1371/journal.pone.0178776. eCollection 2017.
Retinoblastoma (RB) is a rare childhood malignant disorder caused by the biallelic inactivation of RB1 gene. Early diagnosis and identification of carriers of heritable RB1 mutations can improve disease outcome and management. In this study, mutational analysis was conducted on fifty-nine matched tumor and peripheral blood samples from 18 bilateral and 41 unilateral unrelated RB cases by a combinatorial approach of Multiplex Ligation-dependent Probe Amplification (MLPA) assay, deletion screening, direct sequencing, copy number gene dosage analysis and methylation assays. Screening of both blood and tumor samples yielded a mutation detection rate of 94.9% (56/59) while only 42.4% (25/59) of mutations were detected if blood samples alone were analyzed. Biallelic mutations were observed in 43/59 (72.9%) of tumors screened. There were 3 cases (5.1%) in which no mutations could be detected and germline mutations were detected in 19.5% (8/41) of unilateral cases. A total of 61 point mutations were identified, of which 10 were novel. There was a high incidence of previously reported recurrent mutations, occurring at 38.98% (23/59) of all cases. Of interest were three cases of mosaic RB1 mutations detected in the blood from patients with unilateral retinoblastoma. Additionally, two germline mutations previously reported to be associated with low-penetrance phenotypes: missense-c.1981C>T and splice variant-c.607+1G>T, were observed in a bilateral and a unilateral proband, respectively. These findings have implications for genetic counselling and risk prediction for the affected families. This is the first published report on the spectrum of mutations in RB patients from Singapore and shows that further improved mutation screening strategies are required in order to provide a definitive molecular diagnosis for every case of RB. Our findings also underscore the importance of genetic testing in supporting individualized disease management plans for patients and asymptomatic family members carrying low-penetrance, germline mosaicism or heritable unilateral mutational phenotypes.
视网膜母细胞瘤（RB）是一种由RB1基因双等位基因失活引起的罕见儿童恶性疾病。早期诊断和识别遗传性RB1突变携带者可改善疾病预后和管理。在本研究中，采用多重连接依赖探针扩增（MLPA）检测、缺失筛查、直接测序、拷贝数基因剂量分析和甲基化检测等组合方法，对来自18例双侧和41例单侧非相关RB病例的59对匹配肿瘤和外周血样本进行了突变分析。对血液和肿瘤样本进行筛查的突变检出率为94.9%（56/59），而仅分析血液样本时，突变检出率仅为42.4%（25/59）。在筛查的43/59（72.9%）的肿瘤中观察到双等位基因突变。有3例（5.1%）未检测到突变，19.5%（8/41）的单侧病例检测到种系突变。共鉴定出61个点突变，其中10个是新的。先前报道的复发性突变发生率很高，在所有病例中占38.98%（23/59）。有趣的是，在单侧视网膜母细胞瘤患者的血液中检测到3例RB1基因镶嵌突变。此外，分别在一名双侧和一名单侧先证者中观察到两个先前报道与低外显率表型相关的种系突变：错义突变-c.1981C>T和剪接变异体-c.607+1G>T。这些发现对受影响家庭的遗传咨询和风险预测具有重要意义。这是关于新加坡RB患者突变谱的首篇发表报告，表明需要进一步改进突变筛查策略，以便为每一例RB提供明确的分子诊断。我们的研究结果还强调了基因检测在支持针对携带低外显率、种系镶嵌或遗传性单侧突变表型的患者及无症状家庭成员的个体化疾病管理计划方面的重要性。
