未经证实:视网膜母细胞瘤(RB)由RB1基因的两个等位基因的突变引发。然而,即使没有RB1突变,也很少发生病例,这表明RB1以外的基因的作用。
UNASSIGNED:目前的研究计划在患有单侧非家族性RB的印度RB患者中利用靶向外显子组测序。纳入75例5岁以下的单侧RB患者。从血液和肿瘤组织中提取基因组DNA。从外周血DNA中,使用PCR和直接测序扩增所有编码区和外显子/内含子区.使用靶向外显子组测序进一步筛选在外周血DNA中没有致病性变体的病例在其肿瘤组织DNA中的突变。三种致病性预测工具(突变量,SIFT,和PolyPhen-2)用于确定非同义变异的致病性。设计了内部生物信息学管道,用于通过靶向外显子组测序进行突变筛选。还进行了蛋白质建模研究以预测突变对蛋白质结构和功能的影响。
未经评估:使用上述方法,我们在外周血DNA中的RB1基因中发现了两个新的变体(g.69673_69674insT和g.48373314C>A)。我们还在八个基因(RB1,ACAD11,GPR151,KCNA1,OTOR,SOX30,ARL11和MYCT1)可能与RB发病机制有关。
UNASSIGNED:本研究扩展了我们目前关于RB基因组格局的知识,并强调了NGS技术检测可能在癌症发生中起重要作用的基因和新变体的重要性。programming,和预后。
Retinoblastoma (RB) is initiated by mutation in both alleles of RB1 gene. However, few cases may occur even in the absence of RB1 mutation suggesting the role of genes other than RB1.
The current study was planned to utilize targeted exome sequencing in Indian RB patients affected with unilateral non-familial RB. 75 unilateral RB patients below 5 years of age were enrolled. Genomic DNA was extracted from blood and tumor tissue. From peripheral blood DNA, all coding and exon/intron regions were amplified using PCR and direct sequencing. Cases which did not harbor pathogenic variants in peripheral blood DNA were further screened for mutations in their tumor tissue DNA using targeted exome sequencing. Three pathogenicity prediction tools (Mutation Taster, SIFT, and PolyPhen-2) were used to determine the pathogenicity of non-synonymous variations. An in-house bioinformatics pipeline was devised for the mutation screening by targeted exome sequencing. Protein modeling studies were also done to predict the effect of the mutations on the protein structure and function.
Using the mentioned approach, we found two novel variants (g.69673_69674insT and g.48373314C>A) in RB1 gene in peripheral blood DNA. We also found novel variants in eight genes (RB1, ACAD11, GPR151, KCNA1, OTOR, SOX30, ARL11, and MYCT1) that may be associated with RB pathogenesis.
The present study expands our current knowledge regarding the genomic landscape of RB and also highlights the importance of NGS technologies to detect genes and novel variants that may play an important role in cancer initiation, progression, and prognosis.