Targeted next generation sequencing of RB1 gene for the molecular diagnosis of Retinoblastoma

Abstract

BackgroundThe spectrum of RB1gene mutations in Retinoblastoma (RB) patients and the necessity of multiple traditional methods for complete variant analysis make the molecular diagnosis a cumbersome, labor-intensive and time-consuming process. Here, we have used targeted next generation sequencing (NGS) approach with in-house analysis pipeline to explore its potential for the molecular diagnosis of RB.MethodsThirty-three patients with RB and their family members were selected randomly. DNA from patient blood and/or tumor was used for RB1 gene targeted sequencing. The raw reads were obtained from Illumina Miseq. An in-house bioinformatics pipeline was developed to detect both single nucleotide variants (SNVs) and small insertions/deletions (InDels) and to distinguish between somatic and germline mutations. In addition, ExomeCNV and Cn. MOPS were used to detect copy number variations (CNVs). The pathogenic variants were identified with stringent criteria, and were further confirmed by conventional methods and cosegregation in families.ResultsUsing our approach, an array of pathogenic variants including SNVs, InDels and CNVs were detected in 85% of patients. Among the variants detected, 63% were germline and 37% were somatic. Interestingly, nine novel pathogenic variants (33%) were also detected in our study.ConclusionsWe demonstrated for the first time that targeted NGS is an efficient approach for the identification of wide spectrum of pathogenic variants in RB patients. This study is helpful for the molecular diagnosis of RB in a comprehensive and time-efficient manner.

Knudson's hypothesis revisited in Indian retinoblastoma patients

“…Two somatic mutations were identified in tumor samples of each patient 35 and 47. In addition, we have carried out the genetic analysis of 50 patients and the results were very much consistent with Knudson's two‐hit hypothesis …”

“…After the first hit has occurred, the second hit can be due to loss of heterozygosity (LoH), promoter methylation or a second mutation in another allele . Among them, LoH had been the most common second hit and our case series involving more than 50 patients also had more LoH …”

miR-590 promotes cell proliferation and invasion in T-cell acute lymphoblastic leukaemia by inhibiting RB1

“…The deletion of RB1 was ever found in a T-ALL patient [18]. Down-regulation of RB1 is involved in various types of cancers, including osteosarcoma [19], Retinoblastoma [20] and lung adenocarcinoma [21]. miR-590 might take part in the carcinogenesis of T-ALL largely by negative regulating RB1.…”

Next-Generation Technologies and Strategies for the Management of Retinoblastoma

“…This combined, cost-effective approach was able to accurately detect point mutations, macrodeletions, and duplications from exon 18 to exon 23, and it could detect the level of mosaicism down to 1% [37]. Targeted NGS using Illumina MiSeq and precision bioinformatic pipelines were also used to identify a spectrum of pathogenic variants in retinoblastoma patients [38]. Using these comprehensive approaches, an array of novel pathogenic variants-including single nucleotide variants, InDels (insertions/deletions), and copy number variations-were detected in RB patients [37,38] and the time and number of assays required for detection of RB1 pathogenic variants were reduced.…”

“…Targeted NGS using Illumina MiSeq and precision bioinformatic pipelines were also used to identify a spectrum of pathogenic variants in retinoblastoma patients [38]. Using these comprehensive approaches, an array of novel pathogenic variants-including single nucleotide variants, InDels (insertions/deletions), and copy number variations-were detected in RB patients [37,38] and the time and number of assays required for detection of RB1 pathogenic variants were reduced. Another recent method using a combination of direct sequencing and multiplex ligation-dependent probe amplification (MLPA) has also enhanced the detection of gross mutations and is able to identify germline mosaicism and genomic abnormalities spanning the promoter, exons and introns in RB1 [39].…”