Advantages of a next generation sequencing targeted approach for the molecular diagnosis of retinoblastoma

Abstract

BackgroundRetinoblastoma (RB) is the most common malignant childhood tumor of the eye and results from inactivation of both alleles of the RB1 gene. Nowadays RB genetic diagnosis requires classical chromosome investigations, Multiplex Ligation-dependent Probe Amplification analysis (MLPA) and Sanger sequencing. Nevertheless, these techniques show some limitations. We report our experience on a cohort of RB patients using a combined approach of Next-Generation Sequencing (NGS) and RB1 custom array-Comparative Genomic Hybridization (aCGH).MethodsA total of 65 patients with retinoblastoma were studied: 29 cases of bilateral RB and 36 cases of unilateral RB. All patients were previously tested with conventional cytogenetics and MLPA techniques. Fifty-three samples were then analysed using NGS. Eleven cases were analysed by RB1 custom aCGH. One last case was studied only by classic cytogenetics. Finally, it has been tested, in a lab sensitivity assay, the capability of NGS to detect artificial mosaicism series in previously recognized samples prepared at 3 different mosaicism frequencies: 10, 5, 1 %.ResultsOf the 29 cases of bilateral RB, 28 resulted positive (96.5 %) to the genetic investigation: 22 point mutations and 6 genomic rearrangements (four intragenic and two macrodeletion). A novel germline intragenic duplication, from exon18 to exon 23, was identified in a proband with bilateral RB. Of the 36 available cases of unilateral RB, 8 patients resulted positive (22 %) to the genetic investigation: 3 patients showed point mutations while 5 carried large deletion. Finally, we successfully validated, in a lab sensitivity assay, the capability of NGS to accurately measure level of artificial mosaicism down to 1 %.ConclusionsNGS and RB1-custom aCGH have demonstrated to be an effective combined approach in order to optimize the overall diagnostic procedures of RB. Custom aCGH is able to accurately detect genomic rearrangements allowing the characterization of their extension. NGS is extremely accurate in detecting single nucleotide variants, relatively simple to perform, cost savings and efficient and has confirmed a high sensitivity and accuracy in identifying low levels of artificial mosaicisms.

Retinoblastoma and Neuroblastoma Predisposition and Surveillance

“…These patients are at risk not only for RB but also in transmitting this predisposition to offspring. Testing by next-generation sequencing will likely increase the rate of detection of germline mosaicism (31,(38)(39)(40).…”

An alternative to array-based diagnostics: a prospectively recruited cohort, comparing arrayCGH to next-generation sequencing to evaluate foetal structural abnormalities

“…Such information may be of clinical value, especially where breakpoints are close to or intersect with clinically-important genes. It has been reported previously that low level mosaicism is easier to identify using a CNVseq, rather than aCGH workflow (Grotta, 2015). The specimen with confined placental mosaicism in this cohort was more easily visualised in the CNVseq karyogram compared to the array result.…”

Next-Generation Technologies and Strategies for the Management of Retinoblastoma

“…This is particularly important because tumor tissue for the analysis of somatic mutations is only available if the eye has been enucleated (i.e., removed); therefore, the accuracy of the peripheral blood test needs to be high in the absence of the tumor mutation(s). NGS and RB1 custom array-comparative genomic hybridization (aCGH) methods were recently used on a cohort of RB patients to optimize diagnostic procedures and to identify genomic abnormalities in retinoblastoma [37]. 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].…”

“…NGS and RB1 custom array-comparative genomic hybridization (aCGH) methods were recently used on a cohort of RB patients to optimize diagnostic procedures and to identify genomic abnormalities in retinoblastoma [37]. 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].…”

“…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].…”