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BMC Cancer
Published in: BMC Cancer 1/2015

Open Access 01-12-2015 | Research article

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

Authors: Simona Grotta, Gemma D’Elia, Rossana Scavelli, Silvia Genovese, Cecilia Surace, Pietro Sirleto, Raffaele Cozza, Antonino Romanzo, Maria Antonietta De Ioris, Paola Valente, Anna Cristina Tomaiuolo, Francesca Romana Lepri, Tiziana Franchin, Laura Ciocca, Serena Russo, Franco Locatelli, Adriano Angioni

Published in: BMC Cancer | Issue 1/2015

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Abstract

Background

Retinoblastoma (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).

Methods

A 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 %.

Results

Of 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 %.

Conclusions

NGS 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.
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Metadata
Title
Advantages of a next generation sequencing targeted approach for the molecular diagnosis of retinoblastoma
Authors
Simona Grotta
Gemma D’Elia
Rossana Scavelli
Silvia Genovese
Cecilia Surace
Pietro Sirleto
Raffaele Cozza
Antonino Romanzo
Maria Antonietta De Ioris
Paola Valente
Anna Cristina Tomaiuolo
Francesca Romana Lepri
Tiziana Franchin
Laura Ciocca
Serena Russo
Franco Locatelli
Adriano Angioni
Publication date
01-12-2015
Publisher
BioMed Central
Published in
BMC Cancer / Issue 1/2015
Electronic ISSN: 1471-2407
DOI
https://doi.org/10.1186/s12885-015-1854-0

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