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

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

Research output: Contribution to journalArticle

5 Citations (Scopus)

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.

Original languageEnglish
Article number841
JournalBMC Cancer
Volume15
Issue number1
DOIs
Publication statusPublished - Nov 4 2015

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Retinoblastoma
Mosaicism
Comparative Genomic Hybridization
Multiplex Polymerase Chain Reaction
Point Mutation
Cytogenetics
Cost Savings
Exons
Nucleotides
Chromosomes
Alleles

Keywords

  • Next-Generation Sequencing
  • RB1 custom aCGH
  • Retinoblastoma

ASJC Scopus subject areas

  • Oncology
  • Cancer Research
  • Genetics

Cite this

Advantages of a next generation sequencing targeted approach for the molecular diagnosis of retinoblastoma. / Grotta, Simona; D'Elia, Gemma; Scavelli, Rossana; Genovese, Silvia; Surace, Cecilia; Sirleto, Pietro; Cozza, Raffaele; Romanzo, Antonino; De Ioris, Maria Antonietta; Valente, Paola; Tomaiuolo, Anna Cristina; Lepri, Francesca Romana; Franchin, Tiziana; Ciocca, Laura; Russo, Serena; Locatelli, Franco; Angioni, Adriano.

In: BMC Cancer, Vol. 15, No. 1, 841, 04.11.2015.

Research output: Contribution to journalArticle

Grotta, S, D'Elia, G, Scavelli, R, Genovese, S, Surace, C, Sirleto, P, Cozza, R, Romanzo, A, De Ioris, MA, Valente, P, Tomaiuolo, AC, Lepri, FR, Franchin, T, Ciocca, L, Russo, S, Locatelli, F & Angioni, A 2015, 'Advantages of a next generation sequencing targeted approach for the molecular diagnosis of retinoblastoma', BMC Cancer, vol. 15, no. 1, 841. https://doi.org/10.1186/s12885-015-1854-0
Grotta, Simona ; D'Elia, Gemma ; Scavelli, Rossana ; Genovese, Silvia ; Surace, Cecilia ; Sirleto, Pietro ; Cozza, Raffaele ; Romanzo, Antonino ; De Ioris, Maria Antonietta ; Valente, Paola ; Tomaiuolo, Anna Cristina ; Lepri, Francesca Romana ; Franchin, Tiziana ; Ciocca, Laura ; Russo, Serena ; Locatelli, Franco ; Angioni, Adriano. / Advantages of a next generation sequencing targeted approach for the molecular diagnosis of retinoblastoma. In: BMC Cancer. 2015 ; Vol. 15, No. 1.
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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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AU - D'Elia, Gemma

AU - Scavelli, Rossana

AU - Genovese, Silvia

AU - Surace, Cecilia

AU - Sirleto, Pietro

AU - Cozza, Raffaele

AU - Romanzo, Antonino

AU - De Ioris, Maria Antonietta

AU - Valente, Paola

AU - Tomaiuolo, Anna Cristina

AU - Lepri, Francesca Romana

AU - Franchin, Tiziana

AU - Ciocca, Laura

AU - Russo, Serena

AU - Locatelli, Franco

AU - Angioni, Adriano

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N2 - 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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