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04-11-2015 | Ophthalmologic cancers | Article

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

Journal: BMC Cancer

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

Publisher: BioMed Central

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.
Literature
1.
Kivelä T. The epidemiological challenge of the most frequent eye cancer: retinoblastoma, an issue of birth and death. Br J Ophthalmol. 2009;93:1129–31.CrossRefPubMed
2.
Seregard S, Lundell G, Svedberg H, Kivelä T. Incidence of retinoblastoma from 1958 to 1998 in Northern Europe: advantages of birth cohort analysis. Ophthalmology. 2004;111:1228–32.CrossRefPubMed
3.
Aerts I, Lumbroso-Le Rouic L, Gauthier-Villars M, Brisse H, Doz F, Desjardins L. Retinoblastoma. Orphanet J Rare Dis. 2006;1:31.CrossRefPubMedPubMedCentral
4.
Dunn JM, Phillips RA, Becker AJ, Gallie BL. Identification of germline and somatic mutations affecting the retinoblastoma gene. Science. 1988;241:1797–800.CrossRefPubMed
5.
Thèriault BL, Dimaras H, Gallie BL, Corson TW. The genomic landscape of retinoblastoma: a review. Clin Experiment Ophthalmol. 2014;42(1):33–52.CrossRefPubMed
6.
Doz F. Retinoblatoma: a review. Arch Pediatr. 2006;13:1329–37.CrossRefPubMed
7.
Valverde JR, Alonso J, Palacios I, Pestana A. RB1 gene mutation up-date, a meta-analysis based on 932 reported mutations available in a searchable database. BMC Genet. 2005;6:53.CrossRefPubMedPubMedCentral
8.
Lohmann D and Gallie B: Retinoblastoma. [http://​www.​ncbi.​nlm.​nih.​gov/​books/​NBK1452]
9.
Liu L, Li Y, Li S, Hu N, He Y, Pong R, et al. Comparison of next-generation sequencing systems. J Biomed Biotechnol. 2012;2012:251364.PubMedPubMedCentral
10.
Quail MA, Smith M, Coupland P, Otto TD, Harris SR, Connor TR, et al. A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC Genomics. 2012;13:341.CrossRefPubMedPubMedCentral
13.
Thorvaldsdóttir H, Robinson JT, Mesirov JP. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform. 2013;14:178–92.CrossRefPubMed
14.
Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nat Biotechnol. 2011;29:24–6.CrossRefPubMedPubMedCentral
17.
Lepri FR, Scavelli R, Digilio MC, Gnazzo M, Grotta S, Dentici ML, et al. Diagnosis of Noonan syndrome and related disorders using target next generation sequencing. BMC Med Genet. 2014;15:14.CrossRefPubMedPubMedCentral
18.
Krawitz PM, Schiska D, Kru¨ger U, Appelt S, Heinrich V, Parkhomchuk D, et al. Screening for single nucleotide variants, small indels and exon deletions with a next-generation sequencing based gene panel approach for Usher syndrome. Mol Genet Geno Med. 2014;2(5):393–401.CrossRef
19.
D’Elia G, Grotta S, Del Bufalo F, De Ioris MA, Surace C, Sirleto P, et al. Two novel cases of trilateral retinoblastoma: genetics and review of the literature. Cancer Genet. 2013;206:398–401.CrossRefPubMed
20.
Skrypnyk C, Bartsch O. Retinoblastoma, pinealoma, and mild overgrowth in a boy with a deletion of RB1 and neighbor genes on chromosome 13q14. Am J Med Genet A. 2004;124A:397–401.CrossRefPubMed
21.
Amare P, Jose J, Chitalkar P, Kurkure P, Pai S, Nair C, et al. Trilateral retinoblastoma with an RB1 deletion inherited from a carrier mother: a case report. Cancer Genet Cytogenet. 1999;111:28–31.CrossRefPubMed
22.
Kivelä T, Tuppurainen K, Riikonen P, Vapalahti M. Retinoblastoma associated with chromosomal 13q14 deletion mosaicism. Ophthalmology. 2003;110:1983–8.CrossRefPubMed
24.
Lohmann DR, Gerick M, Brandt B, Oelschläger U, Lorenz B, Passarge E, et al. Constitutional RB1-gene mutations in patients with isolated unilateral retinoblastoma. Am J Hum Genet. 1997;61:282–94.CrossRefPubMedPubMedCentral
25.
Dehainault C, Garancher A, Castéra L, Cassoux N, Aerts I, Doz F, et al. The survival gene MED4 explains low penetrance retinoblastoma in patients with large RB1 deletion. Hum Mol Genet. 2014;23:5243–50.CrossRefPubMed
26.
Richter S, Vandezande K, Chen N, Zhang K, Sutherland J, Anderson J, et al. Sensitive and efficient detection of RB1 gene mutations enhances care for families with retinoblastoma. Am J Hum Genet. 2003;72:253–69.CrossRefPubMed
27.
Rushlow D, Piovesan B, Zhang K, Prigoda-Lee NL, Marchong MN, Clark RD, et al. Detection of mosaic RB1 mutations in families with retinoblastoma. Hum Mutat. 2009;30:842–51.CrossRefPubMed
28.
Chen Z, Moran K, Richards-Yutz J, Toorens E, Gerhart D, Ganguly T, et al. Enhanced sensitivity for detection of low-level germline mosaic RB1 mutations in sporadic retinoblastoma cases using deep semiconductor sequencing. Hum Mutat. 2014;35:384–91.CrossRefPubMed
29.
Cowell JK, Smith T, Bia B. Frequent constitutional C to T mutations in CGA-arginine codons in the RB1 gene produce premature stop codons in patients with bilateral (hereditary) retinoblastoma. Eur J Hum Genet. 1994;2:281–90.PubMed
30.
Liu Z, Song Y, Bia B, Cowell JK. Germline mutations in the RB1 gene in patients with hereditary retinoblastoma. Genes Chromosomes Cancer. 1995;14:277–84.CrossRefPubMed
31.
Braggio E, Bonvicino CR, Vargas FR, Ferman S, Eisenberg AL, Seuánez HN. Identification of three novel RB1 mutations in Brazilian patients with retinoblastoma by “exon by exon” PCR mediated SSCP analysis. J Clin Pathol. 2004;57:585–90.CrossRefPubMedPubMedCentral
32.
Blanquet V, Turleau C, Gross-Morand MS, Sénamaud-Beaufort C, Doz F, Besmond C. Spectrum of germline mutations in the RB1 gene: a study of 232 patients with hereditary and non hereditary retinoblastoma. Hum Mol Genet. 1995;4:383–8.CrossRefPubMed
33.
Houdayer C, Gauthier-Villars M, Laugé A, Pagès-Berhouet S, Dehainault C, Caux-Moncoutier V, et al. Comprehensive screening for constitutional RB1 mutations by DHPLC and QMPSF. Hum Mutat. 2004;23:193–202.CrossRefPubMed
34.
Abouzeid H, Munier FL, Thonney F, Schorderet DF. Ten novel RB1 gene mutations in patients with retinoblastoma. Mol Vis. 2007;13:1740–5.PubMed
35.
Weir-Thompson E, Condie A, Leonard RC, Prosser J. A familial RB1 mutation detected by the HOT technique is homozygous in a second primary neoplasm. Oncogene. 1991;6:2353–6.PubMed