title: Advantages of a next generation sequencing targeted approach for the molecular diagnosis of retinoblastoma.
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.
authors.affiliation: Laboratory of Medical Genetics, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant'Onofrio 4, 00165, Rome, Italy. simona.grotta841@gmail.com.|Present address: S. Pietro Fatebenefratelli Hospital, UOSD Medical Genetics, Rome, Italy. simona.grotta841@gmail.com.
authors.forename: Simona
authors.initials: S
authors.lastname: Grotta
authors.affiliation: Laboratory of Medical Genetics, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant'Onofrio 4, 00165, Rome, Italy. gemma.delia@opbg.net.
authors.forename: Gemma
authors.initials: G
authors.lastname: D'Elia
authors.affiliation: Illumina, Inc, San Diego, CA, 92122, USA. rscavelli@illumina.com.
authors.forename: Rossana
authors.initials: R
authors.lastname: Scavelli
authors.affiliation: Laboratory of Medical Genetics, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant'Onofrio 4, 00165, Rome, Italy. silvia.genovese@opbg.net.
authors.forename: Silvia
authors.initials: S
authors.lastname: Genovese
authors.affiliation: Laboratory of Medical Genetics, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant'Onofrio 4, 00165, Rome, Italy. cecilia.surace@opbg.net.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Surace
authors.affiliation: Laboratory of Medical Genetics, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant'Onofrio 4, 00165, Rome, Italy. pietro.sirleto@opbg.net.
authors.forename: Pietro
authors.initials: P
authors.lastname: Sirleto
authors.affiliation: Department of Pediatric Hematology-Oncology and Stem Cell Transplantation, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant'Onofrio 4, Rome, Italy. raffaele.cozza@opbg.net.
authors.forename: Raffaele
authors.initials: R
authors.lastname: Cozza
authors.affiliation: Ophtalmology Unit, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant'Onofrio 4, Rome, Italy. antonino.romanzo@opbg.net.
authors.forename: Antonino
authors.initials: A
authors.lastname: Romanzo
authors.affiliation: Department of Pediatric Hematology-Oncology and Stem Cell Transplantation, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant'Onofrio 4, Rome, Italy. mariaa.deioris@opbg.net.
authors.forename: Maria Antonietta
authors.initials: MA
authors.lastname: De Ioris
authors.affiliation: Ophtalmology Unit, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant'Onofrio 4, Rome, Italy. paola.valente@opbg.net.
authors.forename: Paola
authors.initials: P
authors.lastname: Valente
authors.affiliation: Laboratory of Medical Genetics, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant'Onofrio 4, 00165, Rome, Italy. acristina.tomaiuolo@opbg.net.
authors.forename: Anna Cristina
authors.initials: AC
authors.lastname: Tomaiuolo
authors.affiliation: Laboratory of Medical Genetics, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant'Onofrio 4, 00165, Rome, Italy. francescaromana.lepri@opbg.net.
authors.forename: Francesca Romana
authors.initials: FR
authors.lastname: Lepri
authors.affiliation: Laboratory of Medical Genetics, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant'Onofrio 4, 00165, Rome, Italy. tiziana.franchin@opbg.net.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Franchin
authors.affiliation: Laboratory of Medical Genetics, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant'Onofrio 4, 00165, Rome, Italy. laura.ciocca@opbg.net.
authors.forename: Laura
authors.initials: L
authors.lastname: Ciocca
authors.affiliation: Laboratory of Medical Genetics, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant'Onofrio 4, 00165, Rome, Italy. serena.russo@opbg.net.
authors.forename: Serena
authors.initials: S
authors.lastname: Russo
authors.affiliation: Department of Pediatric Hematology-Oncology and Stem Cell Transplantation, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant'Onofrio 4, Rome, Italy. franco.locatelli@opbg.net.|University of Pavia, Pavia, Italy. franco.locatelli@opbg.net.
authors.forename: Franco
authors.initials: F
authors.lastname: Locatelli
authors.affiliation: Laboratory of Medical Genetics, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant'Onofrio 4, 00165, Rome, Italy. adriano.angioni@opbg.net.
authors.forename: Adriano
authors.initials: A
authors.lastname: Angioni
mesh_terms: D000483:Alleles; D002869:Chromosome Aberrations; D055028:Comparative Genomic Hybridization; D005091:Exons / Q000235:genetics; D005260:Female; D017353:Gene Deletion; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D009154:Mutation; D057089:Pathology, Molecular; D012175:Retinoblastoma / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D016160:Retinoblastoma Protein / Q000235:genetics*
chemical_list: D016160:Retinoblastoma Protein