title: Enhanced sensitivity for detection of low-level germline mosaic RB1 mutations in sporadic retinoblastoma cases using deep semiconductor sequencing.
abstract: Sporadic retinoblastoma (RB) is caused by de novo mutations in the RB1 gene. Often, these mutations are present as mosaic mutations that cannot be detected by Sanger sequencing. Next-generation deep sequencing allows unambiguous detection of the mosaic mutations in lymphocyte DNA. Deep sequencing of the RB1 gene on lymphocyte DNA from 20 bilateral and 70 unilateral RB cases was performed, where Sanger sequencing excluded the presence of mutations. The individual exons of the RB1 gene from each sample were amplified, pooled, ligated to barcoded adapters, and sequenced using semiconductor sequencing on an Ion Torrent Personal Genome Machine. Six low-level mosaic mutations were identified in bilateral RB and four in unilateral RB cases. The incidence of low-level mosaic mutation was estimated to be 30% and 6%, respectively, in sporadic bilateral and unilateral RB cases, previously classified as mutation negative. The frequency of point mutations detectable in lymphocyte DNA increased from 96% to 97% for bilateral RB and from 13% to 18% for unilateral RB. The use of deep sequencing technology increased the sensitivity of the detection of low-level germline mosaic mutations in the RB1 gene. This finding has significant implications for improved clinical diagnosis, genetic counseling, surveillance, and management of RB.
authors.affiliation: Genetic Diagnostic Laboratory, Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Chen
authors.forename: Kimberly
authors.initials: K
authors.lastname: Moran
authors.forename: Jennifer
authors.initials: J
authors.lastname: Richards-Yutz
authors.forename: Erik
authors.initials: E
authors.lastname: Toorens
authors.forename: Daniel
authors.initials: D
authors.lastname: Gerhart
authors.forename: Tapan
authors.initials: T
authors.lastname: Ganguly
authors.forename: Carol L
authors.initials: CL
authors.lastname: Shields
authors.forename: Arupa
authors.initials: A
authors.lastname: Ganguly
mesh_terms: D000483:Alleles; D002138:Calibration; D002675:Child, Preschool; D004252:DNA Mutational Analysis; D005091:Exons; D005260:Female; D017353:Gene Deletion; D020440:Gene Duplication; D016161:Genes, Retinoblastoma; D005838:Genotype; D018095:Germ-Line Mutation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D012175:Retinoblastoma / Q000175:diagnosis / Q000235:genetics*; D016160:Retinoblastoma Protein / Q000235:genetics*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
chemical_list: D016160:Retinoblastoma Protein
keywords: RB1; mosaic mutation; next-generation sequencing; sporadic retinoblastoma