April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Rapid Sequencing Analysis Of The RB1 Gene In Families With Retinoblastoma
Author Affiliations & Notes
  • Uma M. Sachdeva
    University of Pennsylvania, Philadelphia, Pennsylvania
  • Mallory K. Mest
    Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania
  • David W. Collins
    Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania
  • Kurtis R. Van Quill
    Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania
  • Joan M. O'Brien
    Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania
  • Footnotes
    Commercial Relationships  Uma M. Sachdeva, None; Mallory K. Mest, None; David W. Collins, None; Kurtis R. Van Quill, None; Joan M. O'Brien, None
  • Footnotes
    Support  NEI Grant EY13812, National Ophthalmic Disease Genotyping Network, Wayne and Gladys Valley Foundation
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 2472. doi:
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      Uma M. Sachdeva, Mallory K. Mest, David W. Collins, Kurtis R. Van Quill, Joan M. O'Brien; Rapid Sequencing Analysis Of The RB1 Gene In Families With Retinoblastoma. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2472.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : To develop a novel, rapid sequencing method to identify mutations in the retinoblastoma (RB1) gene in patients and families with heritable retinoblastoma

Methods: : Genomic DNA isolated from peripheral blood of patients with heritable retinoblastoma was received from the National Ophthalmic Disease Genotyping Network and was examined for mutations in the RB1 gene using novel cross-validated approaches for rapid sequencing analysis. Protocols were developed to examine the promoter, splice sites, and exon sequences of the RB1 gene for point mutations and small insertions or deletions by automated Sanger sequencing and to detect large deletions or duplications through multiplex ligation-dependent probe amplification (MLPA). By automating and combining these processes, we have developed a novel, high-throughput method to rapidly detect mutations in families with heritable retinoblastoma.

Results: : Unique primers were designed to sequence the RB1 promoter 215 bases upstream of the initiation codon, the 27 protein-coding exons, and the splice site junctions of the RB1 gene in over 90 samples from families with heritable retinoblastoma. Using Biomek® 3000 (Beckman Coulter, Brea, CA) liquid handling workstations, we analyzed multiple replicates of each sample in 384-well plates using cross-validated, blinded sequencing runs. We were able to detect the genetic alterations most associated with risk for retinoblastoma, including heterozygous point mutations, small insertions, and deletions, with over 95% sensitivity and specificity, providing results within one week. Using probes that hybridized at or near 23 of the 27 RB1 exons, we used MLPA to detect large heterozygous deletions and amplifcations with similar sensitivity and specificity. Together, these techniques can detect the major types of genetic alterations at the RB1 locus and return results within one week, providing rapid access to genetic information to inform treatment decisions and family counseling.

Conclusions: : Through the combination of high-throughput automated exon and promoter sequencing and MLPA, common and novel mutations at the RB1 locus can be rapidly detected within one week from peripheral blood samples of affected families. These tests can be used to confirm known familial mutations in children of retinoblastoma patients, to exclude the presence of known familial mutations, or to identify novel mutations.

Keywords: retinoblastoma • genetics • mutations 

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