Enhanced detection of low-level mosaic mutations in RB1 gene in Sporadic Unilateral RB by Ion Torrent semiconductor sequencing: Risk of second cancer. Z. Chen1, S. Walther1, K. Moran1, D. Gerhart2, T. Ganguly2, A. Ganguly1 1) Genetic Diagnostic Laboratory, Department of Genetics, University of Pennsylvania, Philadelphia, PA; 2) DNA Sequencing Facility, Department of Genetics, Perelman school of Medicine, University of Pennsylvania.

   BACKGROUND: Retinoblastoma (RB) is the most common intraocular cancer in children. Sporadic RB is caused by de novo germline mutations in RB1 gene in ~80% of newly diagnosed bilateral cases and ~15% of unilateral cases. De novo germline mutation may be the result of either a pre-conception mutation in a parental germ cell, or an early post-zygotic mutation giving rise to somatic and/or germline mosaicism. Detection of a mosaic mutation, which is challenging, alters the future risk of second cancer for the affected individual. Deep sequencing methods are significantly more efficient in detecting low-level mosaic mutations missed by traditional sequencing. METHODS: We performed deep sequencing of RB1 gene on DNA isolated from blood of 69 RB probands with unilateral disease, with 93 somatic mutations identified in tumors, but without any germline mutation. The individual exons of RB1 gene with known somatic mutations were amplified using DNA of the respective individual. The amplicons were pooled in equimolar concentrations, barcoded libraries prepared and sequenced on the Ion PGM using 316 chip and 100b sequencing kit. Sequencing was performed in duplicate. To estimate the level of background sequencing error, all the tested exons were amplified using DNA from three healthy individuals, pooled, and sequenced following the same procedure. Sequence data was analyzed by Torrent Suite (Life Technologies) and NextGENe software (Softgenetics). RESULTS: About 250 Mb data were generated on a single 316 chip run and the average coverage for each exon was above 10,000. Five low-level mosaic mutations were identified. Sanger sequencing missed three of these mutations and two were detectable upon re-examination of the chromatograms. These variants were called with high statistical significance (p< 0.0001). Thus, the incidence of mosaic germline mutation is estimated to be 7.2% in unilateral RB probands. DISCUSSION: Use of deep sequencing platform, Ion PGM, yielded highly sensitive detection of low-level mosaic mutations in RB1 gene. While previous methods using allele specific PCR had predicted presence of ~3% mosaic mutations in unilateral RB, the current platform indicates that the rate can be as high as 7%. Considering, 15% as the standard germline mutation frequency in sporadic unilateral RB, this is a significant increase. This finding changes the genetic counseling for risk of second cancer in the proband and for future affected offsprings.

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