A Rapid and Sensitive Next-Generation Sequencing Method to Detect RB1 Mutations Improves Care for Retinoblastoma Patients and Their Families

Abstract

Retinoblastoma is a childhood eye malignancy that can lead to the loss of vision, eye(s), and sometimes life. The tumors are initiated by inactivating mutations in both alleles of the tumor-suppressor gene, RB1, or, rarely, by MYCN amplification. Timely identification of a germline RB1 mutation in blood samples or either somatic RB1 mutation or MYCN amplification in tumors is important for effective care and management of retinoblastoma patients and their families. However, current procedures to thoroughly test RB1 mutations are complicated and lengthy. Herein, we report a next-generation sequencing-based method capable of detecting point mutations, small indels, and large deletions or duplications across the entire RB1 gene and amplification of MYCN gene on a single platform. From DNA extraction to clinical interpretation requires only 3 days, enabling early molecular diagnosis of retinoblastoma and optimal treatment outcomes. This method can also detect low-level mosaic mutations in blood samples that can be missed by routine Sanger sequencing. In addition, it can differentiate between RB1 mutation- and MYCN amplification-driven retinoblastomas. This rapid, comprehensive, and sensitive method for detecting RB1 mutations and MYCN amplification can readily identify RB1 mutation carriers and thus improve the management and genetic counseling for retinoblastoma patients and their families.

Figure 1

Coverage analysis of the RB1 next-generation sequencing test. A: The average coverage of each exon of RB1 gene, exons plus 2 bp splice junctions, the RB1 promoter, and the entire RB1 gene was plotted (from eight retinoblastoma blood samples). The line represents the coverage depth of 30×. B: Percentage of RB1 coverage on exons, the RB1 promoter, and the entire RB1 gene was plotted versus the coverage depth. The white, gray, and black bars represent the percentage of coverage on RB1 exons, the RB1 promoter, and the entire RB1 gene, respectively.

Figure 2

Copy number analysis of retinoblastoma samples. A: The entire RB1 gene, with vertical lines representing RB1 exons and introns between, is shown on the top. B–E: The light blue traces represent adjusted depth at all RB1 positions; the dark blue line represents the smoothed copy number (CN); and RB1 exons are labeled as four exon islands: exons 1 to 2, 3 to 6, 7 to 17, and 18 to 27. B: No copy number change in a retinoblastoma tumor sample. C: Heterozygous internal deletion of exons 2 to 6 in a retinoblastoma cell line Y79. D: Heterozygous deletion of entire RB1 gene in a retinoblastoma tumor sample. E: Homozygous deletion of exon 3 to 27 and multiplication of promoter-exon 2 of RB1 gene in a retinoblastoma tumor sample.

Figure 3

Evaluation of the RB1 next-generation sequencing test on mosaic mutation detection. A: Establishment of cutoffs for the mosaicism calling. Genomic DNA of two samples were mixed together with various proportions of each sample at 0%, 5%, 10%, 25%, 50%, 75%, 90%, 95%, and 100%, respectively. Measured mosaicism rate (y axis) was plotted against its nominal rate (x axis) on the basis of the admixture proportion of two samples. Quality scores were 10, 40, 100, 200, and 400 for <10%, 11% to 25%, 26% to 50%, 51% to 75%, and >76% of measured mosaicism, respectively. B: Detection of a mosaic mutation in a retinoblastoma blood sample. The frequency of the nucleotide observed at the position (y axis) was plotted versus Phred quality score (x axis). A mosaic splice mutation c.940-2A>T was detected in the retinoblastoma sample 20. C: Detection of a mosaic mutation in a blood sample from an unaffected relative of a retinoblastoma patient. A mosaic nonsense mutation c.508G>T (p.Glu170^∗) was detected in sample 21.

Figure 4

Quantification of MYCN copy number using the RB1 next-generation sequencing (NGS) test. A:MYCN copy number of 25 blood samples was measured with the RB1 NGS test. B:MYCN copy number of Y79 and KCN cell lines was measured. C: Admixture experiment was performed with different amounts of Y79 DNA spiked into normal control DNA of NA12878. The MYCN copy number was measured by the RB1 NGS test (solid line) as well as by the MYCN real-time quantitative PCR (qPCR) assay (broken line), and the result was plotted against the percentage of spiked-in Y79 DNA.