Test Description

RB1 coding exons 1-27 and well into the 5’ and 3’ ends of all the introns and untranslated regions are analyzed by sequencing. Gross deletion/duplication analysis determines gene copy number for coding exons 1-27. Clinically significant intronic findings beyond 5 base pairs are always reported. Intronic variants of unknown or unlikely clinical significance are not reported beyond 5 base pairs from the splice junction. Genomic deoxyribonucleic acid (gDNA) is isolated from the patient’s specimen using standardized methodology and quantified. Sequence enrichment of the targeted coding exons and adjacent intronic nucleotides is carried out by a bait-capture methodology, using long biotinylated oligonucleotide probes followed by polymerase chain reaction (PCR) and next generation sequencing (NGS). Sanger sequencing is performed for any regions missing or with insufficient read depth coverage for reliable heterozygous variant detection. Potentially homozygous variants, variants in regions complicated by pseudogene interference, and variant calls not satisfying depth of coverage and variant allele frequency quality thresholds are verified by Sanger sequencing. Gross deletion/duplication analysis of RB1 using multiplex ligation-dependent probe amplification (MLPA) and/or targeted chromosomal microarray is also performed.

1. Rushlow D, et al. Detection of mosaic RB1 mutations in families with retinoblastoma. Hum Mutat. 2009:30(5):842-51.

2. Nichols KE, et al. Recent advances in retinoblastoma genetic research. Curr Opin Ophthalmol.2009;20(5):351-5.