Mosaicism and intronic variants in RB1 gene revealed by next generation sequencing in a cohort of Spanish retinoblastoma patients

Constitutional variants in the RB1 gene predispose individuals to the development of Retinoblastoma (RB) and the occurrence of second tumors in adulthood. Detection of causal RB1 gene variants is essential to establish the genetic diagnosis and to performing familial studies and counseling. In our cohort of 579 Spanish RB patients, 15% of cases suspected to have a genetic origin remained negative after traditional Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) of RB1 gene, likely due to the possibility of mosaicism or non-coding variants. A specific next-generation sequencing (NGS) gene panel was designed to analyze the complete sequence of the RB1 gene. While many familial RB cases showed variants through Sanger and MLPA, the analysis of 65 available sporadic RB patients using the NGS gene panel identified a causative variant in an additional 6 of 26 (23%) bilateral cases and 6 of 39 (15.4%) unilateral cases. Seven of these cases exhibited different degrees of mosaicism (26%, 20%, 15.8%, 8%, 6%, 5.9% and 3%) while 5 cases had heterozygous deep intronic variants, all of them previously described in RB patients. Additional cases with suspected variants, not detected in blood but present in tumor tissue, were also analyzed using NGS PCR amplicons, and mosaicism was confirmed in other 10 sporadic cases. Altogether, the use of NGS increased the diagnostic yield, particularly for patients with sporadic RB in 10 bilateral cases and in 12 unilateral cases.