Sanger sequencing
RB1 mutation testing, with special attention toward exon 25, was performed using conventional Sanger sequencing on all DNA samples originating from FFPE blocks of the patient and on the peripherial blood DNA samples of their parents. The purified PCR products were sequenced using both forward and reverse primers (which were used for the PCR amplification) using the BigDye Terminator v3.1 Cycle Sequencing kit. The samples were analysed on the ABI PRISM 310 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA).