Enhanced Sensitivity for Detection of Low‐Level Germline Mosaic RB 1 Mutations in Sporadic Retinoblastoma Cases Using Deep Semiconductor Sequencing

Sporadic retinoblastoma ( RB ) is caused by de novo mutations in the RB 1 gene. Often, these mutations are present as mosaic mutations that cannot be detected by S anger sequencing. Next‐generation deep sequencing allows unambiguous detection of the mosaic mutations in lymphocyte DNA . Deep sequencing of the RB 1 gene on lymphocyte DNA from 20 bilateral and 70 unilateral RB cases was performed, where S anger sequencing excluded the presence of mutations. The individual exons of the RB 1 gene from each sample were amplified, pooled, ligated to barcoded adapters, and sequenced using semiconductor sequencing on an I on T orrent P ersonal G enome M achine. Six low‐level mosaic mutations were identified in bilateral RB and four in unilateral RB cases. The incidence of low‐level mosaic mutation was estimated to be 30% and 6%, respectively, in sporadic bilateral and unilateral RB cases, previously classified as mutation negative. The frequency of point mutations detectable in lymphocyte DNA increased from 96% to 97% for bilateral RB and from 13% to 18% for unilateral RB . The use of deep sequencing technology increased the sensitivity of the detection of low‐level germline mosaic mutations in the RB 1 gene. This finding has significant implications for improved clinical diagnosis, genetic counseling, surveillance, and management of RB .