Detection of mosaic RB1 mutations in families with retinoblastoma

The RB1 gene mutation detection rate in 1,020 retinoblastoma families was increased by the use of highly sensitive allele specific-PCR (AS-PCR) to detect low-level mosaicism for 11 recurrent RB1 CGA>TGA nonsense mutations. For bilaterally affected probands, AS-PCR increased the RB1 mutation detection sensitivity from 92.6% to 94.8%. Both RB1 oncogenic changes were detected in 92.7% of sporadic unilateral tumors (357/385); 14.6% (52/357) of unilateral probands with both tumor mutations identified carried one of the tumor mutations in blood. Mosaicism was evident in 5.5% of bilateral probands (23 of 421), in 3.8% of unilateral probands (22 of 572), and in one unaffected mother of a unilateral proband. Half of the mosaic mutations were only detectable by AS-PCR for the 11 recurrent CGA>TGA mutations, and not by standard sequencing. This suggests that significant numbers of low-level mosaics with other classes of RB1 mutations remain unidentified by current technology. We show that the use of linkage analysis in a two-generation retinoblastoma family resulted in the erroneous conclusion that a child carried the parental mutation, because the founder parent was mosaic for the RB1 mutation. Of 142 unaffected parental pairs tested, only one unaffected parent of a proband (0.7%) showed somatic mosaicism for the proband's mutation, in contrast to an overall 4.5% somatic mosaicism rate for retinoblastoma probands, suggesting that mosaicism for an RB1 mutation is highly likely to manifest as retinoblastoma.