Abstract
Methods: :
A man with a history of isolated bilateral retinoblastoma was tested for a germline RB1 mutation in the blood, using quantitative multiplex-PCR (QM-PCR), reverse-transcriptase PCR (RT-PCR), long-range PCR and sequencing analysis, as previously described. [Richter 2003] Once his mutation was identified, blood from his adult son, who was not affected with retinoblastoma but has been diagnosed with lymphoma, was tested for the presence of the same mutation.
Results: :
Using QM-PCR, the father’s RB1 mutation was identified as a deletion of exons 8-11 (g.59650_65506 del5857). RT-PCR showed that 50% of his mRNA transcripts skipped exons 8-11. Long range PCR showed a deletion of 5.8 kb of genomic DNA from the splice acceptor site preceding exon 8 to the beginning of intron 11. The son was found to carry a deletion around the same size as his father’s, but in a different region of the RB1 gene, from the middle of intron 6 to the splice acceptor region preceding exon 8 (g.54165_59641del5477). RT-PCR showed a normal transcript, as well as a mutant transcript with in-frame skipping of exon 7. Additionally, DNA showed that the normal exon 8 splice accepter site was destroyed by a deletion of nucleotides CAGAA and insertion of nucleotides TAT (g.59648_59652delCAGAAinsTAT), leading to probable activation of a cryptic splice acceptor site 6 bases downstream in exon 8 (splice score 84.8). Use of this cryptic splice acceptor site would lead to the inclusion of exon 8 (with an in-frame deletion of its first 6 base pairs) in the RB1 transcript.
Conclusions: :
The new position of the genomic deletion in the son is believed to have occurred during meiosis as a result of an aberrant cross-over event involving the father’s deleted allele, leading to an mRNA transcript with an in-frame deletion of exon 7 and six bases of exon 8. Such a meiotic event likely caused a "null" RB1 mutation to convert to a low penetrance retinoblastoma mutation in the second generation. However, the risk of retinoblastoma in future offspring is still significant.
Keywords: retinoblastoma • genetics • gene screening