We have analyzed RNA from retinoblastoma patients and unaffected carriers with various RB1 gene mutations to determine the patterns of missplicing and associations with phenotypic expression. Most sequence alterations in or in the neighborhood of conserved splice signals that we tested resulted in simple exon skipping (15 mutations) or intron inclusion (new acceptor AG-sites, four mutations) as expected. Two mutations resulted in skipping of a neighboring exon (exon 11), a complex pattern indicating competition for correct lariat formation. We observed no activation of a cryptic splice site but found that a recurrent missense mutation in exon 7 creates a new splice site (two families). RT-PCR analysis enabled us to confirm the presence and to characterize the transcriptional consequences of gross insertions and deletions in the RB1 gene in six patients, including two patients with mutational mosaicism. We also used RT-PCR analysis to search for unknown mutations in 15 patients and identified three oncogenic point mutations deep in introns. Two of these mutations are recurrent thus indicating that, despite the vast extent of the introns of the RB1 gene, few bases are effective targets for oncogenic mutations. When analyzing associations between phenotypic expression (16 families) and mutational consequences we observed no link to the presence or absence of a premature termination codon in the mutant transcript. However, the location of a mutation relative to the splice sequence has a strong and consistent influence on phenotypic expression.

Retinoblastoma (Rb) is initiated by germline mutations in the RB1 gene. Up to date, no mutation was identified in exons 26 and 27. We have identified a 2 bp frameshift insertion in exon 27 of the RB1 gene (RBg.177008_177009dup) in a boy with unilateral Rb and his healthy father that has occurred de novo on the allele transmitted by the father's father. RT-PCR showed that the mutant +2 bp transcript is present in RNA from peripheral leukocytes after short-term culture. The level of the mutant transcript was low compared to the normal transcript indicating abnormal expression of the variant allele. The mutant transcript was further reduced after puromycin treatment suggesting that NMD is not involved. Although oncogenic mutations in the terminal exons of the RB1 gene are rare molecular testing is important as those terminal mutations can be associated with incomplete penetrance and cause high recurrence risk in family members.