Patterns of missplicing caused byRB1gene mutations in patients with retinoblastoma and association with phenotypic expression

Abstract

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.

Impact of BRCA1 and BRCA2 variants on splicing: clues from an allelic imbalance study

“…Second, it also appears that deep intronic mutations are not an important part of the molecular spectrum of BRCA genes. To our knowledge, there is no deep intronic mutation reported to date in BRCA1/2 and this is perfectly in line with data reported on Impact of BRCA1/2 variants on splicing V Caux-Moncoutier et al other genes such as NF1 4 and RB1, 34 showing that deep intronic mutations account for a mere 1% of their mutational spectrum. The mutation detection rate for BRCA1 and BRCA2 among HBOC families in France was close to 14% in 2006 (http://www.e-cancer.fr/v1/fichiers/public/figures_rapport_ oncogenetique_2003_2006.pdf), meaning not only that other genes are probably involved but also that a substantial number of BRCA1/2 mutations have yet to be found outside the coding sequences and intron/exon boundaries, which are screened in routine diagnostic settings.…”

Genome-wide discovery of human splicing branchpoints

“…For example, we identified a branchpoint nucleotide within the RB1 gene that when mutated, results in the skipping of the downstream exon in patients with retinoblastoma ( Fig. 6C; Houdayer et al 2008;Zhang et al 2008). Similarly, we identified a branchpoint within the MET oncogene that when deleted, results in skipping of exons encoding the juxtamembrane domain, resulting in MET activation in lung adenocarcinoma (Onozato et al 2009).…”

Detection of mosaicRB1mutations in families with retinoblastoma

“…For bilateral probands with no mutation found in blood, we tested the patient's tumor-if available-and fresh blood by reverse transcriptase-PCR (RT-PCR) to help detect an RB1 mutation. RT-PCR was also performed for intronic changes of uncertain significance [Zhang et al, 2008]. For unilateral tumors, we searched for two RB1 inactivating events, using QM-PCR, sequence , and promoter methylation analysis, and then tested blood for each tumor change, including AS-PCR for any AS-PCR detectable mutation in the tumor.…”