Skipping of BRCA2 exon 3 (∆E3) is a naturally occurring splicing event, complicating clinical classification of variants that may alter ∆E3 expression. This study used multiple evidence types to assess pathogenicity of 85 variants in/near BRCA2 exon 3. Bioinformatically predicted spliceogenic variants underwent mRNA splicing analysis using minigenes and/or patient samples. ∆E3 was measured using quantitative analysis. A mouse embryonic stem cell (mESC) based assay was used to determine the impact of 18 variants on mRNA splicing and protein function. For each variant, population frequency, bioinformatic predictions, clinical data, and existing mRNA splicing and functional results were collated. Variant class was assigned using a gene-specific adaptation of ACMG/AMP guidelines, following a recently proposed points-based system. mRNA and mESC analysis combined identified six variants with transcript and/or functional profiles interpreted as loss of function. Cryptic splice site use for acceptor site variants generated a transcript encoding a shorter protein that retains activity. Overall, 69/85 (81%) variants were classified using the points-based approach. Our analysis shows the value of applying gene-specific ACMG/AMP guidelines using a points-based approach and highlights the consideration of cryptic splice site usage to appropriately assign PVS1 code strength.

• Keywords
• ACMG/AMP classification, BRCA2, dPCR, functional analysis, quantitation, splicing,
• MeSH
• Alternative Splicing MeSH
• Genes, BRCA2 * MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• RNA Splice Sites * MeSH
• Mice MeSH
• BRCA2 Protein genetics   metabolism   MeSH
• RNA Splicing MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• BRCA2 protein, human MeSH Browser
• RNA, Messenger MeSH
• RNA Splice Sites * MeSH
• BRCA2 Protein MeSH

U2AF65 (U2AF2) and PUF60 (PUF60) are splicing factors important for recruitment of the U2 small nuclear ribonucleoprotein to lariat branch points and selection of 3' splice sites (3'ss). Both proteins preferentially bind uridine-rich sequences upstream of 3'ss via their RNA recognition motifs (RRMs). Here, we examined 36 RRM substitutions reported in cancer patients to identify variants that alter 3'ss selection, RNA binding and protein properties. Employing PUF60- and U2AF65-dependent 3'ss previously identified by RNA-seq of depleted cells, we found that 43% (10/23) and 15% (2/13) of independent RRM mutations in U2AF65 and PUF60, respectively, conferred splicing defects. At least three RRM mutations increased skipping of internal U2AF2 (~9%, 2/23) or PUF60 (~8%, 1/13) exons, indicating that cancer-associated RRM mutations can have both cis- and trans-acting effects on splicing. We also report residues required for correct folding/stability of each protein and map functional RRM substitutions on to existing high-resolution structures of U2AF65 and PUF60. These results identify new RRM residues critical for 3'ss selection and provide relatively simple tools to detect clonal RRM mutations that enhance the mRNA isoform diversity.

• Keywords
• 3′ splice site, Functional genomics, PUF60, SF3B4, U2AF2, cancer, differential scanning fluorimetry, driver mutation, exon inclusion, gel shift assay, lariat branch point, leukemia, mRNA, pre-mRNA splicing,
• Publication type
• Journal Article MeSH

UNLABELLED: Mutations affecting splicing underlie the development of many human genetic diseases, but rather rarely through mechanisms of pseudoexon activation. Here, we describe a novel c.1092T>A mutation in the iduronate-2-sulfatase (IDS) gene detected in a patient with significantly decreased IDS activity and a clinical diagnosis of mild mucopolysaccharidosis II form. The mutation created an exonic de novo acceptor splice site and resulted in a complex splicing pattern with multiple pseudoexon activation in the patient's fibroblasts. Using an extensive series of minigene splicing experiments, we showed that the competition itself between the de novo and authentic splice site led to the bypass of the authentic one. This event then resulted in activation of several cryptic acceptor and donor sites in the upstream intron. As this was an unexpected and previously unreported mechanism of aberrant pseudoexon inclusion, we systematically analysed and disproved that the patient's mutation induced any relevant change in surrounding splicing regulatory elements. Interestingly, all pseudoexons included in the mature transcripts overlapped with the IDS alternative terminal exon 7b suggesting that this sequence represents a key element in the IDS pre-mRNA architecture. These findings extend the spectrum of mechanisms enabling pseudoexon activation and underscore the complexity of mutation-induced splicing aberrations. KEY MESSAGE: Novel exonic IDS gene mutation leads to a complex splicing pattern. Mutation activates multiple pseudoexons through a previously unreported mechanism. Multiple cryptic splice site (ss) activation results from a bypass of authentic ss. Authentic ss bypass is due to a competition between de novo and authentic ss.

• Keywords
• Complex splicing aberration, De novo splice site, IDS, Pseudoexon, Splice site competition,
• MeSH
• Point Mutation MeSH
• Exons MeSH
• Glycoproteins genetics   MeSH
• Introns MeSH
• Humans MeSH
• RNA, Messenger genetics   MeSH
• RNA Splice Sites MeSH
• Adolescent MeSH
• Mucopolysaccharidosis II genetics   MeSH
• Mutation MeSH
• RNA Splicing MeSH
• Check Tag
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glycoproteins MeSH
• IDS protein, human MeSH Browser
• RNA, Messenger MeSH
• RNA Splice Sites MeSH

The prevalent c.903+469T>C mutation in MTRR causes the cblE type of homocystinuria by strengthening an SRSF1 binding site in an ESE leading to activation of a pseudoexon. We hypothesized that other splicing regulatory elements (SREs) are also critical for MTRR pseudoexon inclusion. We demonstrate that the MTRR pseudoexon is on the verge of being recognized and is therefore vulnerable to several point mutations that disrupt a fine-tuned balance between the different SREs. Normally, pseudoexon inclusion is suppressed by a hnRNP A1 binding exonic splicing silencer (ESS). When the c.903+469T>C mutation is present two ESEs abrogate the activity of the ESS and promote pseudoexon inclusion. Blocking the 3'splice site or the ESEs by SSOs is effective in restoring normal splicing of minigenes and endogenous MTRR transcripts in patient cells. By employing an SSO complementary to both ESEs, we were able to rescue MTRR enzymatic activity in patient cells to approximately 50% of that in controls. We show that several point mutations, individually, can activate a pseudoexon, illustrating that this mechanism can occur more frequently than previously expected. Moreover, we demonstrate that SSO blocking of critical ESEs is a promising strategy to treat the increasing number of activated pseudoexons.

• MeSH
• Cell Line MeSH
• Exons * MeSH
• Ferredoxin-NADP Reductase genetics   metabolism   MeSH
• HEK293 Cells MeSH
• Homocystinuria enzymology   genetics   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Anemia, Megaloblastic enzymology   genetics   MeSH
• RNA Splice Sites MeSH
• Mutation * MeSH
• Oligonucleotides * MeSH
• Regulatory Sequences, Ribonucleic Acid * MeSH
• RNA Splicing * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Ferredoxin-NADP Reductase MeSH
• methionine synthase reductase MeSH Browser
• RNA Splice Sites MeSH
• Oligonucleotides * MeSH
• Regulatory Sequences, Ribonucleic Acid * MeSH

Mutations in the first nucleotide of exons (E(+1)) mostly affect pre-mRNA splicing when found in AG-dependent 3' splice sites, whereas AG-independent splice sites are more resistant. The AG-dependency, however, may be difficult to assess just from primary sequence data as it depends on the quality of the polypyrimidine tract. For this reason, in silico prediction tools are commonly used to score 3' splice sites. In this study, we have assessed the ability of sequence features and in silico prediction tools to discriminate between the splicing-affecting and non-affecting E(+1) variants. For this purpose, we newly tested 16 substitutions in vitro and derived other variants from literature. Surprisingly, we found that in the presence of the substituting nucleotide, the quality of the polypyrimidine tract alone was not conclusive about its splicing fate. Rather, it was the identity of the substituting nucleotide that markedly influenced it. Among the computational tools tested, the best performance was achieved using the Maximum Entropy Model and Position-Specific Scoring Matrix. As a result of this study, we have now established preliminary discriminative cut-off values showing sensitivity up to 95% and specificity up to 90%. This is expected to improve our ability to detect splicing-affecting variants in a clinical genetic setting.

• MeSH
• Agammaglobulinemia genetics   MeSH
• Point Mutation * MeSH
• Exons MeSH
• Genetic Diseases, X-Linked genetics   MeSH
• HeLa Cells MeSH
• Humans MeSH
• RNA Splice Sites * MeSH
• Models, Genetic MeSH
• Molecular Sequence Data MeSH
• Computer Simulation MeSH
• Agammaglobulinaemia Tyrosine Kinase MeSH
• Sequence Analysis, DNA MeSH
• RNA Splicing MeSH
• Software * MeSH
• Protein-Tyrosine Kinases genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA Splice Sites * MeSH
• Agammaglobulinaemia Tyrosine Kinase MeSH
• Protein-Tyrosine Kinases MeSH

Deep intronic mutations are often ignored as possible causes of human diseases. A deep intronic mutation in the MTRR gene, c.903+469T>C, is the most frequent mutation causing the cblE type of homocystinuria. It is well known to be associated with pre-mRNA mis-splicing, resulting in pseudoexon inclusion; however, the pathological mechanism remains unknown. We used minigenes to demonstrate that this mutation is the direct cause of MTRR pseudoexon inclusion, and that the pseudoexon is normally not recognized due to a suboptimal 5' splice site. Within the pseudoexon we identified an exonic splicing enhancer (ESE), which is activated by the mutation. Cotransfection and siRNA experiments showed that pseudoexon inclusion depends on the cellular amounts of SF2/ASF and in vitro RNA-binding assays showed dramatically increased SF2/ASF binding to the mutant MTRR ESE. The mutant MTRR ESE sequence is identical to an ESE of the alternatively spliced MST1R proto-oncogene, which suggests that this ESE could be frequently involved in splicing regulation. Our study conclusively demonstrates that an intronic single nucleotide change is sufficient to cause pseudoexon activation via creation of a functional ESE, which binds a specific splicing factor. We suggest that this mechanism may cause genetic disease much more frequently than previously reported.

• MeSH
• Chlorocebus aethiops MeSH
• COS Cells MeSH
• Exons genetics   MeSH
• Ferredoxin-NADP Reductase genetics   MeSH
• Homocystinuria classification   enzymology   genetics   MeSH
• Introns genetics   MeSH
• Nuclear Proteins metabolism   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• RNA Splice Sites genetics   MeSH
• Molecular Sequence Data MeSH
• Mutation genetics   MeSH
• Mutant Proteins genetics   MeSH
• RNA-Binding Proteins metabolism   MeSH
• Proto-Oncogene Mas MeSH
• Base Sequence MeSH
• Serine-Arginine Splicing Factors MeSH
• RNA Splicing genetics   MeSH
• Protein Binding MeSH
• Vitamin B 12 metabolism   MeSH
• Computational Biology MeSH
• Enhancer Elements, Genetic genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Ferredoxin-NADP Reductase MeSH
• Nuclear Proteins MeSH
• MAS1 protein, human MeSH Browser
• RNA, Messenger MeSH
• methionine synthase reductase MeSH Browser
• RNA Splice Sites MeSH
• Mutant Proteins MeSH
• RNA-Binding Proteins MeSH
• Proto-Oncogene Mas MeSH
• Serine-Arginine Splicing Factors MeSH
• Vitamin B 12 MeSH

The p53 tumour suppressor protein lies at the crossroads of multiple cellular response pathways that control the fate of the cell in response to endogenous or exogenous stresses and inactivation of the p53 tumour suppressor signalling pathway is seen in most human cancers. Such aberrant p53 activity may be caused by mutations in the TP53 gene sequence producing truncated or inactive mutant proteins, or by aberrant production of other proteins that regulate p53 activity, such as gene amplification and overexpression of MDM2 or viral proteins that inhibit or degrade p53. Recent studies have also suggested that inherited genetic polymorphisms in the p53 pathway influence tumour formation, progression and/or response to therapy. In some cases, these variants are clearly associated with clinico-pathological variables or prognosis of cancer, whereas in other cases the evidence is less conclusive. Here, we review the evidence that common polymorphisms in various aspects of p53 biology have important consequences for overall tumour susceptibility, clinico-pathology and prognosis. We also suggest reasons for some of the reported discrepancies in the effects of common polymorphisms on tumourigenesis, which relate to the complexity of effects on tumour formation in combination with other oncogenic changes and other polymorphisms. It is likely that future studies of combinations of polymorphisms in the p53 pathway will be useful for predicting tumour susceptibility in the human population and may serve as predictive biomarkers of tumour response to standard therapies.

• MeSH
• Exons genetics   MeSH
• Genetic Predisposition to Disease * MeSH
• Humans MeSH
• Mutation genetics   MeSH
• Tumor Suppressor Protein p53 chemistry   genetics   metabolism   MeSH
• Neoplasms genetics   therapy   MeSH
• Polymorphism, Genetic * MeSH
• Amino Acid Sequence MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Tumor Suppressor Protein p53 MeSH