Biomedical subjects

Igor Vorechovský

Publications and source records attributed to Igor Vorechovský.

6 recordsLinked to original sources

PubMed2006

Aberrant 3' splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization.

The frequency distribution of mutation-induced aberrant 3' splice sites (3'ss) in exons and introns is more complex than for 5' splice sites, largely owing to sequence constraints upstream of intron/exon boundaries. As a result, prediction of their localization remains a challenging task. Here, nucleotide sequences of previously reported 218 aberrant 3'ss activated by disease-causing mutations in 131 human genes were compared with their authentic counterparts using currently available splice site prediction tools. Each tested algorithm distinguished authentic 3'ss from cryptic sites more effectively than from de novo sites. The best discrimination between aberrant and authentic 3'ss was achieved by the maximum entropy model. Almost one half of aberrant 3'ss was activated by AG-creating mutations and approximately 95% of the newly created AGs were selected in vivo. The overall nucleotide structure upstream of aberrant 3'ss was characterized by higher purine content than for authentic sites, particularly in position -3, that may be compensated by more stringent requirements for positive and negative nucleotide signatures centred around position -11. A newly developed online database of aberrant 3'ss will facilitate identification of splicing mutations in a gene or phenotype of interest and future optimization of splice site prediction tools.

Algorithms↗

PubMed2006

Phenotypic consequences of branch point substitutions.

The branch point sequence (BPS) is a conserved splicing signal important for spliceosome assembly and lariat intron formation. BPS mutations may result in aberrant pre-mRNA splicing and genetic disorders, but their phenotypic consequences have been difficult to predict, largely due to a highly degenerate nature of the BPS consensus. Here, we have examined the splicing pattern of nine reporter pre-mRNAs that have previously been shown to give rise to human hereditary diseases as a result of single-nucleotide substitutions in the predicted BPS. Increased exon skipping and intron retention observed in vivo were recapitulated for each mutated pre-mRNA, but the reproducibility of cryptic splice site activation was lower. BP mutations in reporter pre-mRNAs frequently induced aberrant 3' splice sites and also activated a cryptic 5' splice site. Systematic mutagenesis of BP adenosines showed that in most pre-mRNAs, the expression of canonical transcripts was lower for BP transitions than BP transversions. Differential splicing outcome for transitions vs. transversions was abrogated or reduced if introns were truncated to 200 nt or less, suggesting that the nature of the BP residue is less critical for interactions across very short introns. Together, these results improve prediction of phenotypic consequences of point mutations upstream of splice acceptor sites and suggest that the overrepresentation of disease-causing adenosine-to-guanosine BP substitutions observed in Mendelian disorders is due to more profound defects of gene expression at the level of pre-mRNA splicing.

Cell Line↗

PubMed2005

Biased exon/intron distribution of cryptic and de novo 3' splice sites.

We compiled sequences of previously published aberrant 3' splice sites (3'ss) that were generated by mutations in human disease genes. Cryptic 3'ss, defined here as those resulting from a mutation of the 3'YAG consensus, were more frequent in exons than in introns. They clustered in approximately 20 nt region adjacent to authentic 3'ss, suggesting that their under-representation in introns is due to a depletion of AG dinucleotides in the polypyrimidine tract (PPT). In contrast, most aberrant 3'ss that were induced by mutations outside the 3'YAG consensus (designated 'de novo') were in introns. The activation of intronic de novo 3'ss was largely due to AG-creating mutations in the PPT. In contrast, exonic de novo 3'ss were more often induced by mutations improving the PPT, branchpoint sequence (BPS) or distant auxiliary signals, rather than by direct AG creation. The Shapiro-Senapathy matrix scores had a good prognostic value for cryptic, but not de novo 3'ss. Finally, AG-creating mutations in the PPT that produced aberrant 3'ss upstream of the predicted BPS in vivo shared a similar 'BPS-new AG' distance. Reduction of this distance and/or the strength of the new AG PPT in splicing reporter pre-mRNAs improved utilization of authentic 3'ss, suggesting that AG-creating mutations that are located closer to the BPS and are preceded by weaker PPT may result in less severe splicing defects.

Base Sequence↗

PubMed2005

Exonization of AluYa5 in the human ACE gene requires mutations in both 3' and 5' splice sites and is facilitated by a conserved splicing enhancer.

Ancient Alu elements have been shown to be included in mature transcripts by point mutations that improve their 5' or 3' splice sites. We have examined requirements for exonization of a younger, disease-associated AluYa5 in intron 16 of the human ACE gene. A single G>C transversion in position -3 of the new Alu exon was insufficient for Alu exonization and a significant inclusion in mRNA was only observed when improving several potential splice donor sites in the presence of 3' CAG. Since complete Alu exonization was not achieved by optimizing traditional splicing signals, including the branch site, we tested whether auxiliary elements in AluYa5 were required for constitutive inclusion. Exonization was promoted by a SELEX-predicted heptamer in Alu consensus sequence 222-228 and point mutations in highly conserved nucleotides of this heptamer decreased Alu inclusion. In addition, we show that Alu exonization was facilitated by a subset of serine/arginine-rich (SR) proteins through activation of the optimized 3' splice site. Finally, the haplotype- and allele-specific ACE minigenes generated similar splicing patterns in both ACE-expressing and non-expressing cells, suggesting that previously reported allelic association with plasma ACE activity and cardiovascular disease is not attributable to differential splicing of introns 16 and 17.

Alu Elements↗

PubMed2005

Intergenic transcripts in genes with phase I introns.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality-control mechanism that detects and degrades aberrant transcripts prematurely terminating translation. NMD may be elicited by intergenic transcripts that contain premature termination codons (PTCs), but chimeric mRNAs of genes that have introns of identical phase would be predicted to lack PTCs and escape NMD. We examined intron phase I-containing HLA class II genes for the presence of intergenic mRNAs and found an extraordinary diversity of correctly spliced and polyadenylated intergenic transcripts. They lacked a significant homology at the chimeric joins and had no PTCs. Their expression levels were very low and positively correlated with the expression of natural transcripts. In contrast, pair-wise mixtures of separately transcribed plasmids carrying full-length HLA-DQB1, -DQA1, -DRB1, and -DRA cDNAs produced only hybrid molecules that lacked canonical exon boundaries, had homologous chimeric joins, and occasionally contained PTCs, implicating in vitro artifacts generated by template switching of Taq polymerase and reverse transcriptase. A differential exon structure of hybrid molecules observed in vitro and in cellular RNA preparations suggests that intergenic mRNAs with canonical exon boundaries arise in vivo during exon joining and/or transcription. Since the observed intergenic mRNAs may encode mixed class II heterodimers that were previously shown to present antigens it will be interesting to determine functional properties of such molecules in future studies.

Amino Acid Sequence↗

PubMed2002

CDH1 mutations are present in both ductal and lobular breast cancer, but promoter allelic variants show no detectable breast cancer risk.

Mutations and diminished expression of the E-cadherin gene (CDH1) have been identified in a number of epithelial malignancies. Although somatic CDH1 mutations were detected in lobular breast cancer with a frequency ranging from 10-56%, CDH1 alterations in more frequent ductal tumors appear to be rare. Here we have analyzed the coding region of CDH1 for mutations using denaturing high performance liquid chromatography and found 4 mutations in 83 ductal carcinomas (5%) and 3 mutations in 25 lobular carcinomas (12%). The germline of 13 patients with familial lobular tumors was also analyzed for mutations, but none were detected. In a case-control study, we also tested whether a variant adenine allele in the promoter polymorphism -161C-->A with a putative influence on the transcriptional activity of CDH1 in vitro confers any detectable risk of breast cancer. No significant difference in the allelic frequency between patients with breast cancer (326/1,152, 28.3%) and controls (190/696, 27.3%, p > 0.05; relative risk 1.05, 95% confidence interval 0.85-1.30) was found. A novel promoter polymorphism was identified at position -152, but the frequency of the variant cytosine allele was also similar in patients with breast cancer and controls (0.71% vs. 0.21%, p = 0.23). Transient transfection experiments using reporter constructs containing the nucleotide substitutions -161C/-152C and -161A/-152T showed only a slight decrease in the transcription activity compared to the wild-type construct. These results do not support CDH1 as a prominent low-penetrance cancer susceptibility gene, but indicate that CDH1 mutations contribute to the progression of both lobular and ductal tumors.

Adenine Nucleotides↗