Evolutionary conservation analysis increases the colocalization of predicted exonic splicing enhancers in the BRCA1 gene with missense sequence changes and in-frame deletions, but not polymorphisms

Abstract

Introduction: Aberrant pre-mRNA splicing can be more detrimental to the function of a gene than changes in the length or nature of the encoded amino acid sequence. Although predicting the effects of changes in consensus 5' and 3' splice sites near intron:exon boundaries is relatively straightforward, predicting the possible effects of changes in exonic splicing enhancers (ESEs) remains a challenge.

Methods: As an initial step toward determining which ESEs predicted by the web-based tool ESEfinder in the breast cancer susceptibility gene BRCA1 are likely to be functional, we have determined their evolutionary conservation and compared their location with known BRCA1 sequence variants.

Results: Using the default settings of ESEfinder, we initially detected 669 potential ESEs in the coding region of the BRCA1 gene. Increasing the threshold score reduced the total number to 464, while taking into consideration the proximity to splice donor and acceptor sites reduced the number to 211. Approximately 11% of these ESEs (23/211) either are identical at the nucleotide level in human, primates, mouse, cow, dog and opossum Brca1 (conserved) or are detectable by ESEfinder in the same position in the Brca1 sequence (shared). The frequency of conserved and shared predicted ESEs between human and mouse is higher in BRCA1 exons (2.8 per 100 nucleotides) than in introns (0.6 per 100 nucleotides). Of conserved or shared putative ESEs, 61% (14/23) were predicted to be affected by sequence variants reported in the Breast Cancer Information Core database. Applying the filters described above increased the colocalization of predicted ESEs with missense changes, in-frame deletions and unclassified variants predicted to be deleterious to protein function, whereas they decreased the colocalization with known polymorphisms or unclassified variants predicted to be neutral.

Conclusion: In this report we show that evolutionary conservation analysis may be used to improve the specificity of an ESE prediction tool. This is the first report on the prediction of the frequency and distribution of ESEs in the BRCA1 gene, and it is the first reported attempt to predict which ESEs are most likely to be functional and therefore which sequence variants in ESEs are most likely to be pathogenic.

Figure 1

Shared and conserved predicted ESEs in the human and mouse BRCA1 genes. The BRCA1 open reading frame (ORF) divided into exons is shown to scale, with shorter analyzed fragments of exons 11 and 16. Conserved exonic splicing enhancers (ESEs) are shown on the top of the ORF, with shared ESEs shown on the bottom. Overlapping ESE motifs are staggered. SF2/ASF ESE motifs are shown in red, with SC35 in blue, SRp40 in green and SRp55 in yellow, also to scale. Cryptic splice sites (CSSs) in exons 5 and 11 are marked. Only the sequence within 125 nucleotides (nt) of exonic splice donor and acceptor consensus sequences has been analysed. Conserved ESEs are those sequences that were identical in human, primates, mouse cow, dog and opossum Brca1. Shared ESEs are those sequences for which ESEfinder predicted a binding site for the same arginine-serine rich protein (SR protein) to the same exon position but the motif sequence was not the same between species.

Figure 2

Prioritizing ESEs in the coding region of BRCA1. Shown is a flow diagram of the process of prioritizing exonic splicing enhancers (ESEs) in the BRCA1 open reading frame for biochemical analysis. Numbers indicate predicted ESEs following each stage.