Diverging alternative splicing fingerprints in the transforming growth factor-β signaling pathway identified in thoracic aortic aneurysms

Abstract

Impaired regulation of the transforming growth factor-β (TGFβ) signaling pathway has been linked to thoracic aortic aneurysm (TAA). Previous work has indicated that differential splicing is a common phenomenon, potentially influencing the function of proteins. In the present study we investigated the occurrence of differential splicing in the TGFβ pathway associated with TAA in patients with bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV). Affymetrix human exon arrays were applied to 81 intima/media tissue samples from dilated (n = 51) and nondilated (n = 30) aortas of TAV and BAV patients. To analyze the occurrence of alternative splicing in the TGFβ pathway, multivariate techniques, including principal component analysis and OPLS-DA (orthogonal partial least squares to latent structures discriminant analysis), were applied on all exons (n = 614) of the TGFβ pathway. The scores plot, based on the splice index of individual exons, showed separate clusters of patients with both dilated and nondilated aorta, thereby illustrating the potential importance of alternative splicing in TAA. In total, differential splicing was detected in 187 exons. Furthermore, the pattern of alternative splicing is clearly differs between TAV and BAV patients. Differential splicing was specific for BAV and TAV patients in 40 and 86 exons, respectively, and splicings of 61 exons were shared between the two phenotypes. The occurrence of differential splicing was demonstrated in selected genes by reverse transcription-polymerase chain reaction. In summary, alternative splicing is a common feature of TAA formation. Our results suggest that dilatation in TAV and BAV patients has different alternative splicing fingerprints in the TGFβ pathway.

Figure 1

PCA (A–C) and OPLS-DA (D–F) of gene level normalized exon expression (splice index) in the TGFβ pathway. The analyses were performed on 81 patients chosen according to medical data and selection based on meta probe set level PCA (Supplemental Figure 1). The Hotelling’s T2 (based on 95% confidence level) tolerance ellipsoid and ellipse are shown in the scores plots of PCA and OPLS-DA, respectively. Three-dimensional scores plot showing the PC1–PC3 plane of nondilated (black) and dilated (red) thoracic aorta samples with BAV and TAV together (A), as well as samples from TAV (B) and BAV (C) patients separately. Two-dimensional scores plot of an OPLS-DA showing the first predictive component (t_p1) and orthogonal component (t_o1) plane of nondilated (black) and dilated (red) thoracic aorta samples with BAV and TAV patients together (D), as well as TAV (E) and BAV (F) patients separately.

Figure 2

Venn diagram and OPLS-DA of gene level–normalized exon expression (splice-index) data included in the TGFβ pathway. (A) Venn diagram based on an FDR-corrected t test of dilated versus nondilated samples in TAV and BAV patients separately. TAV-specific (blue), BAV-specific (green), and common (red) exons are marked in the diagram. (B) Combined model scatter plot based on TAV dilated versus nondilated and BAV dilated versus nondilated OPLS-DA models. The exons found to be significant in (A) are color coded accordingly in the combined model scatter plot. The black diagonal is aimed for interpretation purposes. Bar plots showing the loadings of each of the significant exons and indicating their contribution to the first PC are shown in (C–E) and color coded according to (A). The confidence levels for each data point were estimated by a jack-knife algorithm. Exons chosen for RT-PCR validation are marked in (B–E).

Figure 3

The expression plot of alternative splicing events of genes chosen for RT-PCR validation. The expression is shown as mean log₂ intensity of splice index data for each probe set (exon) for dilated (blue) and nondilated (red) samples in TAV and BAV patients separately. The isoforms known from the RefSeq database are shown on top with exons present in the particular isoform (gray box) and if absent (white box). The exons that were validated with RT-PCR are marked with green boxes in the RefSeq plot, by arrows in the expression plots and zoomed in to the particular exon of interest in the lower part of the figure. The RefSeq exons are position-matched with the expression plots probe sets (exons) below. FN1 is shown in (A) and LTBP3 in (B). The three isoforms in (B) do not show any difference at core level. The significance level (*) was calculated according to two methods, one null-hypothesis–driven t test for one variable at a time, by which the resulting P values have been corrected according to the FDR q-method, as well as a multivariate method by which jack-knife confidence levels and loading values have been calculated from OPLS-DA models (Figure 2, Table 1).

Figure 4

OPLS-DA of gene level normalized exon expression (splice index) of the filtered data set for all human exons. The exons are color coded according to significant exons included in TGFβ analysis in Figure 2: TAV-specific (blue), BAV-specific (green), and common (red) probe sets. (A) Combined model scatter plot based on TAV dilated versus nondilated and BAV dilated versus nondilated OPLS-DA models. The black diagonal is aimed for interpretation purposes. Bar plots showing the loadings of each of the significant exons indicating their contribution to the first PC are shown in (B–D) and color coded according to (A). The confidence levels for each data point were estimated by a jack-knife algorithm. Exons chosen for RT-PCR validation are marked in (A–D).

Figure 5

RT-PCR validation of alternative splicing events. A set of exons found to be differentially expressed by statistical analyses (Figure 2) were chosen for RT-PCR validation of alternative splicing. (A) FN1_EDA, (B) LTBP3_E19 and (C) FN1_EDB show alternative splicing, and FN1_E09 (D) is not alternatively spliced and serves as a control. The gray boxes represent flanking exons in closest proximity to the exon that was investigated (marked in green around the box). Red arrows represent the position of the primers for RT-PCR. The length of the PCR product is shown in numbers. Black arrows indicate the position of the two isoforms on the gel.