Proteomics of transformed lymphocytes from a family with familial pulmonary arterial hypertension

Abstract

Rationale: Not all family members with BMPR2 mutations develop pulmonary arterial hypertension (PAH), implying that additional modifier genes or proteins are necessary for full expression of the disease.

Objectives: To determine whether protein expression is altered in patients with familial PAH (FPAH) compared with obligate carriers and nondiseased control subjects.

Methods: Protein extracts from transformed blood lymphocytes from four patients with FPAH, three obligate carriers, and three married-in control subjects from one family with a known BMPR2 mutation (exon 3 T354G) were labeled with either Cy3 or Cy5. Cy3/5 pairs were separated by standard two-dimensional differential gel electrophoresis using a Cy2-labeled internal standard of all patient samples. Log volume ratios were analyzed using a linear mixed-effects model. Proteins were identified by matrix-assisted laser desorption ionization, time-of-flight mass spectrometry (MALDI-TOF MS) and tandem TOF/TOF MS/MS.

Measurements and main results: Hierarchical clustering, heat-map, and principal components analysis revealed marked changes in protein expression in patients with FPAH when compared with obligate carriers. Significant changes were apparent in expression of 16 proteins (P < 0.05) when affected patients were compared with obligates: nine showed a significant increase and seven showed a significant reduction.

Conclusions: A series of novel proteins with altered expression were found that could distinguish affected patients from obligate carriers and married-in controls in a single family with a BMPR2 mutation. These differences provide new information highlighting proteins that may be involved in the mechanism(s) that differentiates those individuals with a BMPR2 mutation who develop FPAH from those who do not.

Figures

Figure 1.

Representative example of sensitivity and resolution from one two-dimensional differential gel electrophoresis (2D-DIGE) gel from the coordinated 10-gel series. (A) False color images are overlaid for comparative purposes only. Shown are the two individual samples coresolved on this gel, one of the obligates labeled with Cy3 and false-colored green, and one of the BMP4-treated control samples (labeled with Cy5 and false-colored red). Both were coresolved on this gel along with an equal aliquot of the Cy2-labeled 20-mix internal standard (false-colored blue), which was also present on the other nine gels (each with two other samples from the 20-sample experiment). For each resolved protein, the abundance values are calculated relative to the internal standard signal within each gel, and then these ratios are normalized between gels to produce the abundance changes depicted in the graphs shown in Figure 5 (Methods). (B) The Sypro Ruby post-stain for the same gel in A is shown. This image is essential to ensure accurate robotic protein excision, and demonstrates the high degree of correspondence between the DIGE and Sypro Ruby images.

Figure 2.

Dendrogram and heat map of protein expression from transformed lymphocytes from affected patients, obligate individuals, and married-in control subjects both with and without a 4-hour treatment with BMP-4. The dendrogram illustrates a distinct compartmentalization in protein expression patterns between the three groups. No differences are found between the samples with and without treatment with BMP-4. The differences in protein expression are clearly seen in the heat map. Expression patterns in selected proteins are visually presented as horizontal lines in an expression matrix using a relative scale ranging from −0.5 (green) to +0.5 (red).

Figure 3.

Principal components analysis (PCA) of protein expression in transformed lymphocytes from affected patients with familial pulmonary arterial hypertension (FPAH), obligate individuals, and married-in control subjects. PCA reduces the complexity of a multidimensional analysis into two principal components, PC1 and PC2, which orthogonally divide the samples based on the two largest sources of variation in the dataset. Values within the circles of the PCA plots are within the 95th percentile confidence interval. All of the points from the affected patients with FPAH lie to the left of the perpendicular while those from the obligate individuals and married-in control subjects lie on the right, indicating a distinct expression pattern in the patients with FPAH versus the obligate individuals and the married-in control subjects. Affected = light green; affected + BMP-4 = dark green; obligates = light blue; obligates + BMP-4 = dark blue; married-in control subjects = pink; married-in control subjects + BMP-4 = red.

Figure 4.

Principal components analysis of protein expression from control transformed lymphocytes matched by age and sex to the individuals in Figure 3. No clustering of points is apparent, indicating that age and sex do not contribute to the different expression patterns seen in Figure 3.

Figure 5.

Standardized log abundance of various protein expression data obtained from two-dimensional differential gel electrophoresis analysis. Affected = light green; affected + BMP-4 = dark green; obligates = light blue; obligates + BMP-4 = dark blue; married-in control subjects = pink; married-in control subjects + BMP-4 = red. Of the proteins identified, only Grb2 showed a trend toward a difference between obligate controls with and without treatment with BMP-4.

Figure 6.

Western blot showing Grb2 expression in unstimulated transformed lymphocytes from obligate individuals (lanes 1–3) and patients with familial pulmonary arterial hypertension (lanes 4–7). Total p42/44 is shown as a loading control.