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Proteomics of Transformed Lymphocytes from a Family with Familial Pulmonary Arterial Hypertension

Departments of ¹ Pathology, ² Medicine, ³ Biochemistry, ⁴ Biostatistics, and ⁵ Genetics, Vanderbilt University Medical Center, Nashville, Tennessee

Correspondence and requests for reprints should be addressed to Barbara Meyrick, Ph.D., Professor of Pathology and Medicine, Vanderbilt University Medical Center, MCN T-1218, Nashville, TN 37232-2650. E-mail: barbara.meyrick{at}vanderbilt.edu

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.

Key Words: two-dimensional differential gel electrophoresis • obligate individuals without FPAH • catalytic activity • MALDI-TOF mass spectrometry

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject Although familial pulmonary arterial hypertension is associated with mutations in BMPR2, not all family members with the mutation develop the disease. Modifier genes and proteins may be necessary for development of the disease. What This Study Adds to the Field Proteomic techniques revealed divergent patterns of protein expression in patients with familial pulmonary arterial hypertension as compared with obligate individuals from the same family with no evidence of disease.

 

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