Connectivity Map Analysis of Nonsense-Mediated Decay–Positive BMPR2-Related Hereditary Pulmonary Arterial Hypertension Provides Insights into Disease Penetrance

Abstract

The molecular mechanisms underlying the reduced penetrance seen in the nonsense-mediated decay-positive (NMD1) BMPR2 mutationassociated hereditary pulmonary arterial hypertension (HPAH) remain unknown. We reasoned that the cellular and genetic mechanisms behind this phenomenon could be uncovered by combining expression profiling with Connectivity Map (cMap) analysis. Cultured lymphocytes from 10 patients with HPAH and 10 matched familial control subjects, all with NMD1 BMPR2 mutations, were subjected to expression analysis. For each group, the expression data were combined before analysis. This generated a signature of 23 up-regulated and 12 down-regulated genes in patients with HPAH compared with control subjects (the "PAH penetrance signature"). Although gene set enrichment analysis of this signature was not uniquely informative, cMap analysis identified drugs with expression signatures similar to the PAH penetrance signature. Several of these drugs were predicted to influence reactive oxygen species (ROS) formation. This hypothesis was tested and confirmed in the same cells initially subjected to the expression analysis using quantitative biochemical detection of ROS concentration. We conclude that expression of the PAH penetrance signature represents an increased risk of developing clinical HPAH and that ROS formation may play a role in pathogenesis of HPAH. These results provide the first molecular insights into NMD1 BMPR2 related HPAH penetrance and highlight the potential utility of cMap analyses in pulmonary research.Keywords: hereditary pulmonary arterial hypertention; ROS; connectivity map; expression signature; BMPR2 Pulmonary arterial hypertension (PAH) is a progressive, fatal disease, and most patients with PAH have a poor prognosis despite standard-of-care therapies (1). PAH is characterized by vascular remodeling of the distal pulmonary arteries (100-200 mM in size) via smooth muscle hypertrophy and intimal endothelial cell proliferation, effectively decreasing the surface area of the pulmonary vasculature (2, 3). The resulting increase in pulmonary vascular resistance leads to the failure of a progressively overloaded right ventricle and, eventually, death. The heritable form of PAH (HPAH) is usually (.80% of the time) due to germline mutations in the Bone Morphogenetic Protein Receptor 2 (BMPR2) (4-7), whereas 5 to 25% of patients diagnosed as having idiopathic (I) PAH have a detectable germline mutation in BMPR2 as well (5,(12)(13)(14)(15).In HPAH, BMPR2 mutations can produce stable transcripts or premature termination codons, resulting in the mutated transcript being rapidly degraded through the nonsense-mediated decay (NMD) pathway (8). NMD is an mRNA surveillance system that degrades transcripts containing premature termination codons to prevent translation of unnecessary or harmful transcripts (9). Thus, patients with PAH with NMD-positive (NMD1) BMPR2 mutations have disease due to haploinsufficiency, whereas patients whose mutations are NMD negative (NMD2) may have disease due to a dominan...

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