Role of BMPR2 Alternative Splicing in HPAH Penetrance
Background—Bone morphogenic protein receptor 2 (BMPR2) gene mutations are the most common cause of heritable PAH (HPAH). However only 20% of mutation carriers get clinical disease. Here we explored the hypothesis that this reduced penetrance is in part due to an alteration in BMPR2 alternative splicing.
Methods and Results—Our data showed that BMPR2 has multiple alternatively spliced variants. Two of these, isoform-A (full-length) and isoform-B (missing exon 12), were expressed in all tissues analyzed. Analysis of cultured lymphocytes (CLs) of 47 BMPR2 mutation-positive HPAH-patients and 35 BMPR2 mutation-positive unaffected-carriers showed that patients had higher levels of isoform-B compared to isoform-A (B/A ratio) than carriers (P=0.002). Furthermore compared to cells with low B/A ratio, cells with high B/A ratio had lower levels of unphosphorylated cofilin following BMP stimulation. Analysis of exon 12 sequences identified an exonic splice enhancer, which binds serine arginine splicing factor 2 (SRSF2). Because SRSF2 promotes exon inclusion, reduced SRSF2 expression would mean that exon 12 would not be included in final BMPR2 mRNA (thus promoting increased isoform-B formation). Western blot analysis showed that SRSF2 expression was lower in cells from patients compared to carriers; and, siRNA-mediated knockdown of SRSF2 in pulmonary microvascular endothelial cells resulted in elevated levels of isoform-B compared to isoform-A, i.e. elevated B/A ratio.
Conclusions—Alterations in BMPR2 isoform ratios may provide an explanation of the reduced penetrance among BMPR2 mutation carriers. This ratio is controlled by an exonic splice enhancer in exon 12 and its associated splicing factor SRSF2.
- Received March 21, 2012.
- Accepted August 3, 2012.
- Copyright © 2012, American Heart Association, Inc. All rights reserved. Unauthorized use prohibited