Role of BMPR2 alternative splicing in heritable pulmonary arterial hypertension penetrance.
Role of BMPR2 alternative splicing in heritable pulmonary arterial hypertension penetrance.
复制标题
BMPR2替代剪接在可遗传的肺动脉高压渗透性中的作用。
DOI：
10.1161/circulationaha.112.106245
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发表时间：
2012-10-09
期刊：
影响因子：
37.8
通讯作者：
Hamid R
中科院分区：
文献类型：
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作者：
Cogan J;Austin E;Hedges L;Womack B;West J;Loyd J;Hamid R
骨形态发生蛋白受体2(BMPR2)基因突变是遗传性PAH(HPAH)最常见的原因。然而，只有20%的突变携带者会患上临床疾病。在这里，我们探索了这样一种假设，即这种外显性降低的部分原因是BMPR2选择性剪接的变化。我们的数据显示，BMPR2有多个选择性剪接变体。其中两个，亚型-A(全长)和亚型-B(缺失外显子12)，在所有被分析的组织中都有表达。对47例BMPR2突变阳性的HPAH患者和35例BMPR2突变阳性的未感染携带者的淋巴细胞培养结果显示，B亚型高于A亚型(B/A比值)，差异有统计学意义(P=0.002)。此外，与低B/A比的细胞相比，高B/A比的细胞在BMP刺激后的非磷酸化粘连蛋白水平较低。对外显子12序列的分析确定了一个外显子剪接增强子，它与丝氨酸精氨酸剪接因子2(SRSF2)结合。由于SRSF2促进外显子包涵体，降低SRSF2的表达将意味着外显子12不会被包括在最终的BMPR2 mRNA中(从而促进异构体-B的形成)。Western印迹分析表明，SRSF2在患者细胞中的表达低于携带者；siRNA介导的SRSF2在肺微血管内皮细胞中的表达下调导致异构体-B的水平高于异构体-A的水平，即B/A比值升高。BMPR2异构体比率的改变可能解释了BMPR2突变携带者外显率降低的原因。这一比例由外显子12的外显子剪接增强子及其相关剪接因子SRSF2控制。
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. 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. 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.