启动子区甲基化状态调控BMPR2基因表达水平影响遗传性肺动脉高压外显率的研究

Heritable pulmonary arterial hypertension (HPAH)is an autosomal dominant trait with incomplete penetrance and an estimated lifetime risk of 10% to 20%. Heterozygous germline mutations of BMPR2 are the most important predisposing factor for HPAH. Haploinsufficiency represents the common molecular mechanism. Low penetrance indicates that many with BMPR2 mutations do not develop HPAH, suggesting a role for, as yet unidentified mechanism in disease penetrance. Mutations result in the activation of nonsense-mediated decay (NMD) pathway, which leads to the degradation of the mutant RNA, thus ensuring that only the WT BMPR2 transcripts will be detected in the real-time assay. Unaffected mutation carrier have higher levels of WT BMPR2 transcripts than HPAH patients. The levels of expression of WT BMPR2 allele transcripts are important in the pathogenesis of HPAH caused by NMD+ mutations. Why does the WT BMPR2 allele have the different transcription level in patients and unaffected carrier? What are the major factors of adjusting the expression level of the WT BMPR2 allele? BMPR2 belongs to transforming growth factor beta receptorII (TGF beta RII) super family. TGF beta RII plays an important role as a tumor suppressor in carcinogenesis. Promoter hypermethylation has been shown to be a common mechanism for inactivation of tumor suppressor genes in cancer. The reduced TGF beta RII expression is highly significantly associated with the methylation event. In particular, aberrant 5' CpG methylation of the gene has explained the down-regulation of the gene at a transcriptional level..We hypothesis that methylation of the BMPR2 promoter may be an important factor in regulating the WT allele transcription level which may serve as one of most important molecular mechanisms in explaining the penetrace of the disease..The aim of this study is to investigate the mechanism of penetrance of BMPR2 mutation carriers by analyzing the methylation status of BMPR2 promoter in the affected and unaffected BMPR2 mutation carriers through bisulfite sequencing PCR and methylation-specific PCR methods;constructing luciferase reporter system to analyze different methylation status of BMPR2 promoter influence protein expression level. If hypermethylation is identified in the affected BMPR2 mutation carrier, it indicates methylation of BMPR2 promoter may affect the transcription lever of WT allele leading to mutation carrier with hypermethylation status are more likely to be a PAH patients, while the hypomethylation status mutation carrier are not..we could predict that methylase inhibitor may improve the transcription level through inhibiting the hypermethylation status of the WT allele of the affected mutation carrier, improving the normal BMPRII protein level, releasing the clinical symptom. This is the first demonstration of an epigenetic basis for the penetrance of HPAH and has pathogenetic and therapeutic implications.

骨形成蛋白II型受体（BMPR2）突变是遗传性肺动脉高压致病原因。突变通过无义介导降解机制，引起单倍剂量不足，是重要发病机制。BMPR2突变均为杂合突变，外显率低。研究提示野生型等位基因表达水平影响突变者是否发展为肺动脉高压。但野生型等位基因BMPR2表达调控机制不清楚。BMPR2所在家族TGF beta RII启动子区高甲基化被认为是抑制该基因活化的普遍机制。因此提出BMPR2启动子甲基化可能对野生型等位基因表达起调控作用，影响表达水平，从而影响突变携带者的外显情况。我中心已累积BMPR2突变患者及无症状突变携带家属各50例。通过比较两者BMPR2表达水平；启动子区甲基化程度；构建荧光素酶报告系统研究甲基化状态（SssI甲基化酶处理）是否影响BMPR2表达活性；探索表观遗传因素是否参与肺动脉高压表型外显。该研究不仅是遗传性肺动脉高压发病机制的重要认识，还提示甲基化酶抑制剂可能具有治疗作用

目的：遗传性肺动脉高压（HPAH）是一种常染色体显性遗传性疾病。BMPR2基因突变是最主要的致病原因。BMPR2的突变类型为杂合突变。突变携带者一对等位基因中仅有一个等位基因发生突变，另一个为野生型。外显率约为10-20%。多数的突变携带者终生不发病。如果我们能够阐明什么因素影响了突变到疾病表型的外显过程，我们很有希望找到一些方法将发病的突变携带者逆转为表型正常的突变携带者。本研究拟揭示BMPR2基因启动子区甲基化水平是否影响等位基因的表达水平，引起HPAH的外显。.研究方法：31个HPAH患者和携带突变表型正常的家属入选本研究。外周血白细胞提取基因组DNA。采用亚硫酸盐测序的方法比较HPAH患者和家属中BMPR2启动子区甲基化水平的差异。克隆BMPR2启动子区800bp，装入荧光素酶报告系统（pGL3-basic和pGL3-promoter），体外采用甲基化转移酶（M. SSSi）孵育，研究不同的BMPR2启动子区甲基化水平是否影响其表达活性。.结果：我们发现在患者和突变携带，表型正常的家属中，BMPR2启动子区的甲基化水平明显不同。HPAH患者存在高甲基化的情况。患者中甲基化水平的中位数为5.5%，而对照组仅为0.7%（P=0.025）。 在其中一个家系中，由于突变的位置恰好位于甲基化检测的区域内。因此，该突变可以作为检测不同等位基因甲基化水平的标志。从这个家系的研究结果表明，在发病的患者中8个野生型等位基因克隆中有3个是高甲基化的，而同样携带突变，表型正常的母亲中20个野生型等位基因克隆中没有一个是高甲基化的（P<0.05）。经M.SssI孵育荧光素酶报告质粒较未孵育的质粒表达活性明显降低，分别是其1/36（pGL3-basic，P<0.001）和1/4（pGL3-promoter， P<0.001）。BMPR2启动子区高甲基化水平抑制其表达活性。.结论：BMPR2启动子区甲基化水平是影响等位基因表达水平的重要因素，并且可能影响HPAH外显。甲基化转移酶抑制剂有望成为新的治疗HPAH的药物。