摘要目的 探讨Ⅱ型骨形成蛋白受体(BMPR2)基因启动子突变与肺动脉高压发病的关系.方法 对1例家族性肺动脉高压(FPAH)、19例特发性肺动脉高压(IPAH)患者和50名健康成人进行BMPR2基因启动子区转录起始点上游-2022 bp的DNA序列测定.分别将突变型和野生型BMPR2基因启动子区片段插入pGL3-basic双荧光素酶报告基因载体中,获得pGL3-BMPR2启动子-突变型重组质粒和pGL3-BMPR2启动子-野生型重组质粒.以2种重组质粒分别转染人肺动脉平滑肌细胞(HPASMC)和人肺动脉内皮细胞(HPAEC),用Veritas微孔板发光检测仪检测突变型和野生型BMPR2基因启动子区片段在2种细胞中的转录调控活性(结果以萤火虫荧光素酶/海肾荧光素酶活性比值表示).应用MAPPER软件分析突变型和野生型BMPR2基因启动子区转录因子结合位点.结果 在1例36岁女性FPAH患者的BMPR2基因启动子区发现杂合子突变-142G>A.突变型BMPR2基因启动子片段在HPASMC和HPAEC中的转录活性(分别为9.58±3.85、59.07±25.54)均明显低于野生型(分别为16.80±3.55、115.58±38.02,均P<0.05),而且缺失转录因子Sp3结合位点.结论 BMPR2基因启动子-142G>A突变可能与FPAH发病有关.

abstractsObjective To investigate the relation between bone morphogenetic protein type Ⅱ receptor (BMPR2) gene promoter mutation and pulmonary arterial hypertension (PAH). Methods Peripheral blood samples were collected from a 36-year-old female patient with familial PAH (FPAH), 19 idiopathic PAH (IPAH) patients, and 50 healthy controls. DNA sequencing was conducted for the position -2022 bp upstream of the promoter transcription start point of BMPR2 gene. Two fragments carrying BMPR2 promoter mutation - 142A and wild - 142G allele were amplified and cloned respectively into the pGL3-basic dual-luciferase reporter gene vector, thus generating two luciferase reporter constructs: pGL3-BMPR2-wild recombinant plasmid ( carrying - 142G allele) and pGL3-BMPR2-mut recombinant plasmid (carrying - 142A allele ). Human pulmonary arterial smooth muscle cells (HPASMCs) and human pulmonary arterial endothelial cells (HPAECs) were cultured and transfected with pGL3-BMPR2-wild and pGL3-BMPR2-mut recombinant plasmids respectively. The transcriptional activity levels of these 2 recombinant plasmids were measured by Veritas Microplate Luminometer, and were calculated as the ratio of firefly luciferase activity to Renilla luciferase activity. The binding sites for transcriptional factors on the flanking sequence of the wild and mutant BMPR2 gene promoter regions were analyzed by using the MAPPER Search Engine. Results A mutation - 142G > A in the promoter region of BMPR2 gene was found in this female patient with FPAH. The transcriptional activity levels of the BMPR2 promoter carrying - 142A allele in the HPASMCs and HPAECs were (9. 58±3. 85 ) and (59. 07±25.54) respectively, both significantly lower than those of the BMPR2 promoter carrying - 142G allele [ ( 16. 80±3.55 ) and ( 115.58±38. 02 ) respectively, beth P <0. 05]. The binding site of specificity protein 3, the potential transcriptional factor, was deleted in the BMPR2 promoter carrying - 142A allele compared to the BMPR2 promoter carrying - 142G allele. Conclusion BMPR2 promoter mutation - 142G > A may be associated with FPAH.