Novel Promoter And Exon Mutations Of The Bone Morphogenetic Protein Type Ⅱ Receptor Gene In Chinese Patients With Pulmonary Arterial Hypertension

Objective To investigate the link between promoter and exon mutations of Bone Morphogenetic Protein Typeâ…¡Receptor(BMPR₂) gene and Chinese patients with pulmonary arterial hypertension(PAH).We proposed that BMPR₂ promoter mutation may associated with pathogenesis of PAH.We plan to identify: 1.Whether there are mutations in the promoter regions of BMPR₂ gene in patients with familial pulmonary arterial hypertension(FPAH) or idiopathic pulmonary arterial hypertension(IPAH)? 2.What's the influence of BMPR₂ promoter mutation on transcriptional activity? 3.Whether novel of BMPR₂ exon mutations are present in Chinese patients with PAH?Methods Twenty patients with either FPAH or IPAH,and fifty healthy controls were collected from Fu Wai Cardiovascular Hospital,Chinese Academy of Medical Sciences and Chinese PLA General Hospital.The patients including one FPAH and nineteen IPAH were diagnosed by right heart catheterization and according to ACCP Evidence-Based Clinical Practice Guidelines.(1)Analysis of transcriptional regulation by BMPR₂ proximal promoter regulatory sequences in patients with PAH.We obtained the 5'flanking sequence of the human BMPR₂ gene through GenBank.PCR primers were designed to amplify 2022 bp of the proximal promoter upstream of the predicted transcription start site in twenty PAH patients and fifty healthy controls.A mutation -142G＞A in the promoter region of BMPR₂ gene was found in one female patient with FPAH.(2) To study the influence of BMPR₂ promoter mutation on transcriptional activity.Two fragments carrying BMPR₂ promoter mutation - 142A and wild - 142G allele were amplified and cloned respectively into upstream of the firefly luciferase reporter gene of pGL3 vector,generating two luciferase reporter constructs of pGL3- BMPR₂-wild (carrying -142G allele) and pGL3- BMPR₂-mut(carrying- 142A allele).The human pulmonary arterial smooth muscle cells(HPASMC) and human pulmonary arterial endothelial cells(HPAEC) were transiently transfected with pGL3-BMPR₂ -wild and pGL3- BMPR₂ -mut.The transcriptional activity of pGL3-BMPR₂-wild and pGL3- BMPR₂-mut was measured by Veritas Microplate Luminometer,and was calculated as the ratio of Firefly to Renilla luciferase activities.The binding sites for transcriptional factors on the flanking sequence of the wild and mutant BMPR₂ gene promoter region were analyzed by using the MAPPER Search Engine.(3) To detect BMPR₂ exon mutations in the patients with PAH.Genomic DNA of the peripheral white blood cells was extracted from the patients with PAH and controls.Primers for the exon 1 to 13 including the lateral intron of BMPR₂ gene were designed.The genomic DNA was amplified by PCR,and then PCR products were sequenced and compared with normal human BMPR₂ gene.Results(1)A mutation -142G＞A in the promoter region of BMPR₂ gene was found in one female patient with Chinese FPAH,and no other mutation was found in any of the 13 exons in the proband.(2)The BMPR₂ promoter carrying -142A allele(The transcriptional activity was 9.58±3.85 and 59.07±25.54 respectively,P＜0.05) showed significantly decreased transcriptional activity compared to BMPR₂ promoter carrying -142G allele(The transcriptional activity was 16.80±3.55 and 115.58±38.02 respectively,P＜0.05) in HPASMC and HPAEC. The potential specificity protein 3(SP3) binding site was abolished in BMPR₂ promoter carrying -142A allele compared to BMPR₂ promoter carrying -142G allele.(3) Three novel heterozygous exon mutations were identified:a nonsense mutation at the nucleotide position 292 in exon 3(Glu98X);a frame shift mutation with deletion of TG at the nucleotide position 608-609 in exon 5 (Leu203fsX15),and a missense mutation single nucleotide substitution in exon 12 (Ser863Asn) in Chinese patients with IPAH.A missense mutation at the nucleotide position 196 leading to single base substitution in exon 2(Cys66Arg) wasn't a novel mutation(Reported by Cahn in 2004).No mutation was found in the BMPR₂ gene promoter of the nineteen IPAH patients.Conclusion(1) A mutation - 142G＞A in the promoter region of BMPR₂ gene was found in one female Chinese patient with FPAH.This is the first report that a BMPR₂ promoter mutation is associated with FPAH.(2)The transcriptional activity level of the BMPR₂ promoter mutation -142G＞A were significantly lower than those of the wild type of BMPR₂ promoter.A abolished potential specificity protein 3(SP3) binding site was found in the BMPR₂ promoter mutation -142G＞A.(3)Three novel heterozygous exon mutations were found in Chinese IPAH patients.The ethnic group difference may be a potential explanation.We conclude that novel promoter and exon mutations of the BMPR₂ may associate with pathogenesis of PAH.