BMPR2Gene Mutations In Children With Pulmonary Arterial Hypertension

ObjectivesBone morphogenetic protein type II receptor was an important pathogenic factor in familial pulmonary hypertension (FPAH) and idiopathic pulmonary arterial hypertension. BMPR2gene mutations in familial, idiopathic pulmonary arterial hypertension patients and congenital heart disease and pulmonary arterial hypertension (CHD/PAH) were reported in foreign studies. Whether there are mutations at the protein-coding region and intron/exon boundaries of the BMPR2gene in patients with CHD/PAH? Whether there are novel mutations in BMPR2exon in patients with FPAH? Therefore, we intend to investigate whether there are mutations or novel mutations at the protein-coding region and intron/exon boundaries of the BMPR2gene in patients with CHD/PAH and FPAH?Part1A Novel Mutation in BMPR2Gene in Children with Congenital Heart Disease and Pulmonary Arterial HypertensionObjective:To determine if patients with congenital heart disease and pulmonary arterial hypertension.Methods:Sixty patients with CHD/PAH were recruited from the Pediatric Cardiology Department and the Cardiac Surgery Department of Provincial Hospital affiliated with Shandong University from February2011to August2011. There was no consanguineous relationship among them. All patients belonged to the Han nationality:39males and27females. Among them,25patients had ventricular septal defect (VSD),5patients had atrial septal defect (ASD),16patients had patent ductus arteriosus (PDA) and20patients had complicated congenital heart diseases. The diagnosis of PAH was confirmed by measuring mean pulmonary arterial pressure with right heart catheterization (>25mmHg). Pulmonary artery pressure was classified into mild (30-40mmHg), moderate (40-70mmHg) and severe (>70mmHg) according to the pulmonary artery systolic pressure. Each patient and control underwent echocardiography and the CHD category of19patients was confirmed by cardiac catheterization.1. The protein-coding region and intron/exon boundaries of the BMPR2gene were amplified by polymerase chain reaction (PCR) using DNA samples from66patients and a panel of66chromosomes from blood of normal donors with matched age and gender. The PCR products were purified and sequenced. Peak charts of sequencing were analyzed with Sequencher4.7Demo software and compared with sequence of BMPR2gene in Gene Bank. A novel missense mutation, a G-to-A transition at position1042in exon8of BMPR2gene, was identified in a female pediatric patient with patial atrioventricular septal defect/anterior mitral valve cleft/pulmonary arterial hypertension (PAVSD/AMVC/PAH).2. The difference in frequency distributions for alleles and genotypes for BMPR2was compared by χ2-test in two groups. Differences between groups were examined for statistical significance using the chi-square test (χ2-test). A P-value<0.05denoted the presence of a statistically significant difference. One single nucleotide polymorphisms (SNP), c.2811G> A, in BMPR2gene was identified in nine patients and ten controls.Results:1. Direct sequencing of the PCR products revealed that one patient with PAVSD/AMVC/PAH carried a novel heterozygous substitution mutation, G1042A, in exon8of BMPR2, which encodes the kinase domain (responsible for phosphorylation). No other mutations were found in any of the13exons. This mutation converted a valine at codon348to an isoleucine. This mutation was not detected in the other CHD/PAH patients or the panel of66chromosomes from normal individuals.2. The patient was a girl aged3years and4months. She came to our hospital because of cyanotic lips after activity for one and a half years. Systolic ejection murmur of level3/6could be heard between her second and third rib along the left sternal edge, with P2hyperfunction and fixed splitting. Twelve-lead electrocardiogram showed right axis deviation and right ventricular hypertrophy. Chest X-ray revealed prominent pulmonary arterysegment, peripheral hypovascularity, and right ventricular enlargement. Echocardiography reported (1) that the diameter of her left atrium was2.65cm, right atrium was3.61cm and right ventricle was2.48cm;(2) that the diameter of the main pulmonary artery was expanded to2.55cm;(3) atrioventricular septal defect;(4) anterior mitralvalve cleft with a large number of reflux;(5) that the pulmonary artery systolic pressure was38mmHg. The diagnosis was partial PAVSD/AMVC/PAH.3. A SNP, c.2811G>A (rs1061157), in BMPR2was identified in9patients and10healthy controls. The SNP was in the coding region, but it had no effect on the encoded amino acid. The frequency of the variant BMPR2c.2811G>A in patients with CHD/PAH was not statistically different from that in the healthy controls.Conclusions:1. A mutation, G1042A, in exon8of BMPR2, was found in one female Chinese patient with PAVSD/AMVC/PAH. This was the first report that a BMPR2exon mutation is associated with CHD、PAH.2. The frequency of the variant BMPR2c.2811G>A in patients with CHD/PAH was not statistically different from that in the healthy controls. The polymorphism in BMPR2may be not associated with PAH.Part2A Novel Mutation in the BMPR2Gene in Children with Familial Pulmonary Arterial HypertensionObjective:To determine if patients with familial pulmonary arterial hypertension (FPAH), due to pulmonary vascular obstructive disease, have mutations in the gene encoding bone morphogenetic protein receptor (BMPR)-2.Methods:A three-generation pedigree of FPAH and87matched controls were collected. All members of the pedigree underwent detailed cinical and laboratory tests. Genomic DNA was isolated from lymphocytes. The protein-coding region and intron/exon boundaries of the BMPR2gene were amplified by polymerase chain reaction (PCR) using DNA samples from the family members and the matched controls with matched age and gender. Direct sequencing of PCR products was performed on both the sense and antisense strands. Peak charts of sequencing were analysed with Sequencher4.7Demo software and compared with sequence of BMPR2gene in Gene Bank.Results:The proband was diagnosed as primary pulmonary hypertension (severe degree), enlargement of the heart, right heart failure, cardiac function Ⅲ. A T-to-C transition at position1039in exon8of the BMPR2gene was identified in the proband which resulted in a Cys347Arg mutation. The results in electrocardiogram and echocardiography from another two members of the pedigree carrying this mutation were nomal. It was a novel mutation by searching the literature and checking database. None BMPR2mutation and abnomal clinical tests were identified in the87normal controls and other members of the pedigree.Conclusions:The Cys347Arg mutation may be responsible for the development of FPAH.