Hemodynamic and clinical onset in patients with hereditary pulmonary arterial hypertension and BMPR2 mutations

Background: Mutations in the bone morphogenetic protein receptor 2 (BMPR2) gene can lead to idiopathic pulmonary arterial hypertension (IPAH). This study prospectively screened for BMPR2 mutations in a large cohort of PAH-patients and compared clinical features between BMPR2 mutation carriers and non-carriers. Methods: Patients have been assessed by right heart catheterization and genetic testing. In all patients a detailed family history and pedigree analysis have been obtained. We compared age at diagnosis and hemodynamic parameters between carriers and non-carriers of BMPR2 mutations. In non-carriers with familial aggregation of PAH further genes/gene regions as the BMPR2 promoter region, the ACVRL1, Endoglin, and SMAD8 genes have been analysed. Results: Of the 231 index patients 22 revealed a confirmed familial aggregation of the disease (HPAH), 209 patients had sporadic IPAH. In 49 patients (86.3% of patients with familial aggregation and 14.3% of sporadic IPAH) mutations of the BMPR2 gene have been identified. Twelve BMPR2 mutations and 3 unclassified sequence variants have not yet been described before. Mutation carriers were significantly younger at diagnosis than non-carriers (38.53 ± 12.38 vs. 45.78 ± 11.32 years, p < 0.001) and had a more severe hemodynamic compromise. No gene defects have been detected in 3 patients with HPAH. Conclusion: This study identified in a large prospectively assessed cohort of PAH- patients new BMPR2 mutations, which have not been described before and confirmed previous findings that mutation carriers are younger at diagnosis with a more severe hemodynamic compromise. Thus, screening for BMPR2 mutations may be clinically useful

Identification of a new intronic BMPR2-mutation and early diagnosis of heritable pulmonary arterial hypertension in a large family with mean clinical follow-up of 12 years.

Mutations in the bone morphogenetic protein receptor 2 (BMPR2) gene can lead to hereditary pulmonary arterial hypertension (HPAH) and are detected in more than 80% of cases with familial aggregation of the disease. Factors determining disease penetrance are largely unknown.A mean clinical follow-up of 12 years was accomplished in 46 family members including echocardiography, stress-Dopplerechocardiography and genetic analysis of TGF-β pathway genes. Right heart catheterization and RNA-analysis was performed in members with pathological findings.Manifest HPAH was diagnosed in 8 members, 4 were already deceased, two died during the follow-up, two are still alive. Normal pulmonary artery systolic pressure at rest but hypertensive response to exercise has been identified in 19 family members. Analysis of BMPR2 transcripts revealed aberrant splicing due to an insertion of an intronic Alu element adjacent to exon 6. All HPAH patients and 12 further asymptomatic family members carried this insertion. During follow-up two family members carrying hypertensive response and the Alu insertion developed manifest HPAH.This is the first report of an intronic BMPR2 mutation due to an Alu element insertion causing HPAH in a large family which has been confirmed on RNA-level. Only those members that carried both hypertensive response and the mutation developed manifest HPAH during follow-up. Our findings highlight the importance of including further methods such as RNA analysis into the molecular genetic diagnostic of PAH patients. They suggest that at least in some families hypertensive response may be an additional risk factor for disease manifestation and penetrance

Polymorphic micro-inversions contribute to the genomic variability of humans and chimpanzees

A combination of inter- and intra-species genome comparisons is required to identify and classify the full spectrum of genetic changes, both subtle and gross, that have accompanied the evolutionary divergence of humans and other primates. In this study, gene order comparisons of 11,518 human and chimpanzee orthologous gene pairs were performed to detect regions of inverted gene order that are potentially indicative of small-scale rearrangements such as inversions. By these means, a total of 71 potential micro-rearrangements were detected, nine of which were considered to represent micro-inversions encompassing more than three genes. These putative inversions were then investigated by FISH and/or PCR analyses and the authenticity of five of the nine inversions, ranging in size from ~800 kb to ~4.4 Mb, was confirmed. These inversions mapped to 1p13.2–13.3, 7p22.1, 7p13–14.1, 18p11.21–11.22 and 19q13.12 and encompass 50, 14, 16, 7 and 16 known genes, respectively. Intriguingly, four of the confirmed inversions turned out to be polymorphic: three were polymorphic in the chimpanzee and one in humans. It is concluded that micro-inversions make a significant contribution to genomic variability in both humans and chimpanzees and inversion polymorphisms may be more frequent than previously realized

Pedigree of the large German family.

<p>This figure represents the pedigree tree of the German family analysed in this study. The index patient of the family is marked by an arrow. All family members with manifest PAH are shown in black. Healthy family members have open symbols and those who were heterozygous for the identified mutation are marked with “Alu”. Those family members with hypertensive response due to exercise have half-filled symbols. Family members with unknown hemodynamic status have open symbols with a question mark inside. A question mark below the pedigree ID indicates that the genetic status in this family member is unknown. An open blue square marks the family members which presented at the beginning of the follow-up with hypertensive response due to exercise and changed their status to manifest PAH (II:12 and III:28). All family members who participated in the clinical and/or genetic analysis are marked by a star. The numbering of the individuals in the pedigree corresponds to the IDs of the family members in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091374#pone-0091374-t001" target="_blank">table 1</a>.</p

Genomic analysis of the intronic region.

<p>The genomic amplification with forward primer located in intron 5 and a reverse primer located in intron 6 resulted in two products of different length identified in several family members (shown is the result of 15 family members). Sequencing analysis of the larger products revealed the insertion of an AluYb8 element with an adjacent duplicated sequence motive in antisense orientation to <i>BMPR2</i> in intron 5 (adjacent to exon 6). A schematic representation of the region and the insertion of the Alu element on one allele and the complete sequence of the Alu element, the duplicated sequence and the beginning of exon 6 are shown.</p

Clinical and genetic assessment in family members.

<p>f =  female, m =  male, n =  no, y =  yes, n.m. =  not measurable, HR =  hypertensive response, NR =  normal response, PAH =  pulmonary arterial hypertension, sec. PH =  secondary pulmonary hypertension, RHC =  right heart catheterization.</p

Genotype-phenotype correlations of Alu carriers and PASP during exercise.

<p>This figure shows the genotype-phenotype correlation between Alu insertion and PASP during exercise, classified as HR and NR. Only patients who carried both HR and Alu insertion developed manifest PAH during follow-up. Patients with the Alu insertion in the <i>BMPR2</i> gene show significantly more frequent Hypertensive response to exercise (10/13 vs. 8/25; Fishers exact test p = 0.016).</p