Evolutionary conservation and mutational spectrum of BMPR2 gene.

A variety of mutations in the bone morphogenetic protein receptor type 2 (BMPR2) have been identified in patients with pulmonary arterial hypertension. In this study, using our BMPR2 mutation database and BMPR-II protein sequences from eight distantly related species, we defined the relationship among evolutionary conservation, mutation frequency and mutation distribution. As a whole, BMPR2 is evolving slower than the average for mammalian protein-encoding genes. As expected, the kinase domain is evolving more slowly than the extracellular ligand-binding and C-terminal domains. A detailed map of evolutionary conservation shows that there are repeating peaks and valleys within the C-terminal domain, representing higher and lower evolutionary conservation. We observed a strong correlation between evolutionary conservation and the distribution of mutations along the gene. All except two, of the nineteen missense mutations occur in absolutely conserved amino acids among the vertebrate homologs. In addition, we identified six mutational hotspots (P<0.05) by comparing the observed distribution of mutations to the pattern expected from a random multinomial distribution. Furthermore, analysis of the sequence environment surrounding the mutations revealed a specific pattern of mutagenesis. Over 22% of all single base-paired substitutions and 30% of all deletions and insertions are situated within tandem or non-tandem direct repeats of at least 5-bp and may be explained by slipped-mispairing model of mutagenesis. Also, over 59% of single base-paired substitutions versus 20% of deletions and insertions are located in perfect palindromic sequences that could produce "hairpin-loop" secondary structures with relatively high thermodynamic stability under physiological conditions. In addition, 3.7% of single base-paired substitutions versus 30% of deletions and insertions are located either within or in close proximity to the Krawczak and Cooper consensus sequence (TG A/G A/G G/T A/C). Further study of the mechanism of mutagenesis in BMPR2 may help identify other potentially mutable sites and differentiate between deleterious mutations and harmless polymorphic variants.

Familial pulmonary arterial hypertension, leucopenia, and atrial septal defect: a probable new familial syndrome with multisystem involvement.

We present two siblings with identical clinical findings that seem to represent a previously unreported familial syndrome. Major findings involve three systems: pulmonary arterial hypertension, cardiac abnormalities including secundum-type atrial septal defect, and the hematopoietic system with intermittent neutropenia, lymphopenia, monocytosis, and anemia. The siblings also shared several minor abnormalities: pectus carinatum, long fingers, proximally placed thumb, broad nasal bridge, and high-arched palate. The male proband also had bilateral inguinal hernias and undescended testes. The same findings in two siblings suggest a genetic cause--either an autosomal recessive disorder or germline mosaicism in one parent for a dominant mutation. Investigations revealed a bone morphogenetic protein receptor 2 polymorphism in intron 4 in only one sibling, which was also present in unaffected maternal relatives.

Polymorphism in the angiotensin II type 1 receptor (AGTR1) is associated with age at diagnosis in pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare, lethal disease associated with single gene disorders, connective tissue disease, exposures to anorexigens, and often, idiopathic etiology. Genes can modify the risk of PAH: (1) monogenic disorders associated with PAH are incompletely penetrant, and (2) not all patients with associated conditions at increased risk for PAH develop the disease. The renin angiotensin aldosterone system (RAAS) provides a set of candidate genes that could modulate pulmonary vascular disease similar to its effects on renal and peripheral vasculature. METHODS: We studied 247 patients with PAH, comprising 177 with idiopathic PAH (IPAH), 63 with PAH/connective tissue disease (CTD), and 7 with PAH associated with anorexigens. Patients were genotyped for 5 common polymorphisms in angiotensinogen (AGT), angiotensin-converting enzyme (ACE), cardiac chymase A (CMA1), angiotensin II type 1 receptor (AGTR1), and aldosterone synthase (CYP11B2). Genotypes were tested for associations with age at diagnosis, hemodynamic parameters at diagnosis, and/or response to acute pulmonary vasodilator testing at diagnosis. RESULTS: Associations were demonstrated for AGTR1 and age at diagnosis in IPAH (p = 0.005). Homozygotes for the 1166C allele (n = 13) were associated with an age at diagnosis 26 years later than those with A/A (n = 139) or A/C (n = 90) genotypes. No associations were demonstrated for AGT, ACE, CMA1, or CYP11B2. CONCLUSIONS: The 1166C polymorphism in AGTR1 appears to be associated with a later age at diagnosis in IPAH, suggesting that this pathway could be involved in the biologic variability that is known to occur in PAH.

Clinical implications of determining BMPR2 mutation status in a large cohort of children and adults with pulmonary arterial hypertension.

BACKGROUND: Bone morphogenetic protein receptor type 2 (BMPR2) mutations occur in idiopathic and familial pulmonary arterial hypertension (IPAH, FPAH); however, the impact of these mutations on clinical assessment and disease severity remains unclear. We investigated the role of BMPR2 mutations on acute vasoreactivity and disease severity in IPAH/FPAH children and adults. METHODS: BMPR2 mutation types were determined in 147 IPAH/FPAH patients. Hemodynamics were obtained at baseline and with acute vasodilator testing. RESULTS: Of 147 patients (69 adults, 78 children; 114 with IPAH, 33 with FPAH), 124 (84%) were BMPR2 mutation-negative, and 23 (16%) were mutation-positive. BMPR2 mutation-positive patients were less likely to respond to acute vasodilator testing than mutation-negative patients (4% vs 33%; p < 0.003; n = 147). BMPR2 mutation-positive children also appeared less likely to respond to acute vasodilator testing than mutation-negative children. BMPR2-positive patients had lower mixed venous saturation (57 +/- 9% vs 62 +/- 10%; p < 0.05) and cardiac index (CI; 2.0 +/- 1.1 vs 2.4 +/- 1.5 liters/min; p < 0.05) than BMPR2-negative patients. CONCLUSIONS: Patients with BMPR2 mutations are less likely to respond to acute vasodilator testing than mutation-negative patients and appear to have more severe disease at diagnosis. Determination of BMPR2 mutations appears to help identify IPAH/FPAH children and adults who are unlikely to respond to acute vasodilator testing and, thus, unlikely to benefit from calcium channel blockade (CCB) treatment.

Bone morphogenetic protein receptor type II C-terminus interacts with c-Src: implication for a role in pulmonary arterial hypertension.

Mutations of bone morphogenetic protein receptor type II (BMPR-II) have been associated with familial and idiopathic pulmonary arterial hypertension (PAH). BMPR-II is a member of the transforming growth factor-beta receptor superfamily. It consists of extracellular, transmembrane, and kinase domains, and a unique C-terminus with mostly unknown function. However, a number of PAH-causing mutations are predicted to truncate the C-terminus, suggesting that this domain plays an important role in the homeostasis of pulmonary vessels. In this study, we sought to elucidate the functional role of this C-terminus by seeking its interacting partners. Using yeast two-hybrid screening, we identified c-Src tyrosine kinase as a binding partner of this C-terminus. In vitro co-immunoprecipitation confirmed their interaction. Mutations truncating the C-terminus disrupted their interaction, while missense mutation within kinase domain reduced their interaction. In addition, BMPR-II and c-Src tyrosine kinase colocalized within intracellular aggregates when overexpressed in HEK293 cells. Moreover, mutations truncating the C-terminus disrupted their colocalization, whereas missense mutation within kinase domain had no effect on their colocalization. Furthermore, BMP ligand stimulation decreased c-Src-activating phosphorylation at Tyrosine 418 in pulmonary smooth muscle cells in both time- and concentration-dependent manners. Mutations that truncated the C-terminus abolished this response. Taken together, these results suggest a model in which proliferative effect of c-Src by vasoactive molecules is balanced by opposing effect of BMP signaling in basal state, and the loss of this balance due to BMPR2 mutations leads to increased c-Src activity and subsequently cell growth.

Prognostic factors for survival in human immunodeficiency virus-associated pulmonary arterial hypertension.

We report a large monocentric case series of 82 patients with human immunodeficiency virus-associated pulmonary arterial hypertension (PAH). No germline mutations of the PPH1 gene (bone morphogenetic protein receptor-II) were found in any of the 19 patients tested. PAH was the direct cause of death in 72% of cases. Survival rates of the overall population at 1, 2, and 3 years were 73, 60, and 47%, respectively. Survival was significantly poorer in patients in New York Heart Association functional class III-IV at the time of diagnosis, as compared with those in functional class I-II with respective rates of 60, 45, and 28% versus 100, 90, 84% at 1, 2, and 3 years (p < 0.0001). Subsequently, we analyzed prognostic factors in patients in functional class III-IV. Univariate analysis indicated that CD4 lymphocyte count of more than 212 cells mm(-3), the use of combination antiretroviral therapy (CART), and epoprostenol infusion were related with a better survival. On multivariate analysis only CD4 lymphocyte count was an independent predictor of survival, presumably because CART and epoprostenol infusion were strongly linked in our patient population. These results suggest that patients with severe human immunodeficiency virus-associated PAH should be considered for long-term epoprostenol infusion in association with CART.