Lisa Wheeler

Sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding BMPR-II, a receptor member of the TGF-beta family

BACKGROUND Primary pulmonary hypertension (PPH), resulting from occlusion of small pulmonary arteries, is a devastating condition. Mutations of the bone morphogenetic protein receptor type II gene (BMPR2), a component of the transforming growth factor beta (TGF-β) family which plays a key role in cell growth, have recently been identified as causing familial PPH. We have searched for BMPR2 gene mutations in sporadic PPH patients to determine whether the same genetic defect underlies the more common form of the disorder. METHODS We investigated 50 unrelated patients, with a clinical diagnosis of PPH and no identifiable family history of pulmonary hypertension, by direct sequencing of the entire coding region and intron/exon boundaries of the BMPR2 gene. DNA from available parent pairs (n=5) was used to assess the occurrence of spontaneous (de novo) mutations contributing to sporadic PPH. RESULTS We found a total of 11 different heterozygous germline mutations of theBMPR2 gene in 13 of the 50 PPH patients studied, including missense (n=3), nonsense (n=3), and frameshift (n=5) mutations each predicted to alter the cell signalling response to specific ligands. Parental analysis showed three occurrences of paternal transmission and two of de novo mutation of theBMPR2 gene in sporadic PPH. CONCLUSION The sporadic form of PPH is associated with germline mutations of the gene encoding the receptor protein BMPR-II in at least 26% of cases. A molecular classification of PPH, based upon the presence or absence ofBMPR2 mutations, has important implications for patient management and screening of relatives.

BMPR2 Haploinsufficiency as the Inherited Molecular Mechanism for Primary Pulmonary Hypertension

Primary pulmonary hypertension (PPH) is a potentially lethal disorder, because the elevation of the pulmonary arterial pressure may result in right-heart failure. Histologically, the disorder is characterized by proliferation of pulmonary-artery smooth muscle and endothelial cells, by intimal hyperplasia, and by in situ thrombus formation. Heterozygous mutations within the bone morphogenetic protein type II receptor (BMPR-II) gene (BMPR2), of the transforming growth factor beta (TGF-beta) cell-signaling superfamily, have been identified in familial and sporadic cases of PPH. We report the molecular spectrum of BMPR2 mutations in 47 additional families with PPH and in three patients with sporadic PPH. Among the cohort of patients, we have identified 22 novel mutations, including 4 partial deletions, distributed throughout the BMPR2 gene. The majority (58%) of mutations are predicted to lead to a premature termination codon. We have also investigated the functional impact and genotype-phenotype relationships, to elucidate the mechanisms contributing to pathogenesis of this important vascular disease. In vitro expression analysis demonstrated loss of BMPR-II function for a number of the identified mutations. These data support the suggestion that haploinsufficiency represents the common molecular mechanism in PPH. Marked variability of the age at onset of disease was observed both within and between families. Taken together, these studies illustrate the considerable heterogeneity of BMPR2 mutations that cause PPH, and they strongly suggest that additional factors, genetic and/or environmental, may be required for the development of the clinical phenotype.

MUTATION IN THE GENE FOR BONE MORPHOGENETIC PROTEIN RECEPTOR II AS A CAUSE OF PRIMARY PULMONARY HYPERTENSION IN A LARGE KINDRED

BACKGROUND Most patients with primary pulmonary hypertension are thought to have sporadic, not inherited, disease. Because clinical disease develops in only 10 to 20 percent of persons carrying the gene for familial primary pulmonary hypertension, we hypothesized that many patients with apparently sporadic primary pulmonary hypertension may actually have familial primary pulmonary hypertension. METHODS In a study conducted over 20 years, we developed a registry of 67 families affected by familial primary pulmonary hypertension. Through patient referrals, extensive family histories, and correlation of family pedigrees, we discovered shared ancestry among five subfamilies. We established the diagnosis of primary pulmonary hypertension by direct evaluation of patients and review of autopsy material and medical records. We assessed some family members for mutations in the gene encoding bone morphogenetic protein receptor II (BMPR2), which has recently been found to cause familial primary pulmonary hypertension. RESULTS We linked five separately identified subfamilies that included 394 known members spanning seven generations, which were traced back to a founding couple in the mid-1800s. Familial primary pulmonary hypertension has been diagnosed in 18 family members, 12 of whom were first thought to have sporadic disease. The conditions of 7 of the 18 were initially misdiagnosed as other cardiopulmonary diseases. Six members affected with familial primary pulmonary hypertension and 6 of 10 at risk for carriage have been undergone genotype analysis, and they have the same mutation in BMPR2, a transversion of thymine to guanine at position 354 in exon 3. CONCLUSIONS Many cases of apparently sporadic primary pulmonary hypertension may be familial. Failure to detect familial primary pulmonary hypertension results from incomplete expression within families, skipped generations, and incomplete family pedigrees. The recent discovery of mutations in BMPR2 should make it possible to identify those with susceptibility to disease.

Alterations in oestrogen metabolism: implications for higher penetrance of familial pulmonary arterial hypertension in females

Mutations in bone morphogenetic protein receptor type 2 (BMPR2) cause familial pulmonary arterial hypertension (FPAH), but the penetrance is reduced and females are significantly overrepresented. In addition, gene expression data implicating the oestrogen-metabolising enzyme CYP1B1 suggests a detrimental role of oestrogens or oestrogen metabolites. We examined genetic and metabolic markers of altered oestrogen metabolism in subjects with a BMPR2 mutation. Genotypes for CYP1B1 Asn453Ser (N453S) were determined for 140 BMPR2 mutation carriers (86 females and 54 males). Nested from those subjects, a case–control study of urinary oestrogen metabolite levels (2-hydroxyoestrogen (2-OHE) and 16α-hydroxyoestrone (16α-OHE1)) was conducted in females (five affected mutation carriers versus six unaffected mutation carriers). Among females, there was four-fold higher penetrance among subjects homozygous for the wild-type genotype (N/N) than those with N/S or S/S genotypes (p = 0.005). Consistent with this finding, the 2-OHE/16α-OHE1 ratio was 2.3-fold lower in affected mutation carriers compared to unaffected mutation carriers (p = 0.006). Our findings suggest that variations in oestrogens and oestrogen metabolism modify FPAH risk. Further investigation of the role of oestrogens in this disease with profound sex bias may yield new insights and, perhaps, therapeutic interventions.

Gross BMPR2 gene rearrangements constitute a new cause for primary pulmonary hypertension

Purpose: Approximately 50% of patients with familial primary pulmonary hypertension (FPPH) have been reported to have mutations within the bone morphogenic protein receptor type 2 (BMPR2) gene. The vast majority of these mutations were identified by PCR amplification and sequencing of individual exons. The aim of our study was to determine if additional BMPR2 mutations not found by exon sequencing alone could account for a significant portion of these negative cases.Methods: We examined DNA samples from 12 families, previously found to be negative for BMPR2 mutations, to identify any large BMPR2 gene rearrangements.Results: Southern blot analysis found large gene rearrangements in four (33%) unrelated kindreds. Further analysis by reverse transcriptase PCR (RT-PCR) of BMPR2 transcripts from two of these kindreds found one to be heterozygous for a exon 10 duplication and the second to be heterozygous for a deletion of exons 4 to 5. Nonhomologous recombination is believed to be the cause of these large insertions/deletions.Conclusion: Our results demonstrate the inherent problems associated with exon-by-exon sequencing and the importance of other screening methods such as Southern blot and RT-PCR in the identification of BMPR2 mutations.

Longitudinal Analysis Casts Doubt on the Presence of Genetic Anticipation in Heritable Pulmonary Arterial Hypertension

RATIONALE Analysis of the age of onset in heritable pulmonary arterial hypertension (HPAH) has led to the hypothesis that genetic anticipation causes younger age of onset and death in subsequent generations. With accrual of pedigree data over multiple decades, we retested this hypothesis using analyses that eliminate the truncation of data that exists with shorter duration of follow-up. OBJECTIVES To analyze the pedigrees of families with mutations in bone morphogenetic protein receptor type 2 (BMPR2), afflicted in two or more generations with HPAH, eliminating time truncation bias by including families for whom we have at least 57 years of data. METHODS We analyzed 355 individuals with BMPR2 mutations from 53 families in the Vanderbilt Pulmonary Hypertension Registry. We compared age at diagnosis or death in affected individuals (n = 249) by generation within families with multigenerational disease. We performed linear mixed effects models and we limited time-truncation bias by restricting date of birth to before 1955. This allowed for 57 years of follow-up (1955-2012) for mutation carriers to develop disease. We also conducted Kaplan-Meier analysis to include currently unaffected mutation carriers (n = 106). MEASUREMENTS AND MAIN RESULTS Differences in age at diagnosis by generation were found in a biased analysis that included all birth years to the present, but this finding was eliminated when the 57-year observation limit was imposed. By Kaplan-Meier analysis, inclusion of currently unaffected mutation carriers strengthens the observation that bias of ascertainment exists when recent generations are included. CONCLUSIONS Genetic anticipation is likely an artifact of incomplete time of observation of kindreds with HPAH due to BMPR2 mutations.

Truncating and missense BMPR2 mutations differentially affect the severity of heritable pulmonary arterial hypertension

BackgroundAutosomal dominant inheritance of germline mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene are a major risk factor for pulmonary arterial hypertension (PAH). While previous studies demonstrated a difference in severity between BMPR2 mutation carriers and noncarriers, it is likely disease severity is not equal among BMPR2 mutations. We hypothesized that patients with missense BMPR2 mutations have more severe disease than those with truncating mutations.MethodsTesting for BMPR2 mutations was performed in 169 patients with PAH (125 with a family history of PAH and 44 with sporadic disease). Of the 106 patients with a detectable BMPR2 mutation, lymphocytes were available in 96 to functionally assess the nonsense-mediated decay pathway of RNA surveillance. Phenotypic characteristics were compared between BMPR2 mutation carriers and noncarriers, as well as between those carriers with a missense versus truncating mutation.ResultsWhile there was a statistically significant difference in age at diagnosis between carriers and noncarriers, subgroup analysis revealed this to be the case only for females. Among carriers, there was no difference in age at diagnosis, death, or survival according to exonic location of the BMPR2 mutation. However, patients with missense mutations had statistically significant younger ages at diagnosis and death, as well as shorter survival from diagnosis to death or lung transplantation than those with truncating mutations. Consistent with this data, the majority of missense mutations were penetrant prior to age 36 years, while the majority of truncating mutations were penetrant after age 36 years.ConclusionIn this cohort, BMPR2 mutation carriers have more severe PAH disease than noncarriers, but this is only the case for females. Among carriers, patients with missense mutations that escape nonsense-mediated decay have more severe disease than those with truncating mutations. These findings suggest that treatment and prevention strategies directed specifically at BMPR2 pathway defects may need to vary according to the type of mutation.

BMPR2 mutations and survival in pulmonary arterial hypertension: an individual participant data meta-analysis

Summary Background Mutations in the gene encoding the bone morphogenetic protein receptor type II (BMPR2) are the commonest genetic cause of pulmonary arterial hypertension (PAH). However, the effect of BMPR2 mutations on clinical phenotype and outcomes remains uncertain. Methods We analysed individual participant data of 1550 patients with idiopathic, heritable, and anorexigen-associated PAH from eight cohorts that had been systematically tested for BMPR2 mutations. The primary outcome was the composite of death or lung transplantation. All-cause mortality was the secondary outcome. Hazard ratios (HRs) for death or transplantation and all-cause mortality associated with the presence of BMPR2 mutation were calculated using Cox proportional hazards models stratified by cohort. Findings Overall, 448 (29%) of 1550 patients had a BMPR2 mutation. Mutation carriers were younger at diagnosis (mean age 35·4 [SD 14·8] vs 42·0 [17·8] years), had a higher mean pulmonary artery pressure (60·5 [13·8] vs 56·4 [15·3] mm Hg) and pulmonary vascular resistance (16·6 [8·3] vs 12·9 [8·3] Wood units), and lower cardiac index (2·11 [0·69] vs 2·51 [0·92] L/min per m2; all p<0·0001). Patients with BMPR2 mutations were less likely to respond to acute vasodilator testing (3% [10 of 380] vs 16% [147 of 907]; p<0·0001). Among the 1164 individuals with available survival data, age-adjusted and sex-adjusted HRs comparing BMPR2 mutation carriers with non-carriers were 1·42 (95% CI 1·15–1·75; p=0·0011) for the composite of death or lung transplantation and 1·27 (1·00–1·60; p=0·046) for all-cause mortality. These HRs were attenuated after adjustment for potential mediators including pulmonary vascular resistance, cardiac index, and vasoreactivity. HRs for death or transplantation and all-cause mortality associated with BMPR2 mutation were similar in men and women, but higher in patients with a younger age at diagnosis (p=0·0030 for death or transplantation, p=0·011 for all-cause mortality). Interpretation Patients with PAH and BMPR2 mutations present at a younger age with more severe disease, and are at increased risk of death, and death or transplantation, compared with those without BMPR2 mutations. Funding Cambridge NIHR Biomedical Research Centre, Medical Research Council, British Heart Foundation, Assistance Publique-Hôpitaux de Paris, INSERM, Université Paris-Sud, Intermountain Research and Medical Foundation, Vanderbilt University, National Center for Advancing Translational Sciences, National Institutes of Health, National Natural Science Foundation of China, and Beijing Natural Science Foundation.

Synergistic heterozygosity for TGFβ1 SNPs and BMPR2 mutations modulates the age at diagnosis and penetrance of familial pulmonary arterial hypertension

Purpose: We hypothesized that functional TGFβ1 SNPs increase TGFβ/BMP signaling imbalance in BMPR2 mutation heterozygotes to accelerate the age at diagnosis, increase the penetrance and SMAD2 expression in familial pulmonary arterial hypertension.Methods: Single nucleotide polymorphism genotypes of BMPR2 mutation heterozygotes, age at diagnosis, and penetrance of familial pulmonary arterial hypertension were compared and SMAD2 expression was studied in lung sections.Results: BMPR2 mutation heterozygotes with least active -509 or codon 10 TGFβ1 SNPs had later mean age at diagnosis of familial pulmonary arterial hypertension (39.5 and 43.2 years) than those with more active genotypes (31.6 and 33.1 years, P = 0.03 and 0.02, respectively). Kaplan-Meier analysis also showed that those with the less active single nucleotide polymorphisms had later age at diagnosis. BMPR2 mutation heterozygotes with nonsense-mediated decay resistant BMPR2 mutations and the least, intermediate and most active -509 TGFβ1 SNP genotypes had penetrances of 33, 72, and 80%, respectively (P = 0.003), whereas those with 0–1, 2, or 3–4 active single nucleotide polymorphism alleles had penetrances of 33, 72, and 75% (P = 0.005). The relative expression of TGFβ1 dependent SMAD2 was increased in lung sections of those with familial pulmonary arterial hypertension compared with controls.Conclusions: The TGFβ1 SNPs studied modulate age at diagnosis and penetrance of familial pulmonary arterial hypertension in BMPR2 mutation heterozygotes, likely by affecting TGFβ/BMP signaling imbalance. This modulation is an example of Synergistic Heterozygosity.

Estrogen Metabolite 16α-Hydroxyestrone Exacerbates Bone Morphogenetic Protein Receptor Type II–Associated Pulmonary Arterial Hypertension Through MicroRNA-29–Mediated Modulation of Cellular Metabolism

Background— Pulmonary arterial hypertension (PAH) is a proliferative disease of the pulmonary vasculature that preferentially affects women. Estrogens such as the metabolite 16&agr;-hydroxyestrone (16&agr;OHE) may contribute to PAH pathogenesis, and alterations in cellular energy metabolism associate with PAH. We hypothesized that 16&agr;OHE promotes heritable PAH (HPAH) via microRNA-29 (miR-29) family upregulation and that antagonism of miR-29 would attenuate pulmonary hypertension in transgenic mouse models of Bmpr2 mutation. Methods and Results— MicroRNA array profiling of human lung tissue found elevation of microRNAs associated with energy metabolism, including the miR-29 family, among HPAH patients. miR-29 expression was 2-fold higher in Bmpr2 mutant mice lungs at baseline compared with controls and 4 to 8-fold higher in Bmpr2 mice exposed to 16&agr;OHE 1.25 &mgr;g/h for 4 weeks. Blot analyses of Bmpr2 mouse lung protein showed significant reductions in peroxisome proliferator–activated receptor-&ggr; and CD36 in those mice exposed to 16&agr;OHE and protein derived from HPAH lungs compared with controls. Bmpr2 mice treated with anti–miR-29 (20-mg/kg injections for 6 weeks) had improvements in hemodynamic profile, histology, and markers of dysregulated energy metabolism compared with controls. Pulmonary artery smooth muscle cells derived from Bmpr2 murine lungs demonstrated mitochondrial abnormalities, which improved with anti–miR-29 transfection in vitro; endothelial-like cells derived from HPAH patient induced pluripotent stem cell lines were similar and improved with anti–miR-29 treatment. Conclusions— 16&agr;OHE promotes the development of HPAH via upregulation of miR-29, which alters molecular and functional indexes of energy metabolism. Antagonism of miR-29 improves in vivo and in vitro features of HPAH and reveals a possible novel therapeutic target.