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1.  Future Directions in Idiopathic Pulmonary Fibrosis Research. An NHLBI Workshop Report 
The median survival of patients with idiopathic pulmonary fibrosis (IPF) continues to be approximately 3 years from the time of diagnosis, underscoring the lack of effective medical therapies for this disease. In the United States alone, approximately 40,000 patients die of this disease annually. In November 2012, the NHLBI held a workshop aimed at coordinating research efforts and accelerating the development of IPF therapies. Basic, translational, and clinical researchers gathered with representatives from the NHLBI, patient advocacy groups, pharmaceutical companies, and the U.S. Food and Drug Administration to review the current state of IPF research and identify priority areas, opportunities for collaborations, and directions for future research. The workshop was organized into groups that were tasked with assessing and making recommendations to promote progress in one of the following six critical areas of research: (1) biology of alveolar epithelial injury and aberrant repair; (2) role of extracellular matrix; (3) preclinical modeling; (4) role of inflammation and immunity; (5) genetic, epigenetic, and environmental determinants; (6) translation of discoveries into diagnostics and therapeutics. The workshop recommendations provide a basis for directing future research and strategic planning by scientific, professional, and patient communities and the NHLBI.
PMCID: PMC3983890  PMID: 24160862
idiopathic pulmonary fibrosis; alveolar epithelial cells; extracellular matrix; interstitial lung disease; inflammation
3.  Prostanoids But Not Oral Therapies Improve Right Ventricular Function in Pulmonary Arterial Hypertension 
JACC. Heart failure  2013;1(4):300-307.
We hypothesized that RV stroke work index (RVSWI) and pulmonary capacitance (PC) would increase after treatment for pulmonary arterial hypertension (PAH) and that prostanoids would have a stronger effect than oral therapy.
Right ventricular (RV) function is a major determinant of outcome in patients with PAH. Little is known about the response of RV function or its hemodynamic determinants to PAH-specific therapy.
We reviewed hemodynamic and health data on 58 patients from an institutional registry and analyzed changes in hemodynamics between diagnostic and first repeat catheterization after initiation of therapy for PAH.
RVSWI and PC increased significantly after therapy (p = 0.007 and 0.02, respectively). Improvement in RV function was limited to patients treated with prostanoid-only therapy (p = 0.04); no improvement was found in patients treated with oral therapy (p = 0.25). Patients with the poorest baseline RV function (lowest tertile) had the greatest improvement post-therapy (p = 0.005 and < 0.001 vs. middle and highest tertiles). The major determinant of RVSWI was change in stroke volume (rs = 0.54, p < 0.001), indicating RVSWI is an accurate reflection of RV function.
RV function improves after therapy with regimens including prostanoids but not oral-only regimens. Patients with the least compensated RV function at diagnosis may derive the most benefit from therapy. Larger studies are needed to determine whether changes in RVSWI after therapy are associated with outcomes.
PMCID: PMC3763862  PMID: 24015376
Tremendous progress has been made in understanding the genetics of heritable pulmonary arterial hypertension (HPAH) since its description in the 1950s. Germline mutations in the gene coding bone morphogenetic receptor type 2 (BMPR2) are detectable in the majority of cases of HPAH, and in a small proportion of cases of idiopathic pulmonary arterial hypertension (IPAH). Recent advancements in gene sequencing methods have facilitated the discovery of additional genes with mutations among those with and without familial PAH (CAV1, KCNK3). HPAH is an autosomal dominant disease characterized by reduced penetrance, variable expressivity, and female predominance. These characteristics suggest that genetic and non-genetic factors modify disease expression, highlighting areas of active investigation. The reduced penetrance makes genetic counseling complex, as the majority of carriers of PAH-related mutations will never be diagnosed with the disease. This issue is increasingly important, as clinical testing for BMPR2 and other mutations is now available for the evaluation of patients and their at risk kin. The possibilities to avoid mutation transmission, such as the rapidly advancing field of pre-implantation genetic testing, highlights the need for all clinicians to understand the genetic features of PAH risk.
PMCID: PMC4102304  PMID: 24037626
bone morphogenetic protein receptor type 2; caveolin-1; KCNK3; CBLN2; pulmonary arterial hypertension; genetics; modifiers; hereditary hemorrhagic telangiectasia
5.  Genetics of Pulmonary Arterial Hypertension 
Tremendous progress has been made in understanding the genetics of hereditable pulmonary arterial hypertension (HPAH) since its description in the 1950s. Germline mutations in the gene coding bone morphogenetic receptor type 2 (BMPR2) are detectable in the majority of cases of HPAH, and in a small proportion of cases of idiopathic pulmonary arterial hypertension (IPAH). HPAH is an autosomal dominant disease characterized by reduced penetrance, variable expressivity, female predominance, and genetic anticipation. These characteristics suggest that endogenous and exogenous factors modify disease expression and areas of emphasis for future investigation. The variable clinical expression makes genetic counseling complex because the majority of carriers of a BMPR2 mutation will not be diagnosed with the disease. This issue will become increasingly important, as clinical testing for BMPR2 mutations is now available for the evaluation of patients and family members with HPAH and IPAH.
PMCID: PMC3737593  PMID: 19634078
Bone morphogenetic protein receptor 2; pulmonary arterial hypertension; genetics; modifiers; hereditary hemorrhagic telangiectasia
6.  Intratracheal Bleomycin Causes Airway Remodeling and Airflow Obstruction in Mice 
Experimental lung research  2012;38(3):135-146.
In addition to parenchymal fibrosis, fibrotic remodeling of the distal airways has been reported in interstitial lung diseases. Mechanisms of airway wall remodeling, which occurs in a variety of chronic lung diseases, are not well defined and current animal models are limited.
We quantified airway remodeling in lung sections from subjects with idiopathic pulmonary fibrosis (IPF) and controls. To investigate intratracheal bleomycin as a potential animal model for fibrotic airway remodeling, we evaluated lungs from C57BL/6 mice after bleomycin treatment by histologic scoring for fibrosis and peribronchial inflammation, morphometric evaluation of subepithelial connective tissue volume density, TUNEL assay, and immunohistochemistry for transforming growth factor β1 (TGFβ1), TGFβ2, and the fibroblast marker S100A4. Lung mechanics were determined at 3 weeks post-bleomycin.
IPF lungs had small airway remodeling with increased bronchial wall thickness compared to controls. Similarly, bleomycin treated mice developed dose-dependent airway wall inflammation and fibrosis and greater airflow resistance after high dose bleomycin. Increased TUNEL+ bronchial epithelial cells and peribronchial inflammation were noted by 1 week, and expression of TGFβ1 and TGFβ2 and accumulation of S100A4+ fibroblasts correlated with airway remodeling in a bleomycin dose-dependent fashion.
IPF is characterized by small airway remodeling in addition to parenchymal fibrosis, a pattern also seen with intratracheal bleomycin. Bronchial remodeling from intratracheal bleomycin follows a cascade of events including epithelial cell injury, airway inflammation, pro-fibrotic cytokine expression, fibroblast accumulation, and peribronchial fibrosis. Thus, this model can be utilized to investigate mechanisms of airway remodeling.
PMCID: PMC4046254  PMID: 22394287
airway; inflammation; remodeling; mouse model; fibroblast
7.  Genome-wide association analysis identifies a susceptibility locus for pulmonary arterial hypertension 
Nature genetics  2013;45(5):518-521.
Pulmonary arterial hypertension (PAH) is a rare and devastating disease, resulting from progressive obliteration of small caliber pulmonary arteries by proliferating vascular cells, and leading to cardiac failure, with an untreated mean survival of less than three years 1,2. PAH can complicate other pathological conditions, or can occur in the context of genetic mutations causing heritable PAH, or can be considered as idiopathic (iPAH), which represents approximately 40% of all PAH 3,4. Low penetrance dominant BMPR2 mutations are found in ~70% of familial PAH (fPAH), and in ~15% of iPAH which are thereafter considered as heritable PAH 5,6. We conducted a Genome-Wide Association Study (GWAS) based on two independent case-control studies for iPAH and fPAH (without BMPR2 mutations) totaling 625 patients and 1,525 healthy individuals, to identify novel genetic factors associated with iPAH and fPAH (i/fPAH) in the absence of BMPR2 mutations. A genome wide significant association was detected at the CBLN2 locus mapping to 18q22.3, the risk allele being associated with an odds ratio for i/fPAH of 1.97 [1.59 – 2.45] (P = 7.47 x 10−10). CBLN2 is expressed in the lung, particularly in pulmonary vascular endothelial cells, and its expression is increased in explanted lungs from PAH patients and in endothelial cells cultured from explanted PAH lungs.
PMCID: PMC3983781  PMID: 23502781
8.  A Novel Channelopathy in Pulmonary Arterial Hypertension 
The New England journal of medicine  2013;369(4):351-361.
Pulmonary arterial hypertension is a devastating disease with high mortality. Familial cases of pulmonary arterial hypertension are usually characterized by autosomal dominant transmission with reduced penetrance, and some familial cases have unknown genetic causes.
We studied a family in which multiple members had pulmonary arterial hypertension without identifiable mutations in any of the genes known to be associated with the disease, including BMPR2, ALK1, ENG, SMAD9, and CAV1. Three family members were studied with whole-exome sequencing. Additional patients with familial or idiopathic pulmonary arterial hypertension were screened for the mutations in the gene that was identified on whole-exome sequencing. All variants were expressed in COS-7 cells, and channel function was studied by means of patch-clamp analysis.
We identified a novel heterozygous missense variant c.608 G→A (G203D) in KCNK3 (the gene encoding potassium channel subfamily K, member 3) as a disease-causing candidate gene in the family. Five additional heterozygous missense variants in KCNK3 were independently identified in 92 unrelated patients with familial pulmonary arterial hypertension and 230 patients with idiopathic pulmonary arterial hypertension. We used in silico bioinformatic tools to predict that all six novel variants would be damaging. Electrophysiological studies of the channel indicated that all these missense mutations resulted in loss of function, and the reduction in the potassium-channel current was remedied by the application of the phospholipase inhibitor ONO-RS-082.
Our study identified the association of a novel gene, KCNK3, with familial and idiopathic pulmonary arterial hypertension. Mutations in this gene produced reduced potassium-channel current, which was successfully remedied by pharmacologic manipulation. (Funded by the National Institutes of Health.)
PMCID: PMC3792227  PMID: 23883380
9.  An evidence-based knowledgebase of pulmonary arterial hypertension to identify genes and pathways relevant to pathogenesis† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c3mb70496c Click here for additional data file.  
Molecular Biosystems  2014;10(4):732-740.
First literature-based, high-quality gene resource focused on pulmonary arterial hypertension (PAH) to identify genes and pathways relevant to PAH pathogenesis.
Pulmonary arterial hypertension (PAH) is a major progressive form of pulmonary hypertension (PH) with more than 4800 patients in the United States. In the last two decades, many studies have identified numerous genes associated with this disease. However, there is no comprehensive research resource for PAH or other PH types that integrates various genetic studies and their related biological information. Thus, the number of associated genes, and their strength of evidence, is unclear. In this study, we tested the hypothesis that a web-based knowledgebase could be used to develop a biological map of highly interrelated, functionally important genes in PAH. We developed the pulmonary arterial hypertension knowledgebase (PAHKB,, a comprehensive database with a user-friendly web interface. PAHKB extracts genetic data from all available sources, including those from association studies, genetic mutation, gene expression, animal model, supporting literature, various genomic annotations, gene networks, cellular and regulatory pathways, as well as microRNAs. Moreover, PAHKB provides online tools for data browsing and searching, data integration, pathway graphical presentation, and gene ranking. In the current release, PAHKB contains 341 human PH-related genes (293 protein coding and 48 non-coding genes) curated from over 1000 PubMed abstracts. Based on the top 39 ranked PAH-related genes in PAHKB, we constructed a core biological map. This core map was enriched with the TGF-beta signaling pathway, focal adhesion, cytokine–cytokine receptor interaction, and MAPK signaling. In addition, the reconstructed map elucidates several novel cancer signaling pathways, which may provide clues to support the application of anti-cancer therapeutics to PAH. In summary, we have developed a system for the identification of core PH-related genes and identified critical signaling pathways that may be relevant to PAH pathogenesis. This system can be easily applied to other pulmonary diseases.
PMCID: PMC3950334  PMID: 24448676
10.  MUC5B Promoter Polymorphism and Interstitial Lung Abnormalities 
The New England journal of medicine  2013;368(23):2192-2200.
A common promoter polymorphism (rs35705950) in MUC5B, the gene encoding mucin 5B, is associated with idiopathic pulmonary fibrosis. It is not known whether this polymorphism is associated with interstitial lung disease in the general population.
We performed a blinded assessment of interstitial lung abnormalities detected in 2633 participants in the Framingham Heart Study by means of volumetric chest computed tomography (CT). We evaluated the relationship between the abnormalities and the genotype at the rs35705950 locus.
Of the 2633 chest CT scans that were evaluated, interstitial lung abnormalities were present in 177 (7%). Participants with such abnormalities were more likely to have shortness of breath and chronic cough and reduced measures of total lung and diffusion capacity, as compared with participants without such abnormalities. After adjustment for covariates, for each copy of the minor rs35705950 allele, the odds of interstitial lung abnormalities were 2.8 times greater (95% confidence interval [CI], 2.0 to 3.9; P<0.001), and the odds of definite CT evidence of pulmonary fibrosis were 6.3 times greater (95% CI, 3.1 to 12.7; P<0.001). Although the evidence of an association between the MUC5B genotype and interstitial lung abnormalities was greater among participants who were older than 50 years of age, a history of cigarette smoking did not appear to influence the association.
The MUC5B promoter polymorphism was found to be associated with interstitial lung disease in the general population. Although this association was more apparent in older persons, it did not appear to be influenced by cigarette smoking. (Funded by the National Institutes of Health and others; number, NCT00005121.)
PMCID: PMC3747636  PMID: 23692170
11.  Pulmonary Arterial Hypertension 
Familial pulmonary arterial hypertension (FPAH) was described 60 years ago, but real progress in understanding its origins and pathogenesis is just beginning. Germline mutations in bone morphogenetic protein receptor type 2 (BMPR2) are responsible for the disease in most families, and also in many sporadic cases of idiopathic PAH. Heritable PAH refers to patients with a positive family history, or with a responsible genetic mutation, and is an autosomal dominant disease that affects females disproportionately, may occur at any age, and is characterized by reduced penetrance and variable expressivity. These characteristics suggest that other endogenous or exogenous factors modify its expression. Several different factors have recently been demonstrated to modify the clinical expression of BMPR2 mutation, including estrogen metabolites and functional polymorphisms in transforming growth factor–β1 and CYP1B1. Furthermore, a linkage study recently identified modifier loci for BMPR2 clinical expression, which suggests an oligogenic model. Clinical testing for BMPR2 mutations is available for families with heritable and idiopathic PAH, and is an evolving model of personalized medicine. Variable age of onset and decreased penetrance confound genetic counseling, because the majority of carriers of a BMPR2 mutation will never develop PAH, but often transmit the risk to their progeny.
PMCID: PMC3131832  PMID: 21543793
pulmonary; pulmonary artery; pulmonary hypertension
12.  Genome-wide association study identifies multiple susceptibility loci for pulmonary fibrosis 
Nature genetics  2013;45(6):613-620.
We performed a genome-wide association study in non-Hispanic white subjects with fibrotic idiopathic interstitial pneumonias (N=1616) and controls (N=4683); replication was assessed in 876 cases and 1890 controls. We confirmed association with TERT and MUC5B on chromosomes 5p15 and 11p15, respectively, the chromosome 3q26 region near TERC, and identified 7 novel loci (PMeta = 2.4×10−8 to PMeta = 1.1×10−19). The novel loci include FAM13A (4q22), DSP (6p24), OBFC1 (10q24), ATP11A (13q34), DPP9 (19p13), and chromosomal regions 7q22 and 15q14-15. Our results demonstrate that genes involved in host defense, cell-cell adhesion, and DNA repair contribute to the risk of fibrotic IIP.
PMCID: PMC3677861  PMID: 23583980
13.  Bronchoscopic Cryobiopsy for the Diagnosis of Diffuse Parenchymal Lung Disease 
PLoS ONE  2013;8(11):e78674.
Although in some cases clinical and radiographic features may be sufficient to establish a diagnosis of diffuse parenchymal lung disease (DPLD), surgical lung biopsy is frequently required. Recently a new technique for bronchoscopic lung biopsy has been developed using flexible cryo-probes. In this study we describe our clinical experience using bronchoscopic cryobiopsy for diagnosis of diffuse lung disease.
A retrospective study of subjects who had undergone bronchoscopic cryobiopsy for evaluation of DPLD at an academic tertiary care center from January 1, 2012 through January 15, 2013 was performed. The procedure was performed using a flexible bronchoscope to acquire biopsies of lung parenchyma. H&E stained biopsies were reviewed by an expert lung pathologist.
Twenty-five eligible subjects were identified. With a mean area of 64.2 mm2, cryobiopsies were larger than that typically encountered with traditional transbronchial forceps biopsy. In 19 of the 25 subjects, a specific diagnosis was obtained. In one additional subject, biopsies demonstrating normal parenchyma were felt sufficient to exclude diffuse lung disease as a cause of dyspnea. The overall diagnostic yield of bronchoscopic cryobiopsy was 80% (20/25). The most frequent diagnosis was usual interstitial pneumonia (UIP) (n = 7). Three of the 25 subjects ultimately required surgical lung biopsy. There were no significant complications.
In patients with suspected diffuse parenchymal lung disease, bronchoscopic cryobiopsy is a promising and minimally invasive approach to obtain lung tissue with high diagnostic yield.
PMCID: PMC3827078  PMID: 24265706
14.  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.
PMCID: PMC3530218  PMID: 22923661
hereditary; pulmonary hypertension; genetics
15.  Shorter survival in familial versus idiopathic pulmonary arterial hypertension is associated with hemodynamic markers of impaired right ventricular function 
Pulmonary Circulation  2013;3(3):589-598.
Although individuals with familial pulmonary arterial hypertension (FPAH) have more severe hemodynamics, compared to individuals with idiopathic PAH (IPAH), it is unclear whether this translates into a survival difference. The influence of right ventricular (RV) function on survival in these groups is also unknown. We reviewed hemodynamic data and health information from a prospective institutional database of 57 FPAH and 66 IPAH patients registered with the Vanderbilt Pulmonary Hypertension Research Cohort. We compared hemodynamics at the time of diagnosis between the two groups and calculated pulmonary arteriolar capacitance (PC) and RV stroke work index (RVSWI). Using survival analysis, we compared freedom from a 5-year composite of death or lung transplantation in FPAH and IPAH patients. The composite outcome of death or transplant at 5 years from diagnosis was significantly increased in FPAH (log rank ). PC and RVSWI were significantly decreased in FPAH, compared to IPAH ( for both). In univariate analysis, PC (odds ratio [OR]: 0.17 [95% confidence interval (95% CI): 0.03–0.83]) and RVSWI (OR: 0.86 [95% CI: 0.77–0.95]) were predictors of mortality, as were cardiac index (OR: 0.17 [95% CI: 0.06–0.51]) and PVR (OR: 1.1 [95% CI: 1.01–1.12]). Among FPAH patients, RVSWI was lower in those who died or received a transplant than in survivors (), while PC was not (). We found significantly worse event-free survival and significantly lower PC and RVSWI in FPAH than in IPAH. In FPAH patients who died or underwent transplantation, RVSWI was lower than that in survivors, suggesting disproportionate RV dysfunction.
PMCID: PMC4070798  PMID: 24618543
pulmonary arterial hypertension; familial pulmonary arterial hypertension; right ventricular function; hemodynamics
16.  Interaction between bone morphogenetic protein receptor type 2 and estrogenic compounds in pulmonary arterial hypertension 
Pulmonary Circulation  2013;3(3):564-577.
The majority of heritable pulmonary arterial hypertension (HPAH) cases are associated with mutations in bone morphogenetic protein receptor type 2 (BMPR2). BMPR2 mutation carries about a 20% lifetime risk of PAH development, but penetrance is approximately three times higher in females. Previous studies have shown a correlation between estrogen metabolism and penetrance, with increased levels of the estrogen metabolite 16α-hydroxyestrone (16αOHE) and reduced levels of the metabolite 2-methoxyestrogen (2ME) associated with increased risk of disease. The goal of this study was to determine whether 16αOHE increased and 2ME decreased penetrance of disease in Bmpr2 mutant mice and, if so, by what mechanism. We found that 16αOHE∶2ME ratio was high in male human HPAH patients. Bmpr2 mutant male mice receiving chronic 16αOHE had doubled disease penetrance, associated with reduced cardiac output. 2ME did not have a significant protective effect, either alone or in combination with 16αOHE. In control mice but not in Bmpr2 mutant mice, 16αOHE suppressed bone morphogenetic protein signaling, probably directly through suppression of Bmpr2 protein. Bmpr2 mutant pulmonary microvascular endothelial cells were insensitive to estrogen signaling through canonical pathways, associated with aberrant intracellular localization of estrogen receptor α. In both control and Bmpr2 mutant mice, 16αOHE was associated with suppression of cytokine expression but with increased alternate markers of injury, including alterations in genes related to thrombotic function, angiogenesis, planar polarity, and metabolism. These data support a causal relationship between increased 16αOHE and increased PAH penetrance, with the likely molecular mechanisms including suppression of BMPR2, alterations in estrogen receptor translocation, and induction of vascular injury and insulin resistance–related pathways.
PMCID: PMC4070799  PMID: 24618541
estrogen; pulmonary hypertension; bmp; metabolism; insulin; wnt.
17.  Genetics and Mediators in Pulmonary Arterial Hypertension 
Clinics in chest medicine  2007;28(1):43-viii.
PMCID: PMC3740514  PMID: 17338927
18.  Genetics and Genomics of Pulmonary Arterial Hypertension 
Pulmonary arterial hypertension (PAH) is a rare disorder that may be hereditable (HPAH), idiopathic (IPAH), or associated with either drug-toxin exposures or other medical conditions. Familial cases have long been recognised and are usually due to mutations in Bone Morphogenetic Protein Receptor type 2 gene (BMPR2), or, much less commonly, 2 other members of the transforming growth factor-beta superfamily, Activin-like Kinase-Type I (ALK1) and Endoglin (ENG), which are associated with hereditary hemorrhagic telangiectasia. In addition, approximately 20% of patients with IPAH carry mutations in BMPR2. We provide a summary of BMPR2 mutations associated with IPAH/HPAH, most of which are unique to each family and are presumed to result in loss of function. We review the finding of missense variants and variants of unknown significance in BMPR2 in IPAH/HPAH, fenfluramine exposure, and PAH associated with congenital heart disease. Clinical testing for BMPR2 mutations is available and may be offered to HPAH and IPAH patients but should be preceded by genetic counselling, since lifetime penetrance is only 10%–20%, and there are currently no known effective preventative measures. Identification of a familial mutation can be valuable in reproductive planning and identifying family members who are not mutation carriers and thus will not require lifelong surveillance. With advances in genomic technology and with international collaborative efforts, genome-wide association studies will be conducted to identify additional genes for HPAH, genetic modifiers for BMPR2 penetrance, and genetic susceptibility to IPAH. In addition, collaborative studies of BMPR2 mutation carriers should enable identification of environmental modifiers, biomarkers for disease development and progression, and surrogate markers for efficacy end points in clinical drug development, thereby providing an invaluable resource for trials of PAH prevention.
PMCID: PMC3725550  PMID: 19555857
Pulmonary Arterial Hypertension; Genetics; Genomics
19.  Whole Exome Sequencing to Identify a Novel Gene (Caveolin-1) Associated with Human Pulmonary Arterial Hypertension 
Heritable and idiopathic pulmonary arterial hypertension (PAH) are phenotypically identical and associated with mutations in several genes related to TGF beta signaling, including bone morphogenetic protein receptor type 2 (BMPR2), activin receptor-like kinase 1 (ALK1), endoglin (ENG), and mothers against decapentaplegic 9 (SMAD9). Approximately 25% of heritable cases lack identifiable mutations in any of these genes.
Methods and Results
We used whole exome sequencing to study a three generation family with multiple affected family members with PAH but no identifiable TGF beta mutation. We identified a frameshift mutation in Caveolin-1 (CAV1), which encodes a membrane protein of caveolae abundant in the endothelium and other cells of the lung. An independent de novo frameshift mutation was identified in a child with idiopathic PAH. Western blot analysis demonstrated a reduction in caveolin-1 protein, while lung tissue immunostaining studies demonstrated a reduction in normal caveolin-1 density within the endothelial cell layer of small arteries.
Our study represents successful elucidation of a dominant Mendelian disorder using whole exome sequencing. Mutations in CAV1 are associated in rare cases with PAH. This may have important implications for pulmonary vascular biology as well as PAH-directed therapeutic development.
PMCID: PMC3380156  PMID: 22474227
bioinfomatics genes; genetics; BMPR2; caveolae; pulmonary hypertension
20.  The Genetic Approach in Pulmonary Fibrosis 
Multiple investigators have undertaken genetic studies in idiopathic pulmonary fibrosis populations in attempts to define genetic links to disease in hopes that this would improve understanding of disease pathogenesis and target pathways for therapy. Multiple genes have been evaluated using a candidate gene approach with limited success, with results suggesting a disease modifier effect rather than a disease causing effect. Using this approach, associations have been observed between idiopathic pulmonary fibrosis and specific polymorphisms in genes encoding interleukin-1 receptor antagonist, tumor necrosis factor-α, and complement receptor 1. Recently investigators have used familial pulmonary fibrosis cohorts to evaluate for genetic mutations associated with idiopathic pulmonary fibrosis. Using one pulmonary fibrosis kindred, a mutation in the gene encoding surfactant protein C was identified as the cause of pulmonary fibrosis in this family. Subsequently, another individual with idiopathic pulmonary fibrosis was identified with a different mutation in surfactant protein C. Though rarely found in patients with idiopathic pulmonary fibrosis, these surfactant protein C mutations highlight the importance of the alveolar epithelium in disease pathogenesis. A recent collaboration between investigators at three major centers has resulted in the largest collection of families with pulmonary fibrosis to date, with hopes that this effort will identify genetic mutations associated with idiopathic pulmonary fibrosis. If genetic links to idiopathic pulmonary fibrosis are defined in this study, then the pathways involved with these genes and gene products can be targeted by investigators to help identify potential treatment options for this disease.
PMCID: PMC2658686  PMID: 16738199
familial pulmonary fibrosis; idiopathic pulmonary fibrosis; surfactant protein C
21.  A Common MUC5B Promoter Polymorphism and Pulmonary Fibrosis 
The New England Journal of Medicine  2011;364(16):1503-1512.
The mutations that have been implicated in pulmonary fibrosis account for only a small proportion of the population risk.
Using a genomewide linkage scan, we detected linkage between idiopathic interstitial pneumonia and a 3.4-Mb region of chromosome 11p15 in 82 families. We then evaluated genetic variation in this region in gel-forming mucin genes expressed in the lung among 83 subjects with familial interstitial pneumonia, 492 subjects with idiopathic pulmonary fibrosis, and 322 controls. MUC5B expression was assessed in lung tissue.
Linkage and fine mapping were used to identify a region of interest on the p-terminus of chromosome 11 that included gel-forming mucin genes. The minor-allele of the single-nucleotide polymorphism (SNP) rs35705950, located 3 kb upstream of the MUC5B transcription start site, was present at a frequency of 34% among subjects with familial interstitial pneumonia, 38% among subjects with idiopathic pulmonary fibrosis, and 9% among controls (allelic association with familial interstitial pneumonia, P = 1.2×10−15; allelic association with idiopathic pulmonary fibrosis, P = 2.5×10−37). The odds ratios for disease among subjects who were heterozygous and those who were homozygous for the minor allele of this SNP were 6.8 (95% confidence interval [CI], 3.9 to 12.0) and 20.8 (95% CI, 3.8 to 113.7), respectively, for familial interstitial pneumonia and 9.0 (95% CI, 6.2 to 13.1) and 21.8 (95% CI, 5.1 to 93.5), respectively, for idiopathic pulmonary fibrosis. MUC5B expression in the lung was 14.1 times as high in subjects who had idiopathic pulmonary fibrosis as in those who did not (P<0.001). The variant allele of rs35705950 was associated with up-regulation in MUC5B expression in the lung in unaffected subjects (expression was 37.4 times as high as in unaffected subjects homozygous for the wild-type allele, P<0.001). MUC5B protein was expressed in lesions of idiopathic pulmonary fibrosis.
A common polymorphism in the promoter of MUC5B is associated with familial interstitial pneumonia and idiopathic pulmonary fibrosis. Our findings suggest that dys-regulated MUC5B expression in the lung may be involved in the pathogenesis of pulmonary fibrosis. (Funded by the National Heart, Lung, and Blood Institute and others.)
PMCID: PMC3379886  PMID: 21506741
Idiopathic pulmonary fibrosis (IPF) is the most common form of the idiopathic interstitial pneumonias (IIP) and remains a disease with a poor prognosis. Familial interstitial pneumonia (FIP) occurs when two or more individuals from a given family have an IIP. FIP cases have been linked to mutations in surfactant protein C (SFTPC), surfactant protein A2 (SFTPA2), telomerase reverse transcriptase (TERT), and telomerase RNA component (TERC). Together, mutations in these four genes likely explain only 15-20% of FIP cases, and are even less frequent in sporadic IPF. However, dysfunctional aspects of the pathways that are involved with these genes are present in sporadic forms of IPF even in the absence of mutations, suggesting common underlying disease mechanisms. By serving as a resource for identifying the current and future genetic links to disease, FIP families hold great promise in defining IPF pathogenesis, potentially suggesting targets for the development of future therapies.
PMCID: PMC3103077  PMID: 21613931
familial interstitial pneumonia; idiopathic pulmonary fibrosis; lung; surfactant protein; telomerase
23.  Percutaneous Vascular Stent Implantation as Treatment for Central Vascular Obstruction due to Fibrosing Mediastinitis 
Circulation  2011;123(13):1391-1399.
Fibrosing Mediastinitis (FM) is a rare complication of infection with Histoplasma capsulatum, which can lead to obstruction of pulmonary and systemic vasculature and large airways, often resulting in significant morbidity and mortality. Medical therapy is ineffective and surgical intervention is often not feasible. Stent implantation offers a potential treatment for vascular obstruction due to FM, but this has not been well studied.
Methods and Results
We conducted a retrospective review of all patients undergoing cardiac catheterization for FM. Anatomic site of stenosis and hemodynamic information before and after intervention, as well as clinical presentation and follow-up data were recorded. From 1996 to 2008, 58 patients underwent cardiac catheterization for FM, with intervention performed in 40 (69%). A total of 77 stents were used to relieve 59 lesions (pulmonary artery = 26, pulmonary vein = 21, superior vena cava = 12). Significant reduction in pressure gradients (p<0.001) and increase in vessel caliber (p<0.001) was seen at all locations. Symptomatic recurrent stenosis requiring further intervention occurred in 11 (28%) patients. Median time to recurrence was 115 months. Thirty-two (87%) of 37 patients for whom follow-up was available reported symptomatic improvement following stent placement. Procedure-related complications occurred in 14 patients (24%). Overall mortality was 19%, with the majority of deaths in patients with bilateral disease. Among patients with bilateral disease, intervention was associated with improved survival at 5 years.
Percutaneous vascular stent implantation is an effective therapy for central vascular obstruction due to FM, providing significant relief of anatomic obstruction and sustained clinical improvement.
PMCID: PMC3095436  PMID: 21422386
stents; stenosis; pulmonary heart disease; *fibrosing mediastinitis; *pulmonary vascular obstruction
24.  BMPR2 expression is suppressed by signaling through the estrogen receptor 
Studies in multiple organ systems have shown cross-talk between signaling through the bone morphogenetic protein receptor type 2 (BMPR2) and estrogen pathways. In humans, pulmonary arterial hypertension (PAH) has a female predominance, and is associated with decreased BMPR2 expression. The goal of this study was to determine if estrogens suppress BMPR2 expression.
A variety of techniques were utilized across several model platforms to evaluate the relationship between estrogens and BMPR2 gene expression. We used quantitative RT-PCR, gel mobility shift, and luciferase activity assays in human samples, live mice, and cell culture.
BMPR2 expression is reduced in lymphocytes from female patients compared with male patients, and in whole lungs from female mice compared with male mice. There is an evolutionarily conserved estrogen receptor binding site in the BMPR2 promoter, which binds estrogen receptor by gel-shift assay. Increased exogenous estrogen decreases BMPR2 expression in cell culture, particularly when induced to proliferate. Transfection of increasing quantities of estrogen receptor alpha correlates strongly with decreasing expression of BMPR2.
BMPR2 gene expression is reduced in females compared to males in live humans and in mice, likely through direct estrogen receptor alpha binding to the BMPR2 promoter. This reduced BMPR2 expression may contribute to the increased prevalence of PAH in females.
PMCID: PMC3310853  PMID: 22348410
BMPR2; estrogen; hormones; expression; pulmonary hypertension
25.  The genetics of pulmonary arterial hypertension in the post-BMPR2 era 
Pulmonary Circulation  2011;1(3):305-319.
Pulmonary arterial hypertension (PAH) is a rapidly progressive and fatal disease for which there is an ever-expanding body of genetic and related pathophysiological information on disease pathogenesis. The most common single culprit gene known is BMPR2, and animal models of the disease in several forms exist. There is a wealth of genetic data regarding modifiers of disease expression, penetrance, and severity. Despite the rapid accumulation of data in the last decade, a complete picture of the molecular pathogenesis of PAH leading to novel therapies is lacking. In this review, we attempt to summarize the current understanding of PAH from the genetic perspective. The most recent PAH demographics are discussed. Heritable PAH in the post-BMPR2 era is examined in detail as the most robust model of PAH genetics in both animal models and human pedigrees. Important downstream molecular pathways and modifiers of disease expression are reviewed in light of what is known about PAH pathogenesis. Current and emerging therapies are examined in light of genetic data. The role of genetic testing in PAH in the post-BMPR2 era is discussed. Finally, directions for future investigations that ideally will fulfill the promise of novel therapeutic or preventive strategies are discussed.
PMCID: PMC3224422  PMID: 22140620
BMPR2; heritable pulmonary arterial hypertension; idiopathic pulmonary arterial hypertension; pulmonary arterial hypertension; right ventricle

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