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.

BACKGROUND

The mutations that have been implicated in pulmonary fibrosis account for only a small proportion of the population risk.

METHODS

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.

RESULTS

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.

CONCLUSIONS

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.)

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.

Background

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.

Conclusions

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.

Background

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.

Methods

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.

Results

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.

Conclusions

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.

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.

The majority of pulmonary arterial hypertension (PAH) is not associated with BMPR2 mutation, and major risk factors for idiopathic PAH are not known. The objective of this study was to identify a gene expression signature for IPAH. To accomplish this, we used Affymetrix arrays to probe expression levels in 86 patient samples, including 22 healthy controls, 20 IPAH patients, 20 heritable PAH patients (HPAH), and 24 BMPR2 mutation carriers that were as yet unaffected (UMC). Culturing the patient cells removes the signatures of drug effects and inflammation which have made interpretation of results from freshly isolated lymphocytes problematic. We found that gene expression signatures from IPAH patients clustered either with HPAH patients or in a single distinct group. There were no groups of genes changed in IPAH that were not also changed in HPAH. HPAH, IPAH, and UMC had common changes in metabolism, actin dynamics, adhesion, cytokines, metabolism, channels, differentiation, and transcription factors. Common to IPAH and HPAH but not UMC were an upregulation of vesicle trafficking, oxidative/nitrosative stress, and cell cycle genes. The transcription factor MSX1, which is known to regulate BMP signaling, was the most upregulated gene (4×) in IPAH patients. These results suggest that IPAH cases have a shared molecular origin, which is closely related to, but distinct from, HPAH. HPAH and IPAH share the majority of altered signaling pathways, suggesting that treatments developed to target the molecular etiology of HPAH will also be effective against IPAH.

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.

The telomerase reverse transcriptase synthesizes new telomeres onto chromosome ends by copying from a short template within its integral RNA component. During telomere synthesis, telomerase adds multiple short DNA repeats successively, a property known as repeat addition processivity. However, the consequences of defects in processivity on telomere length maintenance are not fully known. Germline mutations in telomerase cause haploinsufficiency in syndromes of telomere shortening, which most commonly manifest in the age-related disease idiopathic pulmonary fibrosis. We identified two pulmonary fibrosis families that share two non-synonymous substitutions in the catalytic domain of the telomerase reverse transcriptase gene hTERT: V791I and V867M. The two variants fell on the same hTERT allele and were associated with telomere shortening. Genealogy suggested that the pedigrees shared a single ancestor from the nineteenth century, and genetic studies confirmed the two families had a common founder. Functional studies indicated that, although the double mutant did not dramatically affect first repeat addition, hTERT V791I-V867M showed severe defects in telomere repeat addition processivity in vitro. Our data identify an ancestral mutation in telomerase with a novel loss-of-function mechanism. They indicate that telomere repeat addition processivity is a critical determinant of telomere length and telomere-mediated disease.

Author Summary

Mutations in the essential telomerase components cause a spectrum of diseases mediated by short telomeres. Most frequently, these disorders manifest in the lung in an age-related disease: idiopathic pulmonary fibrosis. Telomerase synthesizes telomere repeats using a specialized reverse transcriptase, hTERT, that copies from a short template within its intrinsic RNA. In order to add long telomere tracts, telomerase adds a single repeat followed by additional repeats successively. This property, known as repeat addition processivity, is unique to the telomerase polymerase. We identified two families that shared two unique variants in the catalytic domain of hTERT: V791I and V867M. The variants co-segregated, indicating they are on the same allele, and were associated with short telomeres. Family history suggested the two families may have a single ancestor, and genetic studies confirmed they had a common founder. Telomerase reconstitution indicated that, although the double mutant did not significantly affect telomerase's ability to add a single telomere repeat, hTERT 791I-867M had severe defects in repeat addition processivity. Our data identify an ancestral mutation in telomerase; this mutation possesses a unique loss-of-function mechanism. Defects in telomere addition processivity are important determinants of telomere length maintenance and of telomere-associated disease.

Dyskeratosis congenita (DC) is a premature ageing syndrome characterised by short telomeres. An X-linked form of DC is caused by mutations in DKC1 which encodes dyskerin, a telomerase component that is essential for telomerase RNA stability. However, mutations in DKC1 are identifiable in only half of X-linked DC families. A four generation family with pulmonary fibrosis and features of DC was identified. Affected males showed the classic mucocutaneous features of DC and died prematurely from pulmonary fibrosis. Although there were no coding sequence or splicing variants, genome wide linkage analysis of 16 individuals across four generations identified significant linkage at the DKC1 locus, and was accompanied by reduced dyskerin protein levels in affected males. Decreased dyskerin levels were associated with compromised telomerase RNA levels and very short telomeres. These data identify decreased dyskerin levels as a novel mechanism of DC, and indicate that intact dyskerin levels, in the absence of coding mutations, are critical for telomerase RNA stability and for in vivo telomere maintenance.

Backgrounds

We tested the hypothesis that right ventricular (RV) pressure overload affects RV function, and further influences left ventricular (LV) geometry that adversely affects LV twist mechanics and segmental function.

Methods and Results

Echocardiographic images were prospectively acquired in 44 (46±12 years; 82%F) patients with evidence of pulmonary hypertension (PH) (estimated pulmonary systolic pressure [PASP] =71±23 mmHg) and in 44 age and gender-matched healthy subjects. Patients with intrinsic LV diseases were excluded. RV lateral wall (RVLAT) longitudinal strain (LS) and interventricular septal (IVS) LS were reduced in PH group compared with controls (-15.9±7.6% vs.-25.5±6.1%, p<0.001 and -17.3±4.4% vs.-20.2±3.9%, p=0.002, respectively), while LV lateral wall (LVLAT) LS was preserved. RVLAT and IVS LS, but not LVLAT LS, correlated with PASP(r=0.56, p<0.01; r=0.32, p<0.01) and LV eccentricity index (LVEI) (r=0.57, p<0.01; r=0.57, p<0.01). IVS and LVLAT circumferential strains (CS) were both reduced in the PH group. Although IVS CS and LVLAT CS correlated with PASP and LVEI, after adjusting CS for LVEI, differences between groups persisted for IVS CS (p<0.01) but not LVLAT CS (p=0.09). LV torsion was decreased in patients with PH compared with controls (9.6±4.9° vs. 14.7±4.9°, p<0.001). LV torsion inversely correlated with PASP (r=-0.39, p<0.01) and LVEI (r=-0.3, p<0.01). LV untwisting rates were similar in both groups (p=0.7).

Conclusions

Chronic RV pressure overload directly affects RV longitudinal systolic deformation. RV pressure overload further influences IVS and LV geometry, which impairs LV torsion and segmental LS and CS, more for the IVS than the free wall of the LV.

Familial pulmonary arterial hypertension (FPAH) is a progressive, fatal disease caused by mutations in the bone morphogenetic protein receptor type 2 gene (BMPR2). FPAH is inherited as an autosomal dominant trait and shows incomplete penetrance in that many with BMPR2 mutations do not develop FPAH suggesting a role for, as yet unidentified, modifier genes in disease penetrance. We hypothesized that variable level of expression of the wild type (WT) BMPR2 allele could act as a modifier and influence penetrance of FPAH. WT BMPR2 levels were determined by real-time PCR analysis in lymphoblastoid (LB) cell lines derived from normal controls and individuals with FPAH. The FPAH kindreds analyzed carried mutations that result in the activation of nonsense mediated decay (NMD) pathway, which leads to the degradation of the mutant RNA thus ensuring that only the WT BMPR2 transcripts will be detected in the real-time assay. Our data show that WT and mutant BMPR2 levels can be reproducibly measured in patient derived LB cell lines and that unaffected mutation carrier derived LB cell lines have higher levels of WT BMPR2 transcripts than FPAH patient derived LB cell lines (p≤0.005). Our findings suggest that the levels of expression of WT BMPR2 allele transcripts is important in the pathogenesis of FPAH caused by NMD+ mutations. Furthermore, our study illustrates a novel application of lymphoblastoid cell lines in the study of PAH, especially important because the affected site, i.e. lung is not available for unaffected mutation carriers.

Background

Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) plays an important role in the development of idiopathic pulmonary arterial hypertension (IPAH), whereas a rise in cytosolic Ca2+ concentration triggers PASMC contraction and stimulates PASMC proliferation. Recently, we demonstrated that upregulation of the TRPC6 channel contributes to proliferation of PASMCs isolated from IPAH patients. This study sought to identify single-nucleotide polymorphisms (SNPs) in the TRPC6 gene promoter that are associated with IPAH and have functional significance in regulating TRPC6 activity in PASMCs.

Methods and Results

Genomic DNA was isolated from blood samples of 237 normal subjects and 268 IPAH patients. Three biallelic SNPs, −361 (A/T), −254(C/G), and −218 (C/T), were identified in the 2000-bp sequence upstream of the transcriptional start site of TRPC6. Although the allele frequencies of the −361 and −218 SNPs were not different between the groups, the allele frequency of the −254(C→G) SNP in IPAH patients (12%) was significantly higher than in normal subjects (6%; P<0.01). Genotype data showed that the percentage of −254G/G homozygotes in IPAH patients was 2.85 times that of normal subjects. Moreover, the −254(C→G) SNP creates a binding sequence for nuclear factor-κB. Functional analyses revealed that the −254(C→G) SNP enhanced nuclear factor-κB–mediated promoter activity and stimulated TRPC6 expression in PASMCs. Inhibition of nuclear factor-κB activity attenuated TRPC6 expression and decreased agonist-activated Ca2+ influx in PASMCs of IPAH patients harboring the −254G allele.

Conclusions

These results suggest that the −254(C→G) SNP may predispose individuals to an increased risk of IPAH by linking abnormal TRPC6 transcription to nuclear factor-κB, an inflammatory transcription factor.

Background

Copy-number variations (CNVs) are structural variations in the genome involving 1 kb to 3 mb of DNA. CNV has been reported within intron 1 of the BMPR2 gene. We propose that CNV could affect phenotype in familial and/or sporadic pulmonary arterial hypertension (PAH) by altering gene expression.

Methods

97 human DNA samples were obtained which included 24 patients with familial PAH, 18 obligate carriers (BMPR2 mutation positive), 20 sporadic PAH patients, and 35 controls. Two sets of primers were designed within the CNV, and two sets of control primers were designed outside the CNV. Quantitative PCR was performed to quantify genomic copies of CNV and control sequences.

Results

A CNV in BMPR2 was present in one African American negative control subject.

Conclusion

We conclude that the CNV in intron 1 in BMPR2 is unlikely to play a role in the pathogenesis of either familial or sporadic PAH.

Trial Registration

NIH NCT00091546.

Rationale: Not all family members with BMPR2 mutations develop pulmonary arterial hypertension (PAH), implying that additional modifier genes or proteins are necessary for full expression of the disease.

Objectives: To determine whether protein expression is altered in patients with familial PAH (FPAH) compared with obligate carriers and nondiseased control subjects.

Methods: Protein extracts from transformed blood lymphocytes from four patients with FPAH, three obligate carriers, and three married-in control subjects from one family with a known BMPR2 mutation (exon 3 T354G) were labeled with either Cy3 or Cy5. Cy3/5 pairs were separated by standard two-dimensional differential gel electrophoresis using a Cy2-labeled internal standard of all patient samples. Log volume ratios were analyzed using a linear mixed-effects model. Proteins were identified by matrix-assisted laser desorption ionization, time-of-flight mass spectrometry (MALDI-TOF MS) and tandem TOF/TOF MS/MS.

Measurements and Main Results: Hierarchical clustering, heat-map, and principal components analysis revealed marked changes in protein expression in patients with FPAH when compared with obligate carriers. Significant changes were apparent in expression of 16 proteins (P < 0.05) when affected patients were compared with obligates: nine showed a significant increase and seven showed a significant reduction.

Conclusions: A series of novel proteins with altered expression were found that could distinguish affected patients from obligate carriers and married-in controls in a single family with a BMPR2 mutation. These differences provide new information highlighting proteins that may be involved in the mechanism(s) that differentiates those individuals with a BMPR2 mutation who develop FPAH from those who do not.

SUMMARY

Little is known about practice patterns regarding the diagnosis and management of idiopathic pulmonary fibrosis (IPF). This study attempts to define the practice patterns of academic pulmonologists caring for patients with idiopathic pulmonary fibrosis. Academic pulmonologists in the United States were surveyed electronically. Completed surveys were received from 272 respondents (representing approximately 10% of academic pulmonologists). The majority agreed that high-resolution computed tomography scanning can establish the diagnosis of idiopathic pulmonary fibrosis, and that surgical lung biopsy is indicated when the diagnosis remains unclear. Bronchoscopy is little utilized. Most respondents treat patients with medications, but there is no consensus regarding treatment regimen. These results suggest there is general consensus regarding the approach to diagnosis, but that there is no consensus about medical management in IPF.

The natural history of idiopathic pulmonary fibrosis (IPF) has been characterized as a steady, predictable decline in lung function over time. Recent evidence suggests that some patients may experience a more precipitous course, with periods of relative stability followed by acute deteriorations in respiratory status. Many of these acute deteriorations are of unknown etiology and have been termed acute exacerbations of IPF. This perspective is the result of an international effort to summarize the current state of knowledge regarding acute exacerbations of IPF. Acute exacerbations of IPF are defined as acute, clinically significant deteriorations of unidentifiable cause in patients with underlying IPF. Proposed diagnostic criteria include subjective worsening over 30 days or less, new bilateral radiographic opacities, and the absence of infection or another identifiable etiology. The potential pathobiological roles of infection, disordered cell biology, coagulation, and genetics are discussed, and future research directions are proposed.

Rationale: Previous studies have shown that approximately 55% of patients with familial pulmonary arterial hypertension (FPAH) have BMPR2 coding sequence mutations. However, direct sequencing does not detect other types of heterozygous mutations, such as exonic deletions/duplications.

Objective: To estimate the frequency of BMPR2 exonic deletions/duplications in FPAH.

Methods: BMPR2 mRNA from lymphoblastoid cell lines of 30 families with PAH and 14 patients with idiopathic PAH (IPAH) was subjected to reverse transcriptase–polymerase chain reaction (RT-PCR) and sequencing. Sequencing of genomic DNA was used to identify point mutations in splice donor/acceptor sites. Multiplex ligation-dependent probe amplification (MLPA) was used to detect exonic deletions/duplications with verification by real-time PCR.

Measurements and Main Results: Eleven (37%) patients with FPAH had abnormally sized RT-PCR products. Four of the 11 patients were found to have splice-site mutations resulting in aberrant splicing, and exonic deletions/duplications were detected in the remaining seven patients. MLPA identified three deletions/duplications that were not detectable by RT-PCR. Coding sequence point mutations were identified in 11 of 30 (37%) patients. Mutations were identified in 21 of 30 (70%) patients with FPAH, with 10 of 21 mutations (48%) being exonic deletions/duplications. Two of 14 (14%) patients with IPAH exhibited BMPR2 point mutations, whereas none showed exonic deletions/duplications.

Conclusions: Our study indicates that BMPR2 exonic deletions/duplications in patients with FPAH account for a significant proportion of mutations (48%) that until now have not been screened for. Because the complementary approach used in this study is rapid and cost effective, screening for BMPR2 deletions/duplications by MLPA and real-time PCR should accompany direct sequencing in all PAH testing.

Rationale: Serotonin is a pulmonary vasoconstrictor and smooth muscle cell mitogen. The serotonin transporter (SERT) is abundant in pulmonary vascular smooth muscle. Compared with the short (S) allele, the long (L) SERT promoter allele is associated with increased SERT transcription and more severe pulmonary hypertension in a cohort of patients with chronic obstructive pulmonary disease, and was more prevalent in a cohort with idiopathic pulmonary arterial hypertension (IPAH), compared with control subjects.

Objective: We hypothesized that the SERT L allele would associate with an earlier age at diagnosis and/or shorter survival interval in pulmonary arterial hypertension (PAH) than the S allele.

Methods: SERT promoters from 166 familial PAH (FPAH), 83 IPAH, and 125 control subjects were sequenced. One hundred twenty-seven of the patients with FPAH had a known mutation in bone morphogenetic protein receptor 2 (BMPR2).

Results: The mean age at diagnosis was 35.8 yr in patients with FPAH and 41.1 yr in patients with IPAH (p = 0.02). There were no significant differences in distribution of the LL, LS, or SS genotypes in IPAH, FPAH, or unaffected BMPR2 mutation carriers. In FPAH, the LL genotype was associated with an earlier age at diagnosis (p < 0.02).

Conclusions: In patients with IPAH, these SERT genotypes do not correlate with age at diagnosis or survival interval. In patients with FPAH, the LL genotype correlates with an earlier age at diagnosis than SL or SS, although survival among the groups was similar. The correlation of the SERT promoter polymorphism with age at diagnosis in FPAH suggests a possible relationship between the SERT and BMPR2.

Rationale: Several lines of evidence suggest that genetic factors and environmental exposures play a role in the development of pulmonary fibrosis.

Objectives: We evaluated families with 2 or more cases of idiopathic interstitial pneumonia among first-degree family members (familial interstitial pneumonia, or FIP), and identified 111 families with FIP having 309 affected and 360 unaffected individuals.

Methods: The presence of probable or definite FIP was based on medical record review in 28 cases (9.1%); clinical history, diffusing capacity of carbon monoxide (DLCO), and chest X-ray in 16 cases (5.2%); clinical history, DLCO, and high-resolution computed tomography chest scan in 191 cases (61.8%); clinical history and surgical lung biopsy in 56 cases (18.1%); and clinical history and autopsy in 18 cases (5.8%).

Results: Older age (68.3 vs. 53.1; p < 0.0001), male sex (55.7 vs. 37.2%; p < 0.0001), and having ever smoked cigarettes (67.3 vs. 34.1%; p < 0.0001) were associated with the development of FIP. After controlling for age and sex, having ever smoked cigarettes remained strongly associated with the development of FIP (odds ratioadj, 3.6; 95% confidence interval, 1.3–9.8). Evidence of aggregation of disease was highly significant (p < 0.001) among sibling pairs, and 20 pedigrees demonstrated vertical transmission, consistent with autosomal dominant inheritance. Forty-five percent of pedigrees demonstrated phenotypic heterogeneity, with some pedigrees demonstrating several subtypes of idiopathic interstitial pneumonia occurring within the same families.

Conclusions: These findings suggest that FIP may be caused by an interaction between a specific environmental exposure and a gene (or genes) that predisposes to the development of several subtypes of idiopathic interstitial pneumonia.

On the basis of earlier reports associating Epstein-Barr Virus (EBV) with half of the cases of idiopathic pulmonary fibrosis (IPF), we hypothesized that chronic infection with EBV or a closely related herpesvirus would be detected in all cases of IPF. We tested lung specimens from 33 IPF patients (8 patients with familial IPF and 25 patients with sporadic IPF) and 25 patients with other diseases as controls for the presence of eight herpesviruses using PCR-based techniques. One or more of four herpesviruses (cytomegalovirus [CMV], EBV, human herpesvirus 7 [HHV-7], and HHV-8) were detected in 32 of 33 (97%) subjects with IPF and in 9 of 25 (36%) controls (P < 0.0001). CMV, EBV, and HHV-8 were found more frequently in IPF patients than in controls (P < 0.05, P < 0.001, and P < 0.01 respectively). Two or more herpesviruses were detected in 19 of 33 (57%) IPF patients and in 2 of 25 (8%) controls (P < 0.001). Two or more herpesviruses and HHV-8 were found more frequently in patients with sporadic IPF than in patients with familial IPF (P < 0.05 for both comparisons), and CMV was found less frequently in patients with sporadic IPF than in patients with familial IPF (P < 0.05). Immunohistochemistry for EBV or HHV-8 antigen showed viral antigen primarily in airway epithelial cells. These data support the concept that a herpesvirus could be a source of chronic antigenic stimulation in IPF.

Background

Prostanoids are known to participate in the process of fibrogenesis. Because lung fibroblasts produce prostanoids and are believed to play a central role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), we hypothesized that fibroblasts (HF) cultured from the lungs of patients with IPF (HF-IPF) have an altered balance between profibrotic (thromboxane [TX]A2) and antifibrotic (prostacyclin [PGI2]) prostaglandins (PGs) when compared with normal human lung fibroblasts (HF-NL).

Methods

We measured inducible cyclooxygenase (COX)-2 gene and protein expression, and a profile of prostanoids at baseline and after IL-1β stimulation.

Results

In both HF-IPF and HF-NL COX-2 expression was undetectable at baseline, but was significantly upregulated by IL-1β. PGE2 was the predominant COX product in IL-1β-stimulated cells with no significant difference between HF-IPF and HF-NL (28.35 [9.09–89.09] vs. 17.12 [8.58–29.33] ng/106 cells/30 min, respectively; P = 0.25). TXB2 (the stable metabolite of TXA2) production was significantly higher in IL-1β-stimulated HF-IPF compared to HF-NL (1.92 [1.27–2.57] vs. 0.61 [0.21–1.64] ng/106 cells/30 min, respectively; P = 0.007) and the ratio of PGI2 (as measured by its stable metabolite 6-keto-PGF1α) to TXB2 was significantly lower at baseline in HF-IPF (0.08 [0.04–0.52] vs. 0.12 [0.11–0.89] in HF-NL; P = 0.028) and with IL-1β stimulation (0.24 [0.05–1.53] vs. 1.08 [0.51–3.79] in HF-NL; P = 0.09).

Conclusion

An alteration in the balance of profibrotic and antifibrotic PGs in HF-IPF may play a role in the pathogeneses of IPF.

Background

Pulmonary arterial hypertension (PAH) is thought to be driven by dysfunction of pulmonary vascular microendothelial cells (PMVEC). Most hereditary PAH is associated with BMPR2 mutations. However, the physiologic and molecular consequences of expression of BMPR2 mutations in PMVEC are unknown.

Methods

In vivo experiments were performed on adult mice with conditional endothelial-specific expression of the truncation mutation Bmpr2delx4+, with age-matched transactivator-only mice as controls. Phenotype was assessed by RVSP, counts of muscularized vessels and proliferating cells, and staining for thromboses, inflammatory cells, and apoptotic cells. The effects of BMPR2 knockdown in PMVEC by siRNA on rates of apoptosis were assessed. Affymetrix expression arrays were performed on PMVEC isolated and cultured from triple transgenic mice carrying the immortomouse gene, a transactivator, and either control, Bmpr2delx4+ or Bmpr2R899X mutation.

Results

Transgenic mice showed increased RVSP and corresponding muscularization of small vessels, with histologic alterations including thrombosis, increased inflammatory cells, increased proliferating cells, and a moderate increase in apoptotic cells. Expression arrays showed alterations in specific pathways consistent with the histologic changes. Bmpr2delx4+ and Bmpr2R899X mutations resulted in very similar alterations in proliferation, apoptosis, metabolism, and adhesion; Bmpr2delx4+ cells showed upregulation of platelet adhesion genes and cytokines not seen in Bmpr2R899X PMVEC. Bmpr2 mutation in PMVEC does not cause a loss of differentiation markers as was seen with Bmpr2 mutation in smooth muscle cells.

Conclusions

Bmpr2 mutation in PMVEC in vivo may drive PAH through multiple, potentially independent, downstream mechanisms, including proliferation, apoptosis, inflammation, and thrombosis.

Background

Autosomal 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.

Methods

Testing 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.

Results

While 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.

Conclusion

In 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.