Bone Marrow-derived Cells Contribute to the Pathogenesis of Pulmonary Arterial Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is a progressive lung disease of the pulmonary microvasculature. Studies suggest that bone marrow (BM)-derived circulating cells may play an important role in its pathogenesis. OBJECTIVES: We used a genetic model of PAH, the Bmpr2 mutant mouse, to study the role of BM-derived circulating cells in its pathogenesis. METHODS: Recipient mice, either Bmpr2(R899X) mutant or controls, were lethally irradiated and transplanted with either control or Bmpr2(R899X) BM cells. Donor cells were traced in female recipient mice by Y chromosome painting. Molecular and function insights were provided by expression and cytokine arrays combined with flow cytometry, colony-forming assays, and competitive transplant assays. MEASUREMENTS AND MAIN RESULTS: We found that mutant BM cells caused PAH with remodeling and inflammation when transplanted into control mice, whereas control BM cells had a protective effect against the development of disease, when transplanted into mutant mice. Donor BM-derived cells were present in the lungs of recipient mice. Functional and molecular analysis identified mutant BM cell dysfunction suggestive of a PAH phenotype soon after activation of the transgene and long before the development of lung pathology. CONCLUSIONS: Our data show that BM cells played a key role in PAH pathogenesis and that the transplanted BM cells were able to drive the lung phenotype in a myeloablative transplant model. Furthermore, the specific cell types involved were derived from hematopoietic stem cells and exhibit dysfunction long before the development of lung pathology.

Recommended joint and meta-analysis strategies for case-control association testing of single low-count variants.

In genome-wide association studies of binary traits, investigators typically use logistic regression to test common variants for disease association within studies, and combine association results across studies using meta-analysis. For common variants, logistic regression tests are well calibrated, and meta-analysis of study-specific association results is only slightly less powerful than joint analysis of the combined individual-level data. In recent sequencing and dense chip based association studies, investigators increasingly test low-frequency variants for disease association. In this paper, we seek to (1) identify the association test with maximal power among tests with well controlled type I error rate and (2) compare the relative power of joint and meta-analysis tests. We use analytic calculation and simulation to compare the empirical type I error rate and power of four logistic regression based tests: Wald, score, likelihood ratio, and Firth bias-corrected. We demonstrate for low-count variants (roughly minor allele count [MAC] < 400) that: (1) for joint analysis, the Firth test has the best combination of type I error and power; (2) for meta-analysis of balanced studies (equal numbers of cases and controls), the score test is best, but is less powerful than Firth test based joint analysis; and (3) for meta-analysis of sufficiently unbalanced studies, all four tests can be anti-conservative, particularly the score test. We also establish MAC as the key parameter determining test calibration for joint and meta-analysis.

Genomic and phenotypic correlates of mosaic loss of chromosome Y in blood.

Mosaic loss of Y (mLOY) is the most common somatic chromosomal alteration detected in human blood. The presence of mLOY is associated with altered blood cell counts and increased risk of Alzheimer's disease, solid tumors, and other age-related diseases. We sought to gain a better understanding of genetic drivers and associated phenotypes of mLOY through analyses of whole genome sequencing of a large set of genetically diverse males from the Trans-Omics for Precision Medicine (TOPMed) program. This approach enabled us to identify differences in mLOY frequencies across populations defined by genetic similarity, revealing a higher frequency of mLOY in the European American (EA) ancestry group compared to those of Hispanic American (HA), African American (AA), and East Asian (EAS) ancestry. Further, we identified two genes ( CFHR1 and LRP6 ) that harbor multiple rare, putatively deleterious variants associated with mLOY susceptibility, show that subsets of human hematopoietic stem cells are enriched for activity of mLOY susceptibility variants, and that certain alleles on chromosome Y are more likely to be lost than others.

Structural variation across 138,134 samples in the TOPMed consortium.

Ever larger Structural Variant (SV) catalogs highlighting the diversity within and between populations help researchers better understand the links between SVs and disease. The identification of SVs from DNA sequence data is non-trivial and requires a balance between comprehensiveness and precision. Here we present a catalog of 355,667 SVs (59.34% novel) across autosomes and the X chromosome (50bp+) from 138,134 individuals in the diverse TOPMed consortium. We describe our methodologies for SV inference resulting in high variant quality and >90% allele concordance compared to long-read de-novo assemblies of well-characterized control samples. We demonstrate utility through significant associations between SVs and important various cardio-metabolic and hemotologic traits. We have identified 690 SV hotspots and deserts and those that potentially impact the regulation of medically relevant genes. This catalog characterizes SVs across multiple populations and will serve as a valuable tool to understand the impact of SV on disease development and progression.

Structural variation across 138,134 samples in the TOPMed consortium.

Ever larger Structural Variant (SV) catalogs highlighting the diversity within and between populations help researchers better understand the links between SVs and disease. The identification of SVs from DNA sequence data is non-trivial and requires a balance between comprehensiveness and precision. Here we present a catalog of 355,667 SVs (59.34% novel) across autosomes and the X chromosome (50bp+) from 138,134 individuals in the diverse TOPMed consortium. We describe our methodologies for SV inference resulting in high variant quality and >90% allele concordance compared to long-read de-novo assemblies of well-characterized control samples. We demonstrate utility through significant associations between SVs and important various cardio-metabolic and hematologic traits. We have identified 690 SV hotspots and deserts and those that potentially impact the regulation of medically relevant genes. This catalog characterizes SVs across multiple populations and will serve as a valuable tool to understand the impact of SV on disease development and progression.

Cytoskeletal defects in Bmpr2-associated pulmonary arterial hypertension.

The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease.

Utilization of prehospital dispatch protocols to identify low-acuity patients.

OBJECTIVES: To describe the experience of a U.S. emergency medical services (EMS) agency utilizing a dispatch algorithm to identify low-acuity patients and determine whether secondary telephone triage by a nurse was associated with subsequent hospital admission among those patients. METHODS: This was a retrospective study of all patients meeting the low-acuity Omega classification by the Medical Priority Dispatch System (MPDS) in a large urban EMS system, conducted in two phases. Patients were excluded from the study if a refusal for transport was obtained, the call was received from a third-party caller, the MPDS system was not used, the patient was being referred from a skilled nursing facility, school, or university nursing office or physician's office, or if the call was referred to the Carolina Poison Center. Patients were enrolled over two phases using two different versions of the MPDS protocol, and in phase 2 patients were offered the option of speaking with an advice-line nurse. The outcome of interest was emergency department disposition, classified as hospital admission or discharge home. Admission to an intensive care unit (ICU) bed was also collected as a subcategory of hospital admission. RESULTS: Of the 1,862 patients in phase 1, 69.3% were discharged home from the emergency department, whereas in phase 2, 73.0% of the 1,078 patients were discharged home. Individuals were most frequently admitted to the hospital across both phases if they had a dispatch determinant of pregnancy, psychiatric/behavioral, fall, sick person. Hospital admission was also associated with receiving an EMS or emergency department procedure. There were 530 patients in phase 2 who underwent secondary triage by an advice-line nurse. Among this cohort of patients, 134 (25.3%) required subsequent hospital admission, with a further three (2.2%) requiring an ICU admission. CONCLUSIONS: This study identified a method for classifying patients during the dispatch period as low-acuity while attempting to ensure that those individuals received the medical care that they needed.

Upregulated heme biosynthesis increases obstructive sleep apnea severity: a pathway-based Mendelian randomization study.

Obstructive sleep apnea (OSA) is a common disorder associated with increased risk of cardiovascular disease and mortality. Iron and heme metabolism, implicated in ventilatory control and OSA comorbidities, was associated with OSA phenotypes in recent admixture mapping and gene enrichment analyses. However, its causal contribution was unclear. In this study, we performed pathway-level transcriptional Mendelian randomization (MR) analysis to investigate the causal relationships between iron and heme related pathways and OSA. In primary analysis, we examined the expression level of four iron/heme Reactome pathways as exposures and four OSA traits as outcomes using cross-tissue cis-eQTLs from the Genotype-Tissue Expression portal and published genome-wide summary statistics of OSA. We identify a significant putative causal association between up-regulated heme biosynthesis pathway with higher sleep time percentage of hypoxemia (p = 6.14 × 10-3). This association is supported by consistency of point estimates in one-sample MR in the Multi-Ethnic Study of Atherosclerosis using high coverage DNA and RNA sequencing data generated by the Trans-Omics for Precision Medicine project. Secondary analysis for 37 additional iron/heme Gene Ontology pathways did not reveal any significant causal associations. This study suggests a causal association between increased heme biosynthesis and OSA severity.

Protein prediction for trait mapping in diverse populations.

Genetically regulated gene expression has helped elucidate the biological mechanisms underlying complex traits. Improved high-throughput technology allows similar interrogation of the genetically regulated proteome for understanding complex trait mechanisms. Here, we used the Trans-omics for Precision Medicine (TOPMed) Multi-omics pilot study, which comprises data from Multi-Ethnic Study of Atherosclerosis (MESA), to optimize genetic predictors of the plasma proteome for genetically regulated proteome-wide association studies (PWAS) in diverse populations. We built predictive models for protein abundances using data collected in TOPMed MESA, for which we have measured 1,305 proteins by a SOMAscan assay. We compared predictive models built via elastic net regression to models integrating posterior inclusion probabilities estimated by fine-mapping SNPs prior to elastic net. In order to investigate the transferability of predictive models across ancestries, we built protein prediction models in all four of the TOPMed MESA populations, African American (n = 183), Chinese (n = 71), European (n = 416), and Hispanic/Latino (n = 301), as well as in all populations combined. As expected, fine-mapping produced more significant protein prediction models, especially in African ancestries populations, potentially increasing opportunity for discovery. When we tested our TOPMed MESA models in the independent European INTERVAL study, fine-mapping improved cross-ancestries prediction for some proteins. Using GWAS summary statistics from the Population Architecture using Genomics and Epidemiology (PAGE) study, which comprises ∼50,000 Hispanic/Latinos, African Americans, Asians, Native Hawaiians, and Native Americans, we applied S-PrediXcan to perform PWAS for 28 complex traits. The most protein-trait associations were discovered, colocalized, and replicated in large independent GWAS using proteome prediction model training populations with similar ancestries to PAGE. At current training population sample sizes, performance between baseline and fine-mapped protein prediction models in PWAS was similar, highlighting the utility of elastic net. Our predictive models in diverse populations are publicly available for use in proteome mapping methods at https://doi.org/10.5281/zenodo.4837327.

Association between prehospital time intervals and ST-elevation myocardial infarction system performance.

BACKGROUND: Among individuals experiencing an ST segment-elevation myocardial infarction, current guidelines recommend that the interval from first medical contact to percutaneous coronary intervention be ≤90 minutes. The objective of this study was to determine whether prehospital time intervals were associated with ST-elevation myocardial infarction system performance, defined as first medical contact to percutaneous coronary intervention. METHODS AND RESULTS: Study patients presented with an acute ST-elevation myocardial infarction diagnosed by prehospital ECG between May 2007 and March 2009. Prehospital time intervals were as follows: 9-1-1 call receipt to ambulance on scene ≤10 minutes, ambulance on scene to 12-lead ECG acquisition ≤8 minutes, on-scene time ≤15 minutes, prehospital ECG acquisition to ST-elevation myocardial infarction team notification ≤10 minutes, and scene departure to patient on cardiac catheterization laboratory table ≤30 minutes. Time intervals were derived and analyzed with descriptive statistics and logistic regression. There were 181 prehospital patients who received percutaneous coronary intervention, with 165 (91.1) having complete data. Logistic regression indicated that table time, response time, and on-scene time were the benchmark time intervals with the greatest influence on the probability of achieving percutaneous coronary intervention in ≤90 minutes. Individuals with a time from scene departure to arrival on cardiac catheterization laboratory table of ≤30 minutes were 11.1 times (3.4 to 36.0) more likely to achieve percutaneous coronary intervention in ≤90 minutes than those with extended table times. CONCLUSIONS: In this patient population, prehospital timing benchmarks were associated with system performance. Although meeting all 5 benchmarks may be an ideal goal, this model may be more useful for identifying areas for system improvement that will have the greatest clinical impact.

Comparison of first-attempt success between tibial and humeral intraosseous insertions during out-of-hospital cardiac arrest.

BACKGROUND: Intraosseous (IO) needle insertion is often utilized in the adult population for critical resuscitation purposes. Standard insertion sites include the proximal humerus and proximal tibia, for which limited comparison data are available. OBJECTIVE: This study compared the frequencies of IO first-attempt success between humeral and tibial sites in out-of-hospital cardiac arrest. METHODS: This observational study was conducted in an urban setting between August 28, 2009, and October 31, 2009, and included all medical cardiac arrest patients for whom resuscitative efforts were performed. Cardiac arrest protocols stipulate that paramedics insert an IO line for initial vascular access. During the first month of the study, the proximal humerus was the preferred primary insertion site, whereas the tibia was preferred throughout the second month. The primary outcome was first-attempt success, defined as secure IO needle position in the marrow cavity and normal fluid flow. Any needle dislodgment during resuscitation was also recorded. The association between first-attempt IO success and initial IO insertion location was analyzed using a test of independent proportions and 95% confidence intervals (CIs) for the difference in proportions. RESULTS: There were 88 cardiac arrest patients receiving IO placement, with 58 (65.9%) patients receiving their initial IO attempt in the tibia. The rate of first-time IO success at the tibia was significantly higher than that observed at the humerus (89.7% vs. 60.0%; p < 0.01). There were 18 initial successes at the humerus; for six (33.3%) of these, the needle became dislodged during resuscitation, compared with 52 initial successes at the tibia, with three (5.8%) dislodgments. The rate of total success for initial IO placements was significantly lower for the humerus (40.0%) compared with that for the tibia (84.5%; p < 0.01) during resuscitation efforts. CONCLUSIONS: In this subset of patients, tibial IO needle placement appeared to be a more effective insertion site than the proximal humerus. Success rates were higher with a lower incidence of needle dislodgments. Further randomized studies are required in order to validate these results.

The association between ambulance hospital turnaround times and patient acuity, destination hospital, and time of day.

INTRODUCTION: The availability of ambulances to respond to emergency calls is related to their ability to return to service from the hospital. Extended hospital turnaround times decrease the number of available unit hours ambulances are deployed, which in turn can increase coverage costs or sacrifice coverage. OBJECTIVE: To determine whether ambulance turnaround times were associated with patient acuity, destination hospital, and time of day. METHODS: This retrospective analysis of ambulance hospital turnaround times utilized 12 months of data from a single, countywide, metropolitan emergency medical services (EMS) service. Turnaround time was defined as the interval between the time of ambulance arrival at the hospital and the time the ambulance became available to respond to another call. Independent variables included patient acuity (low [BLS nonemergency transport], medium [ALS care and nonemergency transport], and high [ALS care and emergency transport]), destination hospital (seven regional hospitals), and time of day (one-hour intervals). Data analysis consisted of descriptive statistics, t-tests, and linear regression. RESULTS: Of the 61,094 patient transports, the mean turnaround time was 35.6 minutes (standard deviation [SD] = 16.5). Turnaround time was significantly associated with patient acuity (p < 0.001). High-acuity calls had a mean turnaround time of 52.5 minutes (SD = 21.5), whereas moderate-acuity and low-acuity calls had mean turnaround times of 42.0 minutes (SD = 16.4) and 32.5 minutes (SD = 14.4), respectively. A statistically significant relationship between destination hospital and turnaround time was found, with the differences in means ranging from 30 seconds to 8 minutes. Similarly, time of day was associated with turnaround time, with the longest turnaround times occurring between 0600 and 1500 hours. CONCLUSION: This study demonstrated that patient acuity, destination hospital, and time of day were associated with variation in ambulance turnaround times. Research describing other system characteristics such as current emergency department census and patient handoff procedures may further demonstrate areas for improvement in HTAT. Results from this analysis may be used to inspire EMS administrators and EMS medical directors to start tracking these times to create a predictive model of EMS staffing needs.

Quantifying changes in the thiol redox proteome upon oxidative stress in vivo.

Antimicrobial levels of reactive oxygen species (ROS) are produced by the mammalian host defense to kill invading bacteria and limit bacterial colonization. One main in vivo target of ROS is the thiol group of proteins. We have developed a quantitative thiol trapping technique termed OxICAT to identify physiologically important target proteins of hydrogen peroxide (H(2)O(2)) and hypochlorite (NaOCl) stress in vivo. OxICAT allows the precise quantification of oxidative thiol modifications in hundreds of different proteins in a single experiment. It also identifies the affected proteins and defines their redox-sensitive cysteine(s). Using this technique, we identified a group of Escherichia coli proteins with significantly (30-90%) oxidatively modified thiol groups, which appear to be specifically sensitive to either H(2)O(2) or NaOCl stress. These results indicate that individual oxidants target distinct proteins in vivo. Conditionally essential E. coli genes encode one-third of redox-sensitive proteins, a finding that might explain the bacteriostatic effect of oxidative stress treatment. We identified a select group of redox-regulated proteins, which protect E. coli against oxidative stress conditions. These experiments illustrate that OxICAT, which can be used in a variety of different cell types and organisms, is a powerful tool to identify, quantify, and monitor oxidative thiol modifications in vivo.

Clonal hematopoiesis associated with epigenetic aging and clinical outcomes.

Clonal hematopoiesis of indeterminate potential (CHIP) is a common precursor state for blood cancers that most frequently occurs due to mutations in the DNA-methylation modifying enzymes DNMT3A or TET2. We used DNA-methylation array and whole-genome sequencing data from four cohorts together comprising 5522 persons to study the association between CHIP, epigenetic clocks, and health outcomes. CHIP was strongly associated with epigenetic age acceleration, defined as the residual after regressing epigenetic clock age on chronological age, in several clocks, ranging from 1.31 years (GrimAge, p < 8.6 × 10-7 ) to 3.08 years (EEAA, p < 3.7 × 10-18 ). Mutations in most CHIP genes except DNA-damage response genes were associated with increases in several measures of age acceleration. CHIP carriers with mutations in multiple genes had the largest increases in age acceleration and decrease in estimated telomere length. Finally, we found that ~40% of CHIP carriers had acceleration >0 in both Hannum and GrimAge (referred to as AgeAccelHG+). This group was at high risk of all-cause mortality (hazard ratio 2.90, p < 4.1 × 10-8 ) and coronary heart disease (CHD) (hazard ratio 3.24, p < 9.3 × 10-6 ) compared to those who were CHIP-/AgeAccelHG-. In contrast, the other ~60% of CHIP carriers who were AgeAccelHG- were not at increased risk of these outcomes. In summary, CHIP is strongly linked to age acceleration in multiple clocks, and the combination of CHIP and epigenetic aging may be used to identify a population at high risk for adverse outcomes and who may be a target for clinical interventions.

Rare Coding Variants Associated With Electrocardiographic Intervals Identify Monogenic Arrhythmia Susceptibility Genes: A Multi-Ancestry Analysis.

BACKGROUND: Alterations in electrocardiographic (ECG) intervals are well-known markers for arrhythmia and sudden cardiac death (SCD) risk. While the genetics of arrhythmia syndromes have been studied, relations between electrocardiographic intervals and rare genetic variation at a population level are poorly understood. METHODS: Using a discovery sample of 29 000 individuals with whole-genome sequencing from Trans-Omics in Precision Medicine and replication in nearly 100 000 with whole-exome sequencing from the UK Biobank and MyCode, we examined associations between low-frequency and rare coding variants with 5 routinely measured electrocardiographic traits (RR, P-wave, PR, and QRS intervals and corrected QT interval). RESULTS: We found that rare variants associated with population-based electrocardiographic intervals identify established monogenic SCD genes (KCNQ1, KCNH2, and SCN5A), a controversial monogenic SCD gene (KCNE1), and novel genes (PAM and MFGE8) involved in cardiac conduction. Loss-of-function and pathogenic SCN5A variants, carried by 0.1% of individuals, were associated with a nearly 6-fold increased odds of the first-degree atrioventricular block (P=8.4×10-5). Similar variants in KCNQ1 and KCNH2 (0.2% of individuals) were associated with a 23-fold increased odds of marked corrected QT interval prolongation (P=4×10-25), a marker of SCD risk. Incomplete penetrance of such deleterious variation was common as over 70% of carriers had normal electrocardiographic intervals. CONCLUSIONS: Our findings indicate that large-scale high-depth sequence data and electrocardiographic analysis identifies monogenic arrhythmia susceptibility genes and rare variants with large effects. Known pathogenic variation in conventional arrhythmia and SCD genes exhibited incomplete penetrance and accounted for only a small fraction of marked electrocardiographic interval prolongation.

Omics-squared: human genomic, transcriptomic and phenotypic data for genetic analysis workshop 19.

BACKGROUND: The Genetic Analysis Workshops (GAW) are a forum for development, testing, and comparison of statistical genetic methods and software. Each contribution to the workshop includes an application to a specified data set. Here we describe the data distributed for GAW19, which focused on analysis of human genomic and transcriptomic data. METHODS: GAW19 data were donated by the T2D-GENES Consortium and the San Antonio Family Heart Study and included whole genome and exome sequences for odd-numbered autosomes, measures of gene expression, systolic and diastolic blood pressures, and related covariates in two Mexican American samples. These two samples were a collection of 20 large families with whole genome sequence and transcriptomic data and a set of 1943 unrelated individuals with exome sequence. For each sample, simulated phenotypes were constructed based on the real sequence data. 'Functional' genes and variants for the simulations were chosen based on observed correlations between gene expression and blood pressure. The simulations focused primarily on additive genetic models but also included a genotype-by-medication interaction. A total of 245 genes were designated as 'functional' in the simulations with a few genes of large effect and most genes explaining < 1 % of the trait variation. An additional phenotype, Q1, was simulated to be correlated among related individuals, based on theoretical or empirical kinship matrices, but was not associated with any sequence variants. Two hundred replicates of the phenotypes were simulated. The GAW19 data are an expansion of the data used at GAW18, which included the family-based whole genome sequence, blood pressure, and simulated phenotypes, but not the gene expression data or the set of 1943 unrelated individuals with exome sequence.

BMP pathway regulation of and by macrophages.

Pulmonary arterial hypertension (PAH) is a disease of progressively increasing pulmonary vascular resistance, associated with mutations of the type 2 receptor for the BMP pathway, BMPR2. The canonical signaling pathway for BMPR2 is through the SMAD family of transcription factors. BMPR2 is expressed in every cell type, but the impact of BMPR2 mutations affecting SMAD signaling, such as Bmpr2delx4+, had only previously been investigated in smooth muscle and endothelium. In the present study, we created a mouse with universal doxycycline-inducible expression of Bmpr2delx4+ in order to determine if broader expression had an impact relevant to the development of PAH. We found that the most obvious phenotype was a dramatic, but patchy, increase in pulmonary inflammation. We crossed these double transgenic mice onto an NF-κB reporter strain, and by luciferase assays on live mice, individual organs and isolated macrophages, we narrowed down the origin of the inflammatory phenotype to constitutive activation of tissue macrophages. Study of bone marrow-derived macrophages from mutant and wild-type mice suggested a baseline difference in differentiation state in Bmpr2 mutants. When activated with LPS, both mutant and wild-type macrophages secrete BMP pathway inhibitors sufficient to suppress BMP pathway activity in smooth muscle cells (SMC) treated with conditioned media. Functionally, co-culture with macrophages results in a BMP signaling-dependent increase in scratch closure in cultured SMC. We conclude that SMAD signaling through BMP is responsible, in part, for preventing macrophage activation in both live animals and in cells in culture, and that activated macrophages secrete BMP inhibitors in sufficient quantity to cause paracrine effect on vascular smooth muscle.

Data for Genetic Analysis Workshop 18: human whole genome sequence, blood pressure, and simulated phenotypes in extended pedigrees.

Genetic Analysis Workshop 18 (GAW18) focused on identification of genes and functional variants that influence complex phenotypes in human sequence data. Data for the workshop were donated by the T2D-GENES Consortium and included whole genome sequences for odd-numbered autosomes in 464 key individuals selected from 20 Mexican American families, a dense set of single-nucleotide polymorphisms in 959 individuals in these families, and longitudinal data on systolic and diastolic blood pressure measured at 1-4 examinations over a period of 20 years. Simulated phenotypes were generated based on the real sequence data and pedigree structures. In the design of the simulation model, gene expression measures from the San Antonio Family Heart Study (not distributed as part of the GAW18 data) were used to identify genes whose mRNA levels were correlated with blood pressure. Observed variants within these genes were designated as functional in the GAW18 simulation if they were nonsynonymous and predicted to have deleterious effects on protein function or if they were noncoding and associated with mRNA levels. Two simulated longitudinal phenotypes were modeled to have the same trait distributions as the real systolic and diastolic blood pressure data, with effects of age, sex, and medication use, including a genotype-medication interaction. For each phenotype, more than 1000 sequence variants in more than 200 genes present on the odd-numbered autosomes individually explained less than 0.01-2.78% of phenotypic variance. Cumulatively, variants in the most influential gene explained 7.79% of trait variance. An additional simulated phenotype, Q1, was designed to be correlated among family members but to not be associated with any sequence variants. Two hundred replicates of the phenotypes were simulated, with each including data for 849 individuals.

Interaction between bone morphogenetic protein receptor type 2 and estrogenic compounds in pulmonary arterial hypertension.

Abstract 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.

Metabolomic analysis of bone morphogenetic protein receptor type 2 mutations in human pulmonary endothelium reveals widespread metabolic reprogramming.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease of the lung vasculature for which the molecular etiologies are unclear. Specific metabolic alterations have been identified in animal models and in PAH patients, though existing data focus mainly on abnormalities of glucose homeostasis. We hypothesized that analysis of the entire metabolome in PAH would reveal multiple other metabolic changes relevant to disease pathogenesis and possible treatment. Layered transcriptomic and metabolomic analyses of human pulmonary microvascular endothelial cells (hPMVEC) expressing two different disease-causing mutations in the bone morphogenetic protein receptor type 2 (BMPR2) confirmed previously described increases in aerobic glycolysis but also uncovered significant upregulation of the pentose phosphate pathway, increases in nucleotide salvage and polyamine biosynthesis pathways, decreases in carnitine and fatty acid oxidation pathways, and major impairment of the tricarboxylic acid (TCA) cycle and failure of anaplerosis. As a proof of principle, we focused on the TCA cycle, predicting that isocitrate dehydrogenase (IDH) activity would be altered in PAH, and then demonstrating increased IDH activity not only in cultured hPMVEC expressing mutant BMPR2 but also in the serum of PAH patients. These results suggest that widespread metabolic changes are an important part of PAH pathogenesis, and that simultaneous identification and targeting of the multiple involved pathways may be a more fruitful therapeutic approach than targeting of any one individual pathway.