Whole-genome sequencing of patients with rare diseases in a national health system.

Most patients with rare diseases do not receive a molecular diagnosis and the aetiological variants and causative genes for more than half such disorders remain to be discovered1. Here we used whole-genome sequencing (WGS) in a national health system to streamline diagnosis and to discover unknown aetiological variants in the coding and non-coding regions of the genome. We generated WGS data for 13,037 participants, of whom 9,802 had a rare disease, and provided a genetic diagnosis to 1,138 of the 7,065 extensively phenotyped participants. We identified 95 Mendelian associations between genes and rare diseases, of which 11 have been discovered since 2015 and at least 79 are confirmed to be aetiological. By generating WGS data of UK Biobank participants2, we found that rare alleles can explain the presence of some individuals in the tails of a quantitative trait for red blood cells. Finally, we identified four novel non-coding variants that cause disease through the disruption of transcription of ARPC1B, GATA1, LRBA and MPL. Our study demonstrates a synergy by using WGS for diagnosis and aetiological discovery in routine healthcare.

Shared and Distinct Genomics of Chronic Thromboembolic Pulmonary Hypertension and Pulmonary Embolism.

Rationale: Chronic thromboembolic pulmonary hypertension involves the formation and nonresolution of thrombus, dysregulated inflammation, angiogenesis, and the development of a small-vessel vasculopathy. Objectives: We aimed to establish the genetic basis of chronic thromboembolic pulmonary hypertension to gain insight into its pathophysiological contributors. Methods: We conducted a genome-wide association study on 1,907 European cases and 10,363 European control subjects. We coanalyzed our results with existing results from genome-wide association studies on deep vein thrombosis, pulmonary embolism, and idiopathic pulmonary arterial hypertension. Measurements and Main Results: Our primary association study revealed genetic associations at the ABO, FGG, F11, MYH7B, and HLA-DRA loci. Through our coanalysis, we demonstrate further associations with chronic thromboembolic pulmonary hypertension at the F2, TSPAN15, SLC44A2, and F5 loci but find no statistically significant associations shared with idiopathic pulmonary arterial hypertension. Conclusions: Chronic thromboembolic pulmonary hypertension is a partially heritable polygenic disease, with related though distinct genetic associations with pulmonary embolism and deep vein thrombosis.

SOX17 Enhancer Variants Disrupt Transcription Factor Binding And Enhancer Inactivity Drives Pulmonary Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare disease characterized by remodeling of the pulmonary arteries, increased vascular resistance, and right-sided heart failure. Genome-wide association studies of idiopathic/heritable PAH established novel genetic risk variants, including conserved enhancers upstream of transcription factor (TF) SOX17 containing 2 independent signals. SOX17 is an important TF in embryonic development and in the homeostasis of pulmonary artery endothelial cells (hPAEC) in the adult. Rare pathogenic mutations in SOX17 cause heritable PAH. We hypothesized that PAH risk alleles in an enhancer region impair TF-binding upstream of SOX17, which in turn reduces SOX17 expression and contributes to disturbed endothelial cell function and PAH development. METHODS: CRISPR manipulation and siRNA were used to modulate SOX17 expression. Electromobility shift assays were used to confirm in silico-predicted TF differential binding to the SOX17 variants. Functional assays in hPAECs were used to establish the biological consequences of SOX17 loss. In silico analysis with the connectivity map was used to predict compounds that rescue disturbed SOX17 signaling. Mice with deletion of the SOX17-signal 1 enhancer region (SOX17-4593/enhKO) were phenotyped in response to chronic hypoxia and SU5416/hypoxia. RESULTS: CRISPR inhibition of SOX17-signal 2 and deletion of SOX17-signal 1 specifically decreased SOX17 expression. Electromobility shift assays demonstrated differential binding of hPAEC nuclear proteins to the risk and nonrisk alleles from both SOX17 signals. Candidate TFs HOXA5 and ROR-α were identified through in silico analysis and antibody electromobility shift assays. Analysis of the hPAEC transcriptomes revealed alteration of PAH-relevant pathways on SOX17 silencing, including extracellular matrix regulation. SOX17 silencing in hPAECs resulted in increased apoptosis, proliferation, and disturbance of barrier function. With the use of the connectivity map, compounds were identified that reversed the SOX17-dysfunction transcriptomic signatures in hPAECs. SOX17 enhancer knockout in mice reduced lung SOX17 expression, resulting in more severe pulmonary vascular leak and hypoxia or SU5416/hypoxia-induced pulmonary hypertension. CONCLUSIONS: Common PAH risk variants upstream of the SOX17 promoter reduce endothelial SOX17 expression, at least in part, through differential binding of HOXA5 and ROR-α. Reduced SOX17 expression results in disturbed hPAEC function and PAH. Existing drug compounds can reverse the disturbed SOX17 pulmonary endothelial transcriptomic signature.

Serum and Pulmonary Expression Profiles of the Activin Signaling System in Pulmonary Arterial Hypertension.

BACKGROUND: Activins are novel therapeutic targets in pulmonary arterial hypertension (PAH). We therefore studied whether key members of the activin pathway could be used as PAH biomarkers. METHODS: Serum levels of activin A, activin B, α-subunit of inhibin A and B proteins, and the antagonists follistatin and follistatin-like 3 (FSTL3) were measured in controls and in patients with newly diagnosed idiopathic, heritable, or anorexigen-associated PAH (n=80) at baseline and 3 to 4 months after treatment initiation. The primary outcome was death or lung transplantation. Expression patterns of the inhibin subunits, follistatin, FSTL3, Bambi, Cripto, and the activin receptors type I (ALK), type II (ACTRII), and betaglycan were analyzed in PAH and control lung tissues. RESULTS: Death or lung transplantation occurred in 26 of 80 patients (32.5%) over a median follow-up of 69 (interquartile range, 50-81) months. Both baseline (hazard ratio, 1.001 [95% CI, 1.000-1.001]; P=0.037 and 1.263 [95% CI, 1.049-1.520]; P=0.014, respectively) and follow-up (hazard ratio, 1.003 [95% CI, 1.001-1.005]; P=0.001 and 1.365 [95% CI, 1.185-1.573]; P<0.001, respectively) serum levels of activin A and FSTL3 were associated with transplant-free survival in a model adjusted for age and sex. Thresholds determined by receiver operating characteristic analyses were 393 pg/mL for activin A and 16.6 ng/mL for FSTL3. When adjusted with New York Heart Association functional class, 6-minute walk distance, and N-terminal pro-B-type natriuretic peptide, the hazard ratios for transplant-free survival for baseline activin A <393 pg/mL and FSTL3 <16.6 ng/mL were, respectively, 0.14 (95% CI, 0.03-0.61; P=0.009) and 0.17 (95% CI, 0.06-0.45; P<0.001), and for follow-up measures, 0.23 (95% CI, 0.07-0.78; P=0.019) and 0.27 (95% CI, 0.09-0.78, P=0.015), respectively. Prognostic values of activin A and FSTL3 were confirmed in an independent external validation cohort. Histological analyses showed a nuclear accumulation of the phosphorylated form of Smad2/3, higher immunoreactivities for ACTRIIB, ALK2, ALK4, ALK5, ALK7, Cripto, and FSTL3 in vascular endothelial and smooth muscle layers, and lower immunostaining for inhibin-α and follistatin. CONCLUSIONS: These findings offer new insights into the activin signaling system in PAH and show that activin A and FSTL3 are prognostic biomarkers for PAH.

Restoration of Foxp3+ Regulatory T Cells by HDAC-Dependent Epigenetic Modulation Plays a Pivotal Role in Resolving Pulmonary Arterial Hypertension Pathology.

Rationale: Immune dysregulation is a common feature of pulmonary arterial hypertension (PAH). Histone deacetylase (HDAC)-dependent transcriptional reprogramming epigenetically modulates immune homeostasis and is a novel disease-oriented approach in modern times. Objectives: To identify a novel functional link between HDAC and regulatory T cells (Tregs) in PAH, aiming to establish disease-modified biomarkers and therapeutic targets. Methods: Peripheral blood mononuclear cells were isolated from patients with idiopathic PAH (IPAH) and rodent models of pulmonary hypertension (PH): monocrotaline rats, Sugen5416-hypoxia rats, and Treg-depleted mice. HDAC inhibitor vorinostat (suberoylanilide hydroxamic acid, SAHA) was used to examine the immune modulatory effects in vivo, ex vivo, and in vitro. Measurements and Main Results: Increased HDAC expression was associated with reduced Foxp3+ Tregs and increased PD-1 (programmed cell death-1) signaling in peripheral blood mononuclear cells from patients with IPAH. SAHA differentially modified a cluster of epigenetic-sensitive genes and induced Foxp3+ Treg conversion in IPAH T cells. Rodent models recapitulated these epigenetic aberrations and T-cell dysfunction. SAHA attenuated PH phenotypes and restored FOXP3 transcription and Tregs in PH rats; interestingly, the effects were more profound in female rats. Selective depletion of CD25+ Tregs in Sugen5416-hypoxia mice neutralized the effects of SAHA. Furthermore, SAHA inhibited endothelial cytokine/chemokine release upon stimulation and subsequent immune chemotaxis. Conclusions: Our results indicated HDAC aberration was associated with Foxp3+ Treg deficiency and demonstrated an epigenetic-mediated mechanism underlying immune dysfunction in PAH. Restoration of Foxp3+ Tregs by HDAC inhibitors is a promising approach to resolve pulmonary vascular pathology, highlighting the potential benefit of developing epigenetic therapies for PAH.

Intracellular Chloride Channels Regulate Endothelial Metabolic Reprogramming in Pulmonary Arterial Hypertension.

Mitochondrial fission and a metabolic switch from oxidative phosphorylation to glycolysis are key features of vascular pathology in pulmonary arterial hypertension (PAH) and are associated with exuberant endothelial proliferation and apoptosis. The underlying mechanisms are poorly understood. We describe the contribution of two intracellular chloride channel proteins, CLIC1 and CLIC4, both highly expressed in PAH and cancer, to mitochondrial dysfunction and energy metabolism in PAH endothelium. Pathological overexpression of CLIC proteins induces mitochondrial fragmentation, inhibits mitochondrial cristae formation, and induces metabolic shift toward glycolysis in human pulmonary artery endothelial cells, consistent with changes observed in patient-derived cells. Interactions of CLIC proteins with structural components of the inner mitochondrial membrane offer mechanistic insights. Endothelial CLIC4 excision and mitofusin 2 supplementation have protective effects in human PAH cells and preclinical PAH. This study is the first to demonstrate the key role of endothelial intracellular chloride channels in the regulation of mitochondrial structure, biogenesis, and metabolic reprogramming in expression of the PAH phenotype.

Cytokines as prognostic biomarkers in pulmonary arterial hypertension.

BACKGROUND: Risk stratification and assessment of disease progression in patients with pulmonary arterial hypertension (PAH) are challenged by the lack of accurate disease-specific and prognostic biomarkers. To date, brain natriuretic peptide (BNP) and/or its N-terminal fragment (NT-proBNP) are the only markers for right ventricular dysfunction used in clinical practice, in association with echocardiographic and invasive haemodynamic variables to predict outcome in patients with PAH. METHODS: This study was designed to identify an easily measurable biomarker panel in the serum of 80 well-phenotyped PAH patients with idiopathic, heritable or drug-induced PAH at baseline and at first follow-up. The prognostic value of identified cytokines of interest was secondly analysed in an external validation cohort of 125 PAH patients. RESULTS: Among the 20 biomarkers studied with the multiplex Ella platform, we identified a three-biomarker panel composed of ß-NGF, CXCL9 and TRAIL that were independently associated with prognosis both at the time of PAH diagnosis and at the first follow-up after initiation of PAH therapy. ß-NGF and CXCL9 were predictors of death or transplantation, whereas high levels of TRAIL were associated with a better prognosis. Furthermore, the prognostic value of the three cytokines was more powerful for predicting survival than usual non-invasive variables (New York Heart Association Functional Class, 6-min walk distance and BNP/NT-proBNP). The results were validated in a fully independent external validation cohort. CONCLUSION: The monitoring of ß-NGF, CXCL9 and TRAIL levels in serum should be considered in the management and treatment of patients with PAH to objectively guide therapeutic options.

Using the Plasma Proteome for Risk Stratifying Patients with Pulmonary Arterial Hypertension.

Rationale: NT-proBNP (N-terminal pro-brain natriuretic peptide), a biomarker of cardiac origin, is used to risk stratify patients with pulmonary arterial hypertension (PAH). Its limitations include poor sensitivity to early vascular pathology. Other biomarkers of vascular or systemic origin may also be useful in the management of PAH. Objectives: Identify prognostic proteins in PAH that complement NT-proBNP and clinical risk scores. Methods: An aptamer-based assay (SomaScan version 4) targeting 4,152 proteins was used to measure plasma proteins in patients with idiopathic, heritable, or drug-induced PAH from the UK National Cohort of PAH (n = 357) and the French EFORT (Evaluation of Prognostic Factors and Therapeutic Targets in PAH) study (n = 79). Prognostic proteins were identified in discovery-replication analyses of UK samples. Proteins independent of 6-minute-walk distance and NT-proBNP entered least absolute shrinkage and selection operator modeling, and the best combination in a single score was evaluated against clinical targets in EFORT. Measurements and Main Results: Thirty-one proteins robustly informed prognosis independent of NT-proBNP and 6-minute-walk distance in the UK cohort. A weighted combination score of six proteins was validated at baseline (5-yr mortality; area under the curve [AUC], 0.73; 95% confidence interval [CI], 0.63-0.85) and follow-up in EFORT (AUC, 0.84; 95% CI, 0.75-0.94; P = 9.96 × 10-6). The protein score risk stratified patients independent of established clinical targets and risk equations. The addition of the six-protein model score to NT-proBNP improved prediction of 5-year outcomes from AUC 0.762 (0.702-0.821) to 0.818 (0.767-0.869) by receiver operating characteristic analysis (P = 0.00426 for difference in AUC) in the UK replication and French samples combined. Conclusions: The plasma proteome informs prognosis beyond established factors in PAH and may provide a more sensitive measure of therapeutic response.

Mining the Plasma Proteome for Insights into the Molecular Pathology of Pulmonary Arterial Hypertension.

Rationale: Pulmonary arterial hypertension (PAH) is characterized by structural remodeling of pulmonary arteries and arterioles. Underlying biological processes are likely reflected in a perturbation of circulating proteins. Objectives: To quantify and analyze the plasma proteome of patients with PAH using inherited genetic variation to inform on underlying molecular drivers. Methods: An aptamer-based assay was used to measure plasma proteins in 357 patients with idiopathic or heritable PAH, 103 healthy volunteers, and 23 relatives of patients with PAH. In discovery and replication subgroups, the plasma proteomes of PAH and healthy individuals were compared, and the relationship to transplantation-free survival in PAH was determined. To examine causal relationships to PAH, protein quantitative trait loci (pQTL) that influenced protein levels in the patient population were used as instruments for Mendelian randomization (MR) analysis. Measurements and Main Results: From 4,152 annotated plasma proteins, levels of 208 differed between patients with PAH and healthy subjects, and 49 predicted long-term survival. MR based on cis-pQTL located in proximity to the encoding gene for proteins that were prognostic and distinguished PAH from health estimated an adverse effect for higher levels of netrin-4 (odds ratio [OR], 1.55; 95% confidence interval [CI], 1.16-2.08) and a protective effect for higher levels of thrombospondin-2 (OR, 0.83; 95% CI, 0.74-0.94) on PAH. Both proteins tracked the development of PAH in previously healthy relatives and changes in thrombospondin-2 associated with pulmonary arterial pressure at disease onset. Conclusions: Integrated analysis of the plasma proteome and genome implicates two secreted matrix-binding proteins, netrin-4 and thrombospondin-2, in the pathobiology of PAH.

Autoimmunity Is a Significant Feature of Idiopathic Pulmonary Arterial Hypertension.

Rationale: Autoimmunity is believed to play a role in idiopathic pulmonary arterial hypertension (IPAH). It is not clear whether this is causative or a bystander of disease and if it carries any prognostic or treatment significance. Objectives: To study autoimmunity in IPAH using a large cross-sectional cohort. Methods: Assessment of the circulating immune cell phenotype was undertaken using flow cytometry, and the profile of serum immunoglobulins was generated using a standardized multiplex array of 19 clinically validated autoantibodies in 473 cases and 946 control subjects. Additional glutathione S-transferase fusion array and ELISA data were used to identify a serum autoantibody to BMPR2 (bone morphogenetic protein receptor type 2). Clustering analyses and clinical correlations were used to determine associations between immunogenicity and clinical outcomes. Measurements and Main Results: Flow cytometric immune profiling demonstrates that IPAH is associated with an altered humoral immune response in addition to raised IgG3. Multiplexed autoantibodies were significantly raised in IPAH, and clustering demonstrated three distinct clusters: "high autoantibody," "low autoantibody," and a small "intermediate" cluster exhibiting high concentrations of ribonucleic protein complex. The high-autoantibody cluster had worse hemodynamics but improved survival. A small subset of patients demonstrated immunoglobulin reactivity to BMPR2. Conclusions: This study establishes aberrant immune regulation and presence of autoantibodies as key features in the profile of a significant proportion of patients with IPAH and is associated with clinical outcomes.

Mendelian randomisation and experimental medicine approaches to interleukin-6 as a drug target in pulmonary arterial hypertension.

BACKGROUND: Inflammation and dysregulated immunity are important in the development of pulmonary arterial hypertension (PAH). Compelling preclinical data supports the therapeutic blockade of interleukin-6 (IL-6) signalling. METHODS: We conducted a phase 2 open-label study of intravenous tocilizumab (8 mg·kg-1) over 6 months in patients with group 1 PAH. Co-primary end-points were safety, defined by incidence and severity of adverse events, and change in pulmonary vascular resistance. Separately, a mendelian randomisation study was undertaken on 11 744 individuals with European ancestry including 2085 patients with idiopathic/heritable disease for the IL-6 receptor (IL6R) variant (rs7529229), known to associate with circulating IL-6R levels. RESULTS: We recruited 29 patients (male/female 10/19; mean±sd age 54.9±11.4 years). Of these, 19 had heritable/idiopathic PAH and 10 had connective tissue disease-associated PAH. Six were withdrawn prior to drug administration; 23 patients received at least one dose of tocilizumab. Tocilizumab was discontinued in four patients owing to serious adverse events. There were no deaths. Despite evidence of target engagement in plasma IL-6 and C-reactive protein levels, both intention-to-treat and modified intention-to-treat analyses demonstrated no change in pulmonary vascular resistance. Inflammatory markers did not predict treatment response. Mendelian randomisation did not support an effect of the lead IL6R variant on risk of PAH (OR 0.99, p=0.88). CONCLUSION: Adverse events were consistent with the known safety profile of tocilizumab. Tocilizumab did not show any consistent treatment effect.

The pathophysiological role of novel pulmonary arterial hypertension gene SOX17.

Pulmonary arterial hypertension (PAH) is a progressive disease predominantly targeting pre-capillary blood vessels. Adverse structural remodelling and increased pulmonary vascular resistance result in cardiac hypertrophy and ultimately failure of the right ventricle. Recent whole-genome and whole-exome sequencing studies have identified SOX17 as a novel risk gene in PAH, with a dominant mode of inheritance and incomplete penetrance. Rare deleterious variants in the gene and more common variants in upstream enhancer sites have both been associated with the disease, and a deficiency of SOX17 expression may predispose to PAH. This review aims to consolidate the evidence linking genetic variants in SOX17 to PAH, and explores the numerous targets and effects of the transcription factor, focusing on the pulmonary vasculature and the pathobiology of PAH.

Plasma metabolomics exhibit response to therapy in chronic thromboembolic pulmonary hypertension.

Pulmonary hypertension is a condition with limited effective treatment options. Chronic thromboembolic pulmonary hypertension (CTEPH) is a notable exception, with pulmonary endarterectomy (PEA) often proving curative. This study investigated the plasma metabolome of CTEPH patients, estimated reversibility to an effective treatment and explored the source of metabolic perturbations.We performed untargeted analysis of plasma metabolites in CTEPH patients compared to healthy controls and disease comparators. Changes in metabolic profile were evaluated in response to PEA. A subset of patients were sampled at three anatomical locations and plasma metabolite gradients calculated.We defined and validated altered plasma metabolite profiles in patients with CTEPH. 12 metabolites were confirmed by receiver operating characteristic analysis to distinguish CTEPH and both healthy (area under the curve (AUC) 0.64-0.94, all p<2×10-5) and disease controls (AUC 0.58-0.77, all p<0.05). Many of the metabolic changes were notably similar to those observed in idiopathic pulmonary arterial hypertension (IPAH). Only five metabolites (5-methylthioadenosine, N1-methyladenosine, N1-methylinosine, 7-methylguanine, N-formylmethionine) distinguished CTEPH from chronic thromboembolic disease or IPAH. Significant corrections (15-100% of perturbation) in response to PEA were observed in some, but not all metabolites. Anatomical sampling identified 188 plasma metabolites, with significant gradients in tryptophan, sphingomyelin, methionine and Krebs cycle metabolites. In addition, metabolites associated with CTEPH and gradients showed significant associations with clinical measures of disease severity.We identified a specific metabolic profile that distinguishes CTEPH from controls and disease comparators, despite the observation that most metabolic changes were common to both CTEPH and IPAH patients. Plasma metabolite gradients implicate cardiopulmonary tissue metabolism of metabolites associated with pulmonary hypertension and metabolites that respond to PEA surgery could be a suitable noninvasive marker for evaluating future targeted therapeutic interventions.

CLIC4/Arf6 Pathway.

RATIONALE: Increased expression of CLIC4 (chloride intracellular channel 4) is a feature of endothelial dysfunction in pulmonary arterial hypertension, but its role in disease pathology is not fully understood. OBJECTIVE: To identify CLIC4 effectors and evaluate strategies targeting CLIC4 signaling in pulmonary hypertension. METHODS AND RESULTS: Proteomic analysis of CLIC4-interacting proteins in human pulmonary artery endothelial cells identified regulators of endosomal trafficking, including Arf6 (ADP ribosylation factor 6) GTPase activating proteins and clathrin, while CLIC4 overexpression affected protein regulators of vesicular trafficking, lysosomal function, and inflammation. CLIC4 reduced BMPRII (bone morphogenetic protein receptor II) expression and signaling as a result of Arf6-mediated reduction in gyrating clathrin and increased lysosomal targeting of the receptor. BMPRII expression was restored by Arf6 siRNA, Arf inhibitor Sec7 inhibitor H3 (SecinH3), and inhibitors of clathrin-mediated endocytosis but was unaffected by chloride channel inhibitor, indanyloxyacetic acid 94 or Arf1 siRNA. The effects of CLIC4 on NF-κB (nuclear factor-kappa B), HIF (hypoxia-inducible factor), and angiogenic response were prevented by Arf6 siRNA and SecinH3. Sugen/hypoxia mice and monocrotaline rats showed elevated expression of CLIC4, activation of Arf6 and NF-κB, and reduced expression of BMPRII in the lung. These changes were established early during disease development. Lung endothelium-targeted delivery of CLIC4 siRNA or treatment with SecinH3 attenuated the disease, reduced CLIC4/Arf activation, and restored BMPRII expression in the lung. Endothelial colony-forming cells from idiopathic pulmonary hypertensive patients showed upregulation of CLIC4 expression and Arf6 activity, suggesting potential importance of this pathway in the human condition. CONCLUSIONS: Arf6 is a novel effector of CLIC4 and a new therapeutic target in pulmonary hypertension.

The zinc transporter ZIP12 regulates the pulmonary vascular response to chronic hypoxia.

The typical response of the adult mammalian pulmonary circulation to a low oxygen environment is vasoconstriction and structural remodelling of pulmonary arterioles, leading to chronic elevation of pulmonary artery pressure (pulmonary hypertension) and right ventricular hypertrophy. Some mammals, however, exhibit genetic resistance to hypoxia-induced pulmonary hypertension. We used a congenic breeding program and comparative genomics to exploit this variation in the rat and identified the gene Slc39a12 as a major regulator of hypoxia-induced pulmonary vascular remodelling. Slc39a12 encodes the zinc transporter ZIP12. Here we report that ZIP12 expression is increased in many cell types, including endothelial, smooth muscle and interstitial cells, in the remodelled pulmonary arterioles of rats, cows and humans susceptible to hypoxia-induced pulmonary hypertension. We show that ZIP12 expression in pulmonary vascular smooth muscle cells is hypoxia dependent and that targeted inhibition of ZIP12 inhibits the rise in intracellular labile zinc in hypoxia-exposed pulmonary vascular smooth muscle cells and their proliferation in culture. We demonstrate that genetic disruption of ZIP12 expression attenuates the development of pulmonary hypertension in rats housed in a hypoxic atmosphere. This new and unexpected insight into the fundamental role of a zinc transporter in mammalian pulmonary vascular homeostasis suggests a new drug target for the pharmacological management of pulmonary hypertension.

Immunoglobulin-driven Complement Activation Regulates Proinflammatory Remodeling in Pulmonary Hypertension.

Rationale: Pulmonary hypertension (PH) is a life-threatening cardiopulmonary disorder in which inflammation and immunity have emerged as critical early pathogenic elements. Although proinflammatory processes in PH and pulmonary arterial hypertension (PAH) are the focus of extensive investigation, the initiating mechanisms remain elusive.Objectives: We tested whether activation of the complement cascade is critical in regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and can serve as a prognostic biomarker of outcome in human PAH.Methods: We used immunostaining of lung tissues from experimental PH models and patients with PAH, analyses of genetic murine models lacking specific complement components or circulating immunoglobulins, cultured human pulmonary adventitial fibroblasts, and network medicine analysis of a biomarker risk panel from plasma of patients with PAH.Measurements and Main Results: Pulmonary perivascular-specific activation of the complement cascade was identified as a consistent critical determinant of PH and PAH in experimental animal models and humans. In experimental hypoxic PH, proinflammatory and pro-proliferative responses were dependent on complement (alternative pathway and component 5), and immunoglobulins, particularly IgG, were critical for activation of the complement cascade. We identified Csf2/GM-CSF as a primary complement-dependent inflammatory mediator. Furthermore, using network medicine analysis of a biomarker risk panel from plasma of patients with PAH, we demonstrated that complement signaling can serve as a prognostic factor for clinical outcome in PAH.Conclusions: This study establishes immunoglobulin-driven dysregulated complement activation as a critical pathobiological mechanism regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and demonstrates complement signaling as a critical determinant of clinical outcome in PAH.

Whole-Blood RNA Profiles Associated with Pulmonary Arterial Hypertension and Clinical Outcome.

Rationale: Idiopathic and heritable pulmonary arterial hypertension (PAH) are rare but comprise a genetically heterogeneous patient group. RNA sequencing linked to the underlying genetic architecture can be used to better understand the underlying pathology by identifying key signaling pathways and stratify patients more robustly according to clinical risk.Objectives: To use a three-stage design of RNA discovery, RNA validation and model construction, and model validation to define a set of PAH-associated RNAs and a single summarizing RNA model score. To define genes most likely to be involved in disease development, we performed Mendelian randomization (MR) analysis.Methods: RNA sequencing was performed on whole-blood samples from 359 patients with idiopathic, heritable, and drug-induced PAH and 72 age- and sex-matched healthy volunteers. The score was evaluated against disease severity markers including survival analysis using all-cause mortality from diagnosis. MR used known expression quantitative trait loci and summary statistics from a PAH genome-wide association study.Measurements and Main Results: We identified 507 genes with differential RNA expression in patients with PAH compared with control subjects. A model of 25 RNAs distinguished PAH with 87% accuracy (area under the curve 95% confidence interval: 0.791-0.945) in model validation. The RNA model score was associated with disease severity and long-term survival (P = 4.66 × 10-6) in PAH. MR detected an association between SMAD5 levels and PAH disease susceptibility (odds ratio, 0.317; 95% confidence interval, 0.129-0.776; P = 0.012).Conclusions: A whole-blood RNA signature of PAH, which includes RNAs relevant to disease pathogenesis, associates with disease severity and identifies patients with poor clinical outcomes. Genetic variants associated with lower SMAD5 expression may increase susceptibility to PAH.

Mendelian randomisation analysis of red cell distribution width in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare disease that leads to premature death from right heart failure. It is strongly associated with elevated red cell distribution width (RDW), a correlate of several iron status biomarkers. High RDW values can signal early-stage iron deficiency or iron deficiency anaemia. This study investigated whether elevated RDW is causally associated with PAH.A two-sample Mendelian randomisation (MR) approach was applied to investigate whether genetic predisposition to higher levels of RDW increases the odds of developing PAH. Primary and secondary MR analyses were performed using all available genome-wide significant RDW variants (n=179) and five genome-wide significant RDW variants that act via systemic iron status, respectively.We confirmed the observed association between RDW and PAH (OR 1.90, 95% CI 1.80-2.01) in a multicentre case-control study (cases n=642, disease controls n=15 889). The primary MR analysis was adequately powered to detect a causal effect (odds ratio) between 1.25 and 1.52 or greater based on estimates reported in the RDW genome-wide association study or from our own data. There was no evidence for a causal association between RDW and PAH in either the primary (ORcausal 1.07, 95% CI 0.92-1.24) or the secondary (ORcausal 1.09, 95% CI 0.77-1.54) MR analysis.The results suggest that at least some of the observed association of RDW with PAH is secondary to disease progression. Results of iron therapeutic trials in PAH should be interpreted with caution, as any improvements observed may not be mechanistically linked to the development of PAH.

Reduced plasma levels of small HDL particles transporting fibrinolytic proteins in pulmonary arterial hypertension.

BACKGROUND: Aberrant lipoprotein metabolism has been implicated in experimental pulmonary hypertension, but the relevance to patients with pulmonary arterial hypertension (PAH) is inconclusive. OBJECTIVE: To investigate the relationship between circulating lipoprotein subclasses and survival in patients with PAH. METHODS: Using nuclear magnetic resonance spectroscopy, 105 discrete lipoproteins were measured in plasma samples from two cohorts of patients with idiopathic or heritable PAH. Data from 1124 plasma proteins were used to identify proteins linked to lipoprotein subclasses. The physical presence of proteins was confirmed in plasma lipoprotein subfractions separated by ultracentrifugation. RESULTS: Plasma levels of three lipoproteins from the small high-density lipoprotein (HDL) subclass, termed HDL-4, were inversely related to survival in both the discovery (n=127) and validation (n=77) cohorts, independent of exercise capacity, comorbidities, treatment, N-terminal probrain natriuretic peptide, C reactive protein and the principal lipoprotein classes. The small HDL subclass rich in apolipoprotein A-2 content (HDL-4-Apo A-2) exhibited the most significant association with survival. None of the other lipoprotein classes, including principal lipoprotein classes HDL and low-density lipoprotein cholesterol, were prognostic. Three out of nine proteins identified to associate with HDL-4-Apo A-2 are involved in the regulation of fibrinolysis, namely, the plasmin regulator, alpha-2-antiplasmin, and two major components of the kallikrein-kinin pathway (coagulation factor XI and prekallikrein), and their physical presence in the HDL-4 subfraction was confirmed. CONCLUSION: Reduced plasma levels of small HDL particles transporting fibrinolytic proteins are associated with poor outcomes in patients with idiopathic and heritable PAH.