BMPR2 Mutations and Iron Metabolism in Pulmonary Arterial Hypertension

Study Overview
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Brief Summary
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Previously characterised PAH patients, including idiopathic, heritable and other forms of group 1 PAH with and without BMPR2 mutation which have already been analysed and are regularly seen in the Center for Pulmonary Hypertension may be contacted to participate in the study. Clinical and laboratory values will be collected prospectively. Patients with IPAH/HPAH and other forms of PAH who are newly diagnosed within the duration of the trial will receive routine diagnostic workup including the routine information about a possible BMPR2 mutation analysis for IPAH/HPAH patients according to guidelines. During their routine visit the patients' medical history will be obtained and physical examination will be conducted. Moreover, an electrocardiogram (ECG), determination of World Health Organization (WHO)-functional class, laboratory testing (NT-proBNP and routine laboratory), echocardiography will be routinely carried out. BMPR2 expression levels will be measured in blood samples. Additionally, laboratory samples will be collected for analysis of further parameters reflecting iron metabolism such as hepcidin, ferritin, iron levels, IL6 and circulating soluble transferrin receptor Levels. In addition, healthy controls will be invited to participate in this study to obtain comparable levels of hepcidin and BMPR2 pathway members.

Detailed Description
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Pulmonary arterial hypertension (PAH) is a rare disease characterized by an increase in pulmonary arterial pressure and pulmonary vascular resistance, which result in right heart hypertrophy and decompensation. It crucially affects exercise capacity, quality of life and prognosis. Idiopathic and heritable forms of PAH (IPAH and HPAH) are often associated with mutations of the bone morphogenetic protein receptor 2 (BMPR2) accompanied by disease development at an earlier age, more severe hemodynamic phenotype and a higher mortality rate. Other forms of PAH also show reduced expression levels of BMPR2, even if no BMPR2 mutation has been identified in these patients. Moreover, the balance of iron metabolism was shown to be disturbed in IPAH patients. IPAH patients suffered from iron deficiency with low levels of serum iron concentrations and while at the same time displaying high levels of the iron uptake regulating hormon hepcidin. The hormone hepcidin, which inhibits iron absorption from the intestine, is upregulated by the BMPR2 signaling pathway (via BMP6). The impact of BMPR2 expression on iron homeostasis, however, has not been investigated yet. Mutation and non-mutation carriers with invasively diagnosed PAH by right heart catheter and under optimized medical therapy will be enrolled in this study. An explicit exclusion criterion is intravenous iron supplementation in the last 2 months to capture their natural iron metabolic status. Subjects will be recruited at the Center for Pulmonary Hypertension at Thoraxklinik Heidelberg University Hospital. The measurement of BMPR2 expression will be performed with real-time polymerase chain reaction. In addition, routine laboratory parameters of iron metabolism and clinical parameters will be statistically correlated with the BMPR2 expression of BMPR2 mutation carriers and non-mutation carriers. Clinical examinations will comprise of routine diagnostic workup. No study specific clinical assessments will be performed. For diagnostic workup, an extended blood analysis for BMPR2 expression will be performed, which is mentioned in the informed consent document. In addition, healthy controls will be invited to participate in this study.Healthy controls will only receive a blood collection to obtain control values for hepcidin, BMPR2 expression rate and levels of BMPR2 pathway members such as Bone Morphogenetic Protein 2 and 6 (BMP2 and BMP6). They will not receive any further examinations. BMPR2 mutation status will not be investigated. The control group will be age and gender matched to non-BMPR2 mutation carriers. Therefore, this study aims to investigate whether PAH patients with a reduced expression rate of BMPR2 have altered serum levels of hepcidin and further iron related metabolites compared to PAH patients with normal expression levels and whether these patients present with more pronounced limitations in clinical parameters. This study could help to understand iron metabolism in PAH and generate new therapeutic targets for the treatment of the disease.

Official Title
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Association Between BMPR2 Mutations and Iron Metabolism in Pulmonary Arterial Hypertension Patients: an Explorative Cross-sectional Study

Conditions
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Pulmonary Arterial Hypertension

Intervention / Treatment
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* Diagnostic Test: hepcidin levels and BMPR2 expression


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria Patients: Informed consent Male or female PAH, including idiopathic, heritable and other forms of group 1 PAH (according to Nice classification) patients 18-80 years of age Invasively diagnosed PAH by right heart catheter (invasively confirmed diagnosis according to the current PAH definition of valid guidelines at time of initial diagnosis) Optimized medical therapy for PAH (such as endothelin-receptor-antagonists, inhaled prostanoids, phosphodiesterase-5-inhibitors, diuretics and if useful, supplemental oxygen) for at least 2 months before entering the study Able to understand and willing to sign the Informed Consent Form Inclusion Criteria Healthy Controls: Informed consent Male or female healthy controls 18-80 years of age Able to understand and willing to sign the Informed Consent Form Exclusion Criteria Patients: Pregnancy or lactation Change in disease-specific medication within 8 weeks before enrolment Intravenous iron supplementation within the preceding 2 months Acute infection Comorbidities affecting iron metabolism such as hemolytic anemias, genetic disorders of hemoglobin, diabetes, systemic cardiovascular disease, sickle cell disease, thalassemia Exclusion Criteria Healthy Controls: Pregnancy or lactation Intravenous iron supplementation within the preceding 2 months Acute infection Heart or lung disease Comorbidities affecting iron metabolism such as hemolytic anemias, genetic disorders of hemoglobin, diabetes, systemic cardiovascular disease, sickle cell disease, thalassemia

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 80 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| BMPR2-mutation carriers<br>Patients affected by pulmonary arterial hypertension (PAH) with already determined BMPR2 mutation status (hereditary PAH, HPAH) or patients with idiopathic PAH (IPAH) and other forms of PAH who are newly diagnosed within the duration of the study and resulted positive for mutation at the routinely-performed (according to current guidelines) BMPR2 analysis. | Diagnostic Test: hepcidin levels and BMPR2 expression<br>* Samples will be collected in the three groups. Hepcidin levels will be measured in serum using ELISA and BMPR2 expression will be assessed as previously described using real time-qPCR<br>|
| non-BMPR2 mutation carriers<br>Patients affected by Pulmonary arterial hypertension (PAH) who resulted negative at the routinely-performed (according to current guidelines) BMPR2 analysis (Idiopathic PAH, IPAH) or patients with Idiopathic PAH (IPAH) and other forms of PAH who are newly diagnosed within the duration of the study and resulted negative for mutation at the routinely-performed (according to current guidelines) BMPR2 analysis. | Diagnostic Test: hepcidin levels and BMPR2 expression<br>* Samples will be collected in the three groups. Hepcidin levels will be measured in serum using ELISA and BMPR2 expression will be assessed as previously described using real time-qPCR<br>|
| healthy controls<br>Healthy controls free of heart and lung disease or any comorbidities affecting iron metabolism. This control group will be age and gender matched to non-BMPR2 mutation carriers. | Diagnostic Test: hepcidin levels and BMPR2 expression<br>* Samples will be collected in the three groups. Hepcidin levels will be measured in serum using ELISA and BMPR2 expression will be assessed as previously described using real time-qPCR<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| The relationship between absolute values of hepcidin levels and BMPR2 expression | assessed as correlation between BMPR2 expression levels and hepcidin levels | at enrollment | 
| The relationship between hepcidin levels and BMPR2 expression | analysis of differences of hepcidin levels in BMPR2 mutation carriers and non-carriers (BMPR2 mutation carriers are assumed to have a lower expression level of BMPR2) | at enrollment | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Correlation of BMPR2 expression levels with ferritin levels | Ferritin levels | at enrollment | 
| Correlation of BMPR2 expression levels with transferrin levels | Transferrin levels | at enrollment | 
| Correlation of BMPR2 expression levels with soluble transferrin receptor saturation and concentration | Soluble transferrin receptor saturation and concentration | at enrollment | 
| Correlation of BMPR2 expression levels with iron levels | Iron levels | at enrollment | 
| Correlation of BMPR2 expression levels with red blood distribution cell width | Red blood distribution cell width | at enrollment | 
| Correlation of BMPR2 expression levels with erythroferrone levels | Erythroferrone levels | at enrollment | 
| Correlation of BMPR2 expression levels with hemoglobin levels | Hemoglobin levels | at enrollment | 
| Correlation of BMPR2 expression levels with hematocrit | Hematocrit | at enrollment | 
| Correlation of BMPR2 expression levels with erythropoietin (EPO) levels | Erythropoietin (EPO) levels | at enrollment | 
| Correlation of BMPR2 expression levels with C-reactive protein levels | C-reactive protein levels | at enrollment | 
| Correlation of BMPR2 expression levels with interleukin 6 (IL6) levels | Interleukin 6 (IL6) levels to approximate inflammation | at enrollment | 
| Correlation of BMPR2 expression levels with NT-proBNP levels | NT-proBNP levels | at enrollment | 
| Correlation of BMPR2 expression levels with BMP2 messenger ribonucleid acid (mRNA) expression levels | BMP2 mRNA expression levels | at enrollment | 
| Correlation of BMPR2 expression levels with BMP6 messenger ribonucleid acid (mRNA) expression levels | BMP6 mRNA expression levels | at enrollment | 
| Correlation of BMPR2 expression levels with overall transcriptomic analysis in blood samples and formalin fixed human PAH lung tissue samples | overall transcriptomic analysis in blood samples and formalin fixed human PAH lung tissue samples | at enrollment | 
| Correlation of BMPR2 expression levels with BMPR2 protein levels | BMPR2 protein levels | at enrollment | 
| Correlation of BMPR2 expression levels with BMP2 protein levels | BMP2 protein levels | at enrollment | 
| Correlation of BMPR2 expression levels with BMP6 protein levels | BMP6 protein levels | at enrollment | 
| Correlation of BMPR2 expression levels with 6-minute walking distance (6-MWD) | 6-minute walking distance (6-MWD) | at enrollment | 
| Correlation of BMPR2 expression levels with BORG Scale of 6-minute walking distance (6-MWD) | Borg Scale of 6-minute walking distance (6-MWD) | at enrollment | 
| Correlation of BMPR2 expression levels with WHO functional class | WHO functional class | at enrollment | 
| Correlation of BMPR2 expression levels with forced vital capacity (FVC) | forced vital capacity (FVC) | at enrollment | 
| Correlation of BMPR2 expression levels with forced expiratory volume in one second (FEV1) | forced expiratory volume in one second (FEV1) | at enrollment | 
| Correlation of BMPR2 expression levels with forced expiratory flow (FEV) | forced expiratory flow (FEV) | at enrollment | 
| Correlation of BMPR2 expression levels with total lung capacity (TLC) | total lung capacity (TLC) | at enrollment | 
| Correlation of BMPR2 expression levels with diffusion-limited carbon monoxide (DLCo) | diffusion-limited carbon monoxide (DLCo) | at enrollment | 
| Correlation of BMPR2 expression levels with residual volume | residual volume, normally accounting for about 25% of total lung capacity | at enrollment | 
| Correlation of BMPR2 expression levels with blood gas analysis including partial pressure of oxygen | partial pressure of oxygen | at enrollment | 
| Correlation of BMPR2 expression levels with blood gas analysis including partial pressure of carbon dioxide | partial pressure of carbon dioxide | at enrollment | 
| Correlation of BMPR2 expression levels with blood gas analysis including oxygen saturation | oxygen saturation | at enrollment | 
| Correlation of BMPR2 expression levels with blood gas analysis including supplemental oxygen yes or no | supplemental oxygen yes or no | at enrollment | 
| Correlation of BMPR2 expression levels with cardiac output (CO) | cardiac output (CO) measured by right heart catheterization | at enrollment | 
| Correlation of BMPR2 expression levels with cardiac index (CI) | cardiac index (CI) measured by right heart catheterization | at enrollment | 
| Correlation of BMPR2 expression levels with pulmonary capillary wedge pressure (PAWP) | pulmonary capillary wedge pressure (PAWP) measured by right heart catheterization | at enrollment | 
| Correlation of BMPR2 expression levels with mixed venous oxygen saturation (SvO2) | mixed venous oxygen saturation (SvO2) measured by right heart catheterization | at enrollment | 
| Correlation of BMPR2 expression levels with right atrium area (RA-area) | right atrium area (RA-area) determined by echocardiography | at enrollment | 
| Correlation of BMPR2 expression levels with right ventricle area (RV-area) | right ventricle area (RV-area) determined by echocardiography | at enrollment | 
| Correlation of BMPR2 expression levels with myocardial performance index (Tei) | myocardial performance index (Tei) determined by echocardiography | at enrollment | 
| Correlation of BMPR2 expression levels with tricuspid annular plane systolic excursion (TAPSE) | tricuspid annular plane systolic excursion (TAPSE) determined by echocardiography | at enrollment | 
| Correlation of BMPR2 Expression with systolic pulmonary artery pressure | systolic pulmonary artery pressure determined by echocardiography | at enrollment | 
| Correlation of BMPR2 Expression with right ventricular function | right ventricular function determined by echocardiography | at enrollment | 
| Correlation of BMPR2 Expression with left ventricular function | left ventricular function determined by echocardiography | at enrollment | 
| Correlation of BMPR2 Expression with right ventricular pressure | systolic, diastolic and mean right ventricular pressure measured by right heart catheterization | at enrollment | 
| Correlation of BMPR2 Expression with pulmonary artery pressure | systolic, diastolic and mean pulmonary artery pressure measured by right heart catheterization | at enrollment | 
| Correlation of BMPR2 Expression with pulmonary vascular resistance | pulmonary vascular resistance measured by right heart catheterization | at enrollment | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Pulmonary Arterial Hypertension, BMPR2, Iron Metabolism, Hepcidin, Mutations