Assessment of the TGF-beta Pathway and Micro-RNA in Pediatric Pulmonary Arterial Hypertension

Study Overview
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Brief Summary
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This is a prospective pilot study to assess the plasma levels of particular proteins involved in the transforming growth factor beta (TGF-β) pathway and its down stream regulators, CHIP, as well as micro RNA molecules in subjects with pulmonary arterial hypertension (PAH) and compare them to control subjects without PAH to see if they can be used as a diagnostic or prognostic marker of PAH and how this compares to other diagnostic biomarkers N-terminal pro-natriuretic peptide (NT Pro-BNP) and C-reactive protein (CRP).

Detailed Description
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Aim 1: This study will correlate proteins in the TGF- β signaling pathway and micro RNA levels with invasive (catheterization) and non-invasive (echocardiography) measurements of pulmonary artery pressures to assess for the presence and severity of PAH and compare these measurements to the established biomarkers of NT Pro BNP and CRP levels. Hypothesis 1: Plasma levels of proteins of the TGF-β pathway; bone morphogenic protein (BMP) 2, 4, 6, 7, 9 and 10 along with activin A and TGF-β1 protein as well as CHIP (carboxyl-terminus of Hsp70-intracting protein), an enzyme that regulates the activations and exports of TGF- β to the nucleus will be significantly different in subjects with PH over control subjects. Hypothesis 2: Plasma levels of proteins in the TGF- β pathway; BMP 2, 4, 6, 7, 9 and 10 along with activin A and TGF-β1 protein as well as CHIP will show better correlation with the presence of PAH and its severity than NT-Pro BNP and CRP levels. Hypothesis 3: The micro-RNA profiles in plasma will be significantly different in subjects with PAHPH over control subjects. Aim 2: To correlate protein/micro-RNA levels with clinical status in PAH subjects as assessed by functional status, exercise testing, and PAH drug regimen to determine if they can correlate with disease severity. Hypothesis 1: Clinical findings in PAH patients will correlate with disease severity and study proteins and micro-RNA levels better than established biomarkers. Aim 3: To correlate evidence of genetic abnormalities through whole exome sequencing especially in regions known or suspected to cause PAH (e.g. BMPR2, ENG, and ALK1 mutations), within the TGF-β pathway or lung development with the tested protein/micro-RNA levels. Hypothesis 1: Genetic evaluation of patients with PAH will show abnormalities within the TGF-β pathway or lung development.

Official Title
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Assessment of the TGF-beta Pathway and Micro-RNA in Pediatric Pulmonary Arterial Hypertension

Conditions
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Pulmonary Arterial Hypertension

Intervention / Treatment
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* Diagnostic Test: Protein Elisa analysis, microRNA analysis, whole exome sequencing


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Pediatric subjects ages 2-17 years Subjects undergoing a clinically indicated cardiac catheterization. Subjects with proven or being evaluated for pulmonary hypertension in WHO classification group 1 or 3† Subjects will be categorized as PAH subjects if they meet the hemodynamic criteria: pulmonary artery pressure >20mmHg, pulmonary vascular resistance index >3 Woods units*m2, and wedge pressures <15mmHg. Subjects can be categorized as control subjects if they do not have PH on catheterization and do not meet any exclusion criteria. Exclusion Criteria: -

Ages Eligible for Study
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Minimum Age: 2 Years
Maximum Age: 17 Years

Sexes Eligible for Study
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All

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| PH subjects<br>Pediatric subjects ages 2-17 years Subjects undergoing a clinically indicated cardiac catheterization. Subjects with proven or being evaluated for pulmonary hypertension in WHO classification group 1 or 3† Subjects will be categorized as PAH subjects if they meet the hemodynamic criteria: pulmonary artery pressure >20mmHg, pulmonary vascular resistance index >3 Woods units*m2, and wedge pressures <15mmHg. | Diagnostic Test: Protein Elisa analysis, microRNA analysis, whole exome sequencing<br>* ELISA Testing Procedure: The circulating levels of BMP 2, 4, 6, 7, 9 and 10, together with activin A and TGF-β as well as CHIP will be measured using streptavidin ELISA assays. Micro-RNA analysis: From 100µL of plasma, we will prepare small RNA libraries using TruSeq Small RNA Sample Preparation kit (Illumina Inc). TGF-B mutation evaluation: Blood from subjects with PH will also be analyzed for known genetic mutations in TGF-B pathway using whole exome sequencing.<br>|
| Control subjects<br>Pediatric subjects ages 2-17 years Subjects undergoing a clinically indicated cardiac catheterization. Subjects can be categorized as control subjects if they do not have PH on catheterization and do not meet any exclusion criteria. | Diagnostic Test: Protein Elisa analysis, microRNA analysis, whole exome sequencing<br>* ELISA Testing Procedure: The circulating levels of BMP 2, 4, 6, 7, 9 and 10, together with activin A and TGF-β as well as CHIP will be measured using streptavidin ELISA assays. Micro-RNA analysis: From 100µL of plasma, we will prepare small RNA libraries using TruSeq Small RNA Sample Preparation kit (Illumina Inc). TGF-B mutation evaluation: Blood from subjects with PH will also be analyzed for known genetic mutations in TGF-B pathway using whole exome sequencing.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Plasma levels of BMP proteins of the TGF-β pathway | bone morphogenic protein (BMP) 2, 4, 6, 7, 9 and 10 levels will be obtained in PH subjects and will be compared to control subjects in effort to describe how those levels are different in subjects with PH over control subjects. All measured amounts of proteins will be in pg/ml. | One day | 
| Plasma Activin A and TGF-β1 level of the TGF-β pathway | Activin A and TGF-β1 plasma levels will be obtained in PH subjects and compared to control subjects in effort to describe how those levels are different in subjects with PH over control subjects. All measured amounts will be will be in pg/ml. | One day | 
| Plasma CHIP levels assessment in PH patients and control subjects. | CHIP (carboxyl-protein terminus of Hsp70-intracting protein) plasma levels will be obtained in PH subjects and compared to control subjects in effort to describe how those levels are different in subjects with PH over control subjects. All measured levels will be in pg/ml | One day | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Plasma levels of proteins in the TGF- β pathway and PH disease severity | BMP 2, 4, 6, 7, 9 and 10 along with activin A and TGF-β1 protein as well as CHIP levels will be correlated with the presence of PAH and its severity and how that compares to the levels of NT-Pro BNP and CRP levels. | One day | 
| MicroRNA levels in PH vs control subjects | The micro-RNA profiles in plasma will be compared between in subjects with PAH and control subjects. | One day | 
| Clinical findings correlation with study proteins/microRNA | Clinical findings in PAH patients will be correlated with disease severity and study protein and micro-RNA levels. | One day | 
| Whole exome sequence analysis in PH patients and protein/microRNA levels | To correlate evidence of genetic abnormalities through whole exome sequencing especially in regions known or suspected to cause PAH (e.g. BMPR2, ENG, and ALK1 mutations), within the TGF-β pathway or lung development with the tested protein/micro-RNA levels. | One day | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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pulmonary arterial hypertension, TGF-beta pathway, microRNA