Molecular Investigation of Genetic Factors in CArDiovascular Diseases Using an BIOresource of Healthy Volunteers

Study Overview
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Brief Summary
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The risk of cardiovascular disease is determined by the complex interplay between an individual's genetic make-up, lifestyle, and the environment. We are investigating three potential genetic risk factors in this observational, cross-sectional, epidemiology pilot study to investigate if and how functional variants identified in large-scale genome wide association studies can explain a predisposition to cardiovascular disease. By determining the molecular mechanisms that are regulated at the EDNRA, PNPLA3 and PROCR CVD risk loci, we hope to translate findings from this study into the clinical setting for better diagnosis, prevention and treatment for patients suffering with cardiovascular disease. Volunteers will enter into one of the study's three arms based on their genotype: EDNRA locus (Arm 1), PNPLA3 locus (Arm 2), or PROCR locus (Arm 3). Members of the Cambridge Bioresource who match for the target alleles will be invited to participate and will enter into one of the three study arms. All study assessment visits will take place at Addenbrooke's Hospital in collaboration with the University of Cambridge. Volunteers will participate in the study for a maximum of 12 months and depending on study arm they are assigned to, they will complete procedures including a medical, demographic and lifestyle factors questionnaire; height, weight and body fat assessments; in addition to blood pressure/heart rate measurements. Minimally invasive procedures including forearm blood flow and venepuncture will be performed to assess the primary objectives of the study. The hypothesis for arm 1 is that the genetic variant we are investigating at the EDNRA gene locus alters the function of the endothelin receptor A leading to an increased risk of coronary artery disease and large artery stroke. For study arm 2, we hypothesize that the genetic variant we are investigating in PNPLA3 will increase the risk of Non-alcoholic fatty liver disease but reduce the risk of Coronary Heart Disease. For study arm 3, we hypothesize that the genetic variant we are investigating in the PROCR locus triggers molecular events that potentially increase the risk of Venous Thrombosis/Venous Thromboembolism nut reducing blood pressure. Furthermore we aim to investigate the anti-inflammatory effects to see if there is an effect on explaining reduced risk of CHD. This study is funded from the BHF Cambridge Center of Excellence and the Wellcome Trust Institutional Strategic Support Fund.

Official Title
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Molecular Investigation of Genetic Factors in CArDiovascular Diseases Using an BIOresource of Healthy Volunteers

Conditions
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Cardiovascular Diseases

Intervention / Treatment
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* Other: Eligibility and lifestyle restrictions check.
* Other: Medical history, demographic and lifestyle factors
* Other: Anthropometric measurements: height, weight, and body fat
* Other: Blood pressure and heart rate
* Other: Forearm blood flow: Visit 1 (EDNRA)
* Other: Forearm blood flow: Visit 2 (EDNRA)
* Other: Blood biochemistry (PNPLA3)
* Other: Blood EPCR function (PROCR)
* Other: Blood platelet coagulation and function (PROCR)
* Other: Blood endothelial permeability (PROCR)
* Other: Blood leukocyte-endothelium adhesion (PROCR)
* Other: Baseline Venepuncture (PNPLA3, Visit 1)
* Other: Deuterium water - Loading dose 1 (PNPLA3, Visit 1)
* Other: Deuterium water - Loading dose 2 (PNPLA3, Visit 1)
* Other: Energy-balanced dinner (PNPLA3, Visit 1)
* Other: Fasting venepuncture (PNPLA3, Visit 2)
* Other: Deuterium water - Maintenance dose (PNPLA3, Visit 2)
* Other: Consumption of high carbohydrate meal (PNPLA3, Visit 2)
* Other: Postprandial Venepuncture (PNPLA3, Visit 2)
* Other: Venepuncture (PROCR)


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Volunteers are homozygous for: Arm 1: The A-allele of rs6841581, they are assigned to the 'case' group. If they are homozygous for the G-allele, they are assigned to the 'control' group Arm 2: The G-allele of rs738409, they are assigned to the 'case' group. If they are homozygous for the C-allele, they are assigned to the 'control' group Arm 3: The G-allele of rs867186, they are assigned to the 'case' group. If they are homozygous for the A-allele, they are assigned to the 'control' group Volunteers are aged between 18-50 years old Volunteers have a BMI: Arm 1: Between18.5-29.9 Arm 2: Between 25.0-39.9 Arm 3: Between 18.5-29.9 Volunteers are willing not to consume products containing alcohol or caffeine 12 hours prior to procedures. Additionally, volunteers must agree to fast before procedures for: Arm 1: At least 4 hours Arm 2: At least 8 hours (for visit 2 only) Arm 3: At least 4 hours Have given written informed consent to participate Exclusion Criteria: Volunteers with chronic diseases, including cardiovascular diseases, liver diseases, type 1 and type 2 diabetes autoimmune diseases and cancer Biological first-degree relatives (parents, brothers, sisters or children) who are or have suffered from one of the conditions described above Current smokers. Ex-smokers are suitable if they stopped smoking >10 years ago Volunteers with a diagnosis of hypertension, or history of consistently high blood pressure readings, >140/90 mmHg Volunteers with a diagnosis of hypercholesterolemia, or history of consistently high cholesterol levels, e.g. total cholesterol level >6 mmol/l Volunteers have ≥3 alcoholic drinks per day

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 50 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 |
| --- | --- |
| EDNRA Sub-study<br>Study population will be split into two groups defined by the allele of EDNRA the participant possesses: Participants Homozygous for the A-allele of EDNRA, are assigned to the 'case' group. Participants that are Homozygous for the G-allele will be assigned to the 'control' group. 20 participants will be recruited to each group, 40 in total. | Other: Eligibility and lifestyle restrictions check.<br>* Eligibility will be assessed at each study visit and participants will be informed in advance of their visit.<br>Other: Medical history, demographic and lifestyle factors<br>* Medical History, participants demographics and lifestyle factors will be assessed by the participant completion of the medical history and ethnicity questionnaires.<br>Other: Anthropometric measurements: height, weight, and body fat<br>* Height measured by stadiometer. Weight and body fat measured by Tanita scale bioelectrical impedance analysis.<br>Other: Blood pressure and heart rate<br>* Parameters will be measured using a validated, automated device while seated and again after 3-5 min standing. All measurements will be done in triplicate.<br>Other: Forearm blood flow: Visit 1 (EDNRA)<br>* Intra-arterial infusion of Phenylephrine at doses of 0.75, 2.5, and 7.5 μg/min for 6 min each (18 min in total). FBF measured in the last 3 minutes. There will be a 30 minute washout with saline followed by Endothelin-1 infusion of at a dose of 5 pmol/min for 90 min.<br>Other: Forearm blood flow: Visit 2 (EDNRA)<br>* Intra-arterial infusion of Sodium nitroprusside infusion at doses of 1, 3, and 10 μg/min for 6 min each (18 min in total). FBF measured in the last 3 minutes. There will be a 20 minute washout with saline followed by infusion of BQ-123 at a dose of 10 nmol/min for 90 min.<br>|
| PNPLA3 Sub-study<br>Study population will be split into two groups defined by the allele of PNPLA3 the participant possesses: Participants Homozygous for the G-allele of PNPLA3, are assigned to the 'case' group. Participants that are Homozygous for the C-allele of PNPLA3 will be assigned to the 'control' group. 60 participants will be recruited to each group, 120 in total. | Other: Eligibility and lifestyle restrictions check.<br>* Eligibility will be assessed at each study visit and participants will be informed in advance of their visit.<br>Other: Medical history, demographic and lifestyle factors<br>* Medical History, participants demographics and lifestyle factors will be assessed by the participant completion of the medical history and ethnicity questionnaires.<br>Other: Anthropometric measurements: height, weight, and body fat<br>* Height measured by stadiometer. Weight and body fat measured by Tanita scale bioelectrical impedance analysis.<br>Other: Blood pressure and heart rate<br>* Parameters will be measured using a validated, automated device while seated and again after 3-5 min standing. All measurements will be done in triplicate.<br>Other: Blood biochemistry (PNPLA3)<br>* 25ml blood will be taken from the participant in a glucose fasting state. Following a high carbohydrate meal, a second 25ml blood sample will be taken. Clinical Biochemistry tests and detailed lipid analysis will be performed.<br>Other: Baseline Venepuncture (PNPLA3, Visit 1)<br>* Up to 25ml Blood will be taken.<br>Other: Deuterium water - Loading dose 1 (PNPLA3, Visit 1)<br>* Loading dose of 3 g/kg body water.<br>Other: Deuterium water - Loading dose 2 (PNPLA3, Visit 1)<br>* Loading dose of 3 g/kg body water.<br>Other: Energy-balanced dinner (PNPLA3, Visit 1)<br>* Provided following baseline bloods and loading dose 1.<br>Other: Fasting venepuncture (PNPLA3, Visit 2)<br>* Up to 25ml Blood will be taken.<br>Other: Deuterium water - Maintenance dose (PNPLA3, Visit 2)<br>* every hour, maintenance dose 0.04 g/kg body water<br>Other: Consumption of high carbohydrate meal (PNPLA3, Visit 2)<br>* Provided following fasting blood sample.<br>Other: Postprandial Venepuncture (PNPLA3, Visit 2)<br>* Up to 25ml Blood will be taken.<br>|
| PROCR Sub-study<br>Study population will be split into two groups defined by the allele of PROCR the participant possesses: Participants Homozygous for the G-allele of PROCR, are assigned to the 'case' group. Participants that are Homozygous for the A-allele of PROCR will be assigned to the 'control' group. 30 participants will be recruited to each group, 60 in total. | Other: Eligibility and lifestyle restrictions check.<br>* Eligibility will be assessed at each study visit and participants will be informed in advance of their visit.<br>Other: Medical history, demographic and lifestyle factors<br>* Medical History, participants demographics and lifestyle factors will be assessed by the participant completion of the medical history and ethnicity questionnaires.<br>Other: Anthropometric measurements: height, weight, and body fat<br>* Height measured by stadiometer. Weight and body fat measured by Tanita scale bioelectrical impedance analysis.<br>Other: Blood pressure and heart rate<br>* Parameters will be measured using a validated, automated device while seated and again after 3-5 min standing. All measurements will be done in triplicate.<br>Other: Blood EPCR function (PROCR)<br>* Blood sample taken will be analysed via ELISA and FACS.<br>Other: Blood platelet coagulation and function (PROCR)<br>* Blood sample taken will be analysed using a Platelet coagulation and function assay.<br>Other: Blood endothelial permeability (PROCR)<br>* Blood sample will be analysed in vitro using a permeability assay kit.<br>Other: Blood leukocyte-endothelium adhesion (PROCR)<br>* Blood sample will be analysed in vitro using a leukocyte-endothelium adhesion assay.<br>Other: Venepuncture (PROCR)<br>* Up to 50ml Blood will be taken.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Forearm Blood Flow (Arterial contractility) | Arm 1 specific measurement to be measured using venous occlusion plethysmography. Outcome measure will compare results between case vs control groups. | 2 years | 
| Blood Biochemistry (Lipoprotein composition/dynamics) | Arm 2 specific measurement collectively comparing the lipid dynamic results between case vs control groups. | 2 years | 
| EPCR levels/shedding | Arm 3 specific measurement comparing results between case vs control groups. | 2 years | 
| Platelet aggregation/function | Arm 3 specific measurement to be measured by platelet coagulation function assay comparing results between case vs control groups. | 2 years | 
| Endothelial permeability | Arm 3 specific measurement to be measured by an endothelial permeability assay comparing results between case vs control groups. | 2 years | 
| Leukocyte-endothelium adhesion | Arm 3 specific measurement to be measured by a leukocyte-endothelium adhesion assay comparing results between case vs control groups. | 2 years | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Blood pressure | All study arms comparing results between case vs control groups. | 2 years | 
| Heart rate | All study arms comparing results between case vs control groups. | 2 years | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Healthy Volunteers, Genetic Risk Markers for Cardiovascular Disease, Non-Alcoholic Fatty Liver Disease, Coronary Disease, Hypertension, Venous Thromboses, Venous Thromboembolism, Coronary Heart Disease, Coronary Artery Disease