Genomics, Environmental Factors and Social Determinants of Cardiovascular Disease in African-Americans Study(GENE-FORECAST): Sodium Intervention Trial (SIT)/GENE-FORECAST SIT
Phase: NA
Conditions: Cardiovascular Disease
Sponsor: National Heart, Lung, and Blood Institute (NHLBI)

 
 Protocol with Statistical Analysis Plan Cover Page:   
 
Official Title: 
Genomics, Environmental Factors and Social Determinants of Cardiovascular 
Disease in African-Americans Study (GENE-FORECAST): Sodium Intervention 
Trial (SIT)/GENE-FORECAST SIT 
NCT number: 
 
NCT04717336 
Document Type:  
Protocol with Statistical Analysis Plan (SAP)  
Document Date: 
29JUL2024 
 

GENE-FORECAST SIT 
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Abbreviated Title: GENE-FORECAST SIT 
NIH IRB #: 000070  
Version Date: 29JUL2024 
Title: Genomics, Environmental Factors and Social Determinants of Cardiovascular Disease in 
African-Americans Study (GENE-FORECAST): Sodium Intervention Trial (SIT)/GENE-
FORECAST SIT 
 
NIH Principal Investigator: Georg Aue, MD, PhD, MBA (E) 
Office of the Clinical Director 
National Heart, Lung, and Blood Institute 
Building 10-CRC, Rm 6-3142 
9000 Rockville Pike 
Bethesda, MD 20892 
Phone: 301-451-7141 
E-mail: aueg@nhlbi.nih.gov 
 
Investigational Agents: 
Drug Name: 
Sodium Chloride 
IND Number: 
Not applicable 
Sponsor: 
Not applicable 
Manufacturer: 
Pine Pharmaceuticals 
 
 
 

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TABLE OF CONTENTS  
TABLE OF CONTENTS ................................................................................................................ 2 
STATEMENT OF COMPLIANCE ................................................................................................ 6 
1 
PROTOCOL SUMMARY ...................................................................................................... 7 
1.1 
Synopsis ........................................................................................................................... 7 
1.2 
Schema ............................................................................................................................. 8 
1.3 
Schedule of Activities (SOA) ........................................................................................... 8 
2 
INTRODUCTION ................................................................................................................ 10 
2.1 
Study Rationale .............................................................................................................. 10 
2.2 
Background .................................................................................................................... 10 
2.3 
Risk/Benefit Assessment ................................................................................................ 11 
2.3.1 
Known Potential Risks ............................................................................................ 11 
2.3.2 
Known Potential Benefits ....................................................................................... 12 
2.3.3 
Assessment of Potential Risks and Benefits ........................................................... 12 
3 
OBJECTIVES AND ENDPOINTS ...................................................................................... 13 
4 
STUDY DESIGN.................................................................................................................. 14 
4.1 
Overall Design ................................................................................................................ 14 
4.2 
Scientific Rationale for Study Design ............................................................................ 15 
4.3 
Justification for Dose ..................................................................................................... 16 
5 
STUDY POPULATION ....................................................................................................... 16 
5.1 
Inclusion Criteria ............................................................................................................ 16 
5.2 
Exclusion Criteria ........................................................................................................... 16 
5.3 
Inclusion of Vulnerable Participants .............................................................................. 17 
5.4 
Lifestyle Considerations ................................................................................................. 17 
5.5 
Screen Failures ............................................................................................................... 17 
5.6 
Strategies for Recruitment and Retention ...................................................................... 17 
5.6.1 
Costs ........................................................................................................................ 18 
5.6.2 
Compensation ......................................................................................................... 18 
6 
STUDY INTERVENTION ................................................................................................... 18 
6.1 
Study Interventions(s) Administration ........................................................................... 18 
6.1.1 
Study Intervention Description ............................................................................... 18 
6.1.2 
Dosing and Administration ..................................................................................... 18 
6.2 
Preparation/Handling/Storage/Accountability ............................................................... 18 
6.2.1 
Acquisition and Accountability .............................................................................. 18 
6.2.2 
Formulation, Appearance, Packaging, and Labeling .............................................. 19 
6.2.3 
Product Storage and Stability.................................................................................. 19 
6.2.4 
Preparation .............................................................................................................. 19 
6.3 
Measures to Minimize Bias: Randomization and Blinding ........................................... 19 
6.4 
Study Intervention Compliance ...................................................................................... 19 
6.5 
Concomitant Therapy ..................................................................................................... 19 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL ..................................................................................... 20 
7.1 
Discontinuation of Study Intervention ........................................................................... 20 
7.2 
Participant Discontinuation/Withdrawal from the Study ............................................... 20 
7.3 
Lost to Follow-up ........................................................................................................... 20 
8 
STUDY ASSESSMENTS AND PROCEDURES ................................................................ 21 

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8.1 
Screening Procedures ..................................................................................................... 21 
8.2 
Efficacy Assessments ..................................................................................................... 21 
8.2.1 
Clinical Evaluations ................................................................................................ 23 
8.2.2 
Biospecimen Evaluations ........................................................................................ 23 
8.2.3 
Correlative Studies for Research/Pharmacokinetic Studies .................................... 27 
8.2.4 
Samples for Genetic/Genomic Analysis ................................................................. 27 
8.3 
Safety and Other Assessments ....................................................................................... 28 
8.4 
Adverse Events and Serious Adverse Events ................................................................. 32 
8.4.1 
Definition of Adverse Event ................................................................................... 32 
8.4.2 
Definition of Serious Adverse Events (SAE) ......................................................... 32 
8.4.3 
Classification of an Adverse Event ......................................................................... 32 
8.4.4 
Time Period and Frequency for Event Assessment and Follow-Up ....................... 34 
8.4.5 
Adverse Event Reporting ........................................................................................ 35 
8.4.6 
Serious Adverse Event Reporting ........................................................................... 35 
8.4.7 
Events of Special Interest........................................................................................ 35 
8.4.8 
Reporting of Pregnancy .......................................................................................... 35 
8.5 
Unanticipated Problems ................................................................................................. 35 
8.5.1 
Definition of Unanticipated Problems (UP) ........................................................... 35 
8.5.2 
Unanticipated Problem Reporting ........................................................................... 36 
8.5.3 
NIH Intramural IRB Reporting of IND Safety Reports .......................................... 36 
9 
STATISTICAL CONSIDERATIONS.................................................................................. 36 
9.1 
Statistical Hypothesis ..................................................................................................... 36 
9.2 
Sample Size Determination ............................................................................................ 36 
9.3 
Populations for Analyses ................................................................................................ 38 
9.3.1 
Evaluable for toxicity .............................................................................................. 38 
9.3.2 
Evaluable for objective response ............................................................................ 38 
9.3.3 
Evaluable Non-Target Disease Response ............................................................... 38 
9.4 
Statistical Analyses ........................................................................................................ 38 
9.4.1 
General Approach ................................................................................................... 38 
9.4.2 
Analysis of the Primary Endpoints ......................................................................... 38 
9.4.3 
Analysis of the Secondary Endpoint(s) ................................................................... 40 
9.4.4 
Safety Analyses ....................................................................................................... 41 
9.4.5 
Baseline Descriptive Statistics ................................................................................ 41 
9.4.6 
Planned Interim Analyses ....................................................................................... 41 
9.4.7 
Sub-Group Analyses ............................................................................................... 41 
9.4.8 
Tabulation of individual Participant Data ............................................................... 41 
9.4.9 
Exploratory Analyses .............................................................................................. 41 
10 REGULATORY AND OPERATIONAL CONSIDERATIONS ......................................... 41 
10.1 
Informed Consent Process .......................................................................................... 41 
10.1.1 Consent/Assent Procedures and Documentation .................................................... 41 
10.1.2 Consent for minors when they reach the age of majority ....................................... 41 
10.1.3 Telephone consent ................................................... Error! Bookmark not defined. 
10.1.4 Telephone assent ..................................................................................................... 42 
10.1.5 Participation of Subjects who are/become Decisionally Impaired ......................... 42 
10.2 
Study Discontinuation and Closure ............................................................................ 42 
10.3 
Confidentiality and Privacy ........................................................................................ 42 

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10.4 
Future use of Stored Specimens and Data .................................................................. 43 
10.5 
Safety Oversight ......................................................................................................... 43 
10.6 
Clinical Monitoring .................................................................................................... 43 
10.7 
Quality Assurance and Quality Control...................................................................... 43 
10.8 
Data Handling and Record Keeping ........................................................................... 43 
10.8.1 Data Collection and Management Responsibilities ................................................ 43 
10.8.2 Study Records Retention ......................................................................................... 44 
10.9 
Protocol Deviations .................................................................................................... 44 
10.9.1 NIH Definition of Protocol Deviation .................................................................... 44 
10.10 
Publication and Data Sharing Policy .......................................................................... 44 
10.10.1 Human Data Sharing Plan ....................................................................................... 44 
10.10.2 Genomic Data Sharing Plan .................................................................................... 44 
10.11 
Collaborative Agreements .......................................................................................... 45 
10.11.1 Agreement Type ...................................................................................................... 45 
10.12 
Conflict of Interest Policy........................................................................................... 45 
11 ABBREVIATIONS .............................................................................................................. 45 
12 REFERENCES ..................................................................................................................... 46 
 
 
 

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STATEMENT OF COMPLIANCE 
The trial will be carried out in accordance with International Conference on Harmonisation Good 
Clinical Practice (ICH GCP) and the following:  
• United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 
CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812)  
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are 
responsible for the conduct, management, or oversight of NIH-funded clinical trials have 
completed Human Subjects Protection and ICH GCP Training. 
The protocol, informed consent form(s), recruitment materials, and all participant materials will 
be submitted to the Institutional Review Board (IRB) for review and approval.  Approval of both 
the protocol and the consent form must be obtained before any participant is enrolled.  Any 
amendment to the protocol will require review and approval by the IRB before the changes are 
implemented to the study.  In addition, all changes to the consent form will be IRB-approved; a 
determination will be made regarding whether a new consent needs to be obtained from 
participants who provided consent, using a previously approved consent form. 
 
 

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1 
PROTOCOL SUMMARY 
1.1 
SYNOPSIS 
Title: 
Genomics, Environmental Factors and Social Determinants of 
Cardiovascular Disease in African Americans Study (GENE-
FORECAST): Sodium Intervention Trial (SIT)/GENE-
FORECAST SIT 
Study Description: 
The objective of this study is to implement a sodium intervention 
investigation to assess the effect of increased dietary sodium intake on  
changes in blood pressure, vascular function, microbiome, whole 
blood epigenome, whole blood and urine transcriptome as outcome 
measures.  The study design will include a double-blind, cross-over 
treatment/placebo trial among 40 former African Americans GENE-
FORECAST participants with normal blood pressure and will last 
7weeks. It is hypothesized that exposure to increased dietary sodium 
will affect blood pressure, whole blood epigenome, whole blood and 
urine transcriptome, vascular function, microbiome and blood 
pressure. 
Objectives: 
 
Primary Objective: 
Define the effect of increased dietary sodium intake on microbiome 
and vascular function. 
Secondary Objective: 
1) Determine if increased sodium affects blood pressure. 
2) Determine if increased sodium affects whole blood epigenome. 
3) Determine if increased sodium affects whole blood and urine 
transcriptome. 
 
 
 
 
Endpoints: 
Primary: 
Vascular function 
Microbiome 
Secondary: 
Blood pressure 
Whole blood epigenome 
Whole blood and urine transcriptome 
Study Population: 
Study population will include 40 former African American 
normotensive men and women ages 21-65 residing in the DC, 
Maryland, Virginia regions who previously participated in the 
GENE-FORECAST protocol. 
Phase: 
<0. 
Description of 
Sites/Facilities 
Enrolling Participants: 
All protocol activities will take place at the NIH Clinical Center. 
 
Description of Study 
Intervention: 
Participants will be enrolled in a double-blind cross-over sodium 
chloride/placebo intervention trial.  Intervention product will be taken 
in capsule form (1 gram per capsule) 3 times per day. 
 

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Study Duration: 
Participants will be enrolled concurrently along with ongoing GENE-
FORECAST protocol.  It is anticipated that it will take 36 months 
from enrollment to completion of data analyses. 
 
Participant Duration: 
It will take 7 weeks for each participant to complete 4 visits to the 
Clinical Center during the trial. 
 
 
 
 
1.2 
SCHEMA 
 
 
 
Figure 1: Randomized double-blind crossover treatment/control flowchart 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Participants will be randomized to a placebo or salt treatment arm for two weeks (Period 1), 
followed by a wash-out period for three-weeks, ending with a cross-over to the alternative 
treatment arm for two weeks (Period 2).  The total length of participation is 7 weeks (Figure 1). 
Participants will be randomly assigned by a computer-generated code. 
 
1.3 
 SCHEDULE OF ACTIVITIES (SOA) 
It is estimated that participants will spend approximately 5 hours at the Clinical Center during 
baseline and approximately 3 hours during each of the subsequent visit. Obtaining and explaining 
the informed consent will take about 20-30 minutes.  Administering the baseline questionnaires 
will take about 1.45 hours and 75 minutes for each subsequent visit. The clinic visit assessment, 
plus vascular function test, will take about 1.30 hours. 
 
Table 1: Estimated length of time for study procedures 
GENE-FORECAST 
recall Participants  
Sodium 
Placebo 
Washout 
period 
(3-week) 
Placebo  
Sodium 
Randomization  
Period 1 
Period 2 
t0 
Baseline 
        t1 
(week2) 
     t3 
(week7) 
        t1 
(week5) 

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 NIH Clinic Visit 
Estimated 
time 4 hours 
Total 
Participants  
Duration of 
Study  
a) Informed consent 
(baseline only) 
20-30 
minutes 
40 
7 wks 
b) Baseline 
Questionnaires  
1.75 hours 
40 
Baseline 
 
c) Phlebotomy 
30 minutes 
40 
7 wks 
d) Physical 
examination 
20 minutes 
40 
7 wks 
e) 24 diet recall 
30 minutes 
40 
7wks 
f) Microbiome 
15 minutes 
40 
7 wks 
g) Psychophysical 
Taste Task: Sucrose 
and Salt Detection 
Thresholds 
30 minutes 
40 
7 wks 
h Psychophysical 
Taste Task: Sucrose 
and Salt Preference 
20 minutes 
40 
7 wks 
i) Vascular Function  1.0 hrs. 
40 
7 wks 
 
 
 
 
 
Visit to the NIH Clinical Center: 
Once potential participant arrives at the Clinical Center, they will report to admissions and be 
greeted by the GENE-FORECAST-SIT staff who will escort him/her to a private room to 
administer informed consent and baseline questionnaires.  GENE-FORECAST-SIT staff 
(Research Nurse/Physician Assistant) will subsequently escort potential participant to 
phlebotomy followed by the collection of microbiome specimens from the GENE-FORECAST-
SIT staff. A physical examination by the Physician Assistant will be performed on the participant, 
they will then have a standard physical exam, including blood pressure measurement and vitals 
concluding with administration of vascular function tests using Endo-PAT and SphygmoCor by 
the Physician Assistant.  Two blood pressure measurements will be obtained from the participant 
by the Clinical Center Nursing Staff and an average recorded.  The blood pressure will be 
measured using a Dinamap and will be obtained from the left arm after 5 minutes of rest with the 
participant in a sitting position with legs uncrossed.  A 24-hour diet recall will be administered 
by staff from the Clinical Center Nutrition Department.  Taste assessment will be administered 
by an Associate Investigator on the study. 
 
Table 2: Specific activities by visit 
Activity 
Visit 1 
(baseline) 
Visit 2 
(after 2-
week 
treatment) 
Visit 3 
(after 3-
week 
washout) 
Visit 4 
(after second 
2-week 
treatment) 
24-Hour nutrient recall  
x 
x 
x 
x 

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Psychophysical Taste 
Task: Sucrose and Salt 
Detection Thresholds 
x 
x 
x 
x 
Psychophysical Taste 
Task: Sucrose and Salt 
Preference 
x 
x 
x 
x 
History and physical 
exam with medication 
history  
x 
x 
x 
x 
Baeke’s physical activity 
questionnaire 
x 
 
 
 
Pittsburg sleep 
questionnaire 
x 
 
 
 
Stool/skin/mouth swabs 
for microbiome analysis 
x 
x 
x 
x 
Urine sample 
x 
x 
x 
x 
Blood sample 
x 
x 
x 
x 
Vascular function 
x 
x 
x 
x 
24-hour blood pressure 
monitor given to 
participant 
x 
 
x 
 
24-hour blood pressure 
monitor returned by 
participant 
 
x 
 
x 
 
2 
INTRODUCTION 
2.1 
STUDY RATIONALE 
Little is known about the relationship between dietary sodium and gut microbiome ecosystem in 
humans; or how it might be altered by incremental changes in sodium intake. The GENE-
FORECAST-SIT study will be the first to test the hypothesis that changes in dietary sodium 
intake are sufficient to influence the composition of the gut microbiome in association with 
sodium-induced changes in vascular function, epigenome, transcriptome and blood pressure in 
African Americans.   
 
2.2 
BACKGROUND  
There is an abundant body of evidence from longitudinal population studies that have established 
a striking relationship between certain dietary patterns and the risk of cardiovascular disease. 
Diets rich in fruits, vegetables, whole grains, fish, nuts and fats such as olive oil have been 
associated with reduced risk of cardiovascular disease and adverse outcomes 1. Similarly, 
controlled dietary intervention trials with the DASH diet have demonstrated that a low-sodium 
diet that is rich in fruits, vegetables, whole grains and low-fat dairy products is an effective 
approach to reducing blood pressure in hypertension patients 2. It is noteworthy that African 
Americans appeared to have the greatest benefit from this dietary intervention. However, the 
molecular mechanisms that mediate the effect of diet on blood pressure and the development of 
cardiovascular disease remain to be further elucidated.  

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It is well established that increased dietary sodium can predispose to an increase in blood 
pressure; particularly in ‘salt-sensitive’ individuals 3. In most individuals there are a powerful 
series of homeostatic systems (renal, neurohumoral, vascular) that function to maintain the 
normal set-point blood pressure across a very broad range of dietary sodium intakes. Yet, a sub-
set of ‘salt-sensitive’ individuals exhibit changes in vascular function and modest elevations in 
blood pressure in response to relatively modest changes in dietary sodium intake 4.  It is 
noteworthy that several studies have suggested an apparent increased prevalence of ‘salt-
sensitive’ blood pressure among African Americans 5.   
Commonly used animal models of hypertension often employ high dietary sodium intake as a 
means of inducing sustained hypertension. The Dahl Salt-Sensitive Rat is a genetic model of 
hypertension that becomes hypertensive upon the administration of a high-sodium diet; whereas 
the genetically related Dahl Salt-Resistant Rat remains normotensive despite the high dietary 
sodium intake. Although there are multiple factors that appear to mediate this genetic 
predisposition to salt-sensitive hypertension, an intriguing recent report has noted a striking 
difference between the gut microbiome in Dahl salt-sensitive vs salt-resistant animals 6. In 
addition, recent studies have demonstrated a relationship between increased dietary sodium 
intake and modulation of the immune system in animal models 7,8. The proposed project seeks to 
further extend the working hypothesis that the effect of dietary sodium on vascular function and 
blood pressure may be modulated by the gut microbiome and its influence on the immune 
system.  The selected dosage of sodium chloride is based on prior research.8-10 
It is becoming increasingly clear that human biology and pathobiology is influenced in part by 
the trillions of microbes that co-exist within our bodies in a mutually beneficial manner. The 
microbe ecosystems that live within the human gastrointestinal tract are shaped by our dietary 
intake that supports the predominance of certain flora relative to others. Conversely, the microbe 
composition of each individual’s gut can influence the digestion and generation of certain 
metabolites from certain foods.  The various patterns of gut microbiome composition have been 
implicated in the regulation of the immune system as well as the pathogenesis of obesity, 
autoimmune disorders and cardiovascular disease 11-13. For example, Stan Hazen’s team has 
shown striking differences in the human microbiome between vegetarians and those who are 
frequent ‘meat-eaters’. This dietary difference is associated with differential generation of a 
microbe metabolite, trimethylamine-oxidase (TMAO).  Increased generation of the microbe 
metabolite, TMAO, is associated with increased risk of cardiovascular disease.  Moreover, it has 
been shown that pharmacologic inhibition of the generation of this gut microbe metabolite 
attenuates atherogenesis in animal models. Taken together, these clinical and animal model 
studies suggest a causal link between dietary patterns, the gut microbiome and cardiovascular 
disease.  
 
2.3 
RISK/BENEFIT ASSESSMENT 
2.3.1 Known Potential Risks 
Risk of participating in the study is minimal given the benefit to the scientific community 
derived from findings related to the effect of salt on vascular, blood pressure and microbiome 
among African Americans. The salt capsules are generally safe and well tolerated but side effects 
can occur -including discomfort while swallowing, increased thirst, indigestion, nausea, 
vomiting, headaches, mild elevations in blood pressure, mild changes in blood chemicals 
(electrolytes), lightheadedness, tiredness, mild swelling of arms/legs, and/or diarrhea.  Although 
it would be very rare to have an allergic reaction to the capsules, any time a new medication is 

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introduced there is a small chance of an allergic reaction which would most commonly manifest 
itself as a rash.  
 
See Section 2.3.3 for detailed description of risks 
 
.  
 
 
2.3.2 Known Potential Benefits 
There is no direct benefit to the participant for participating in the study. However, 
findings generated from the study will add to the scientific body of knowledge regarding 
the effect of salt among African Americans who have a disproportionately higher 
prevalence of cardiovascular disease 
2.3.3 Assessment of Potential Risks and Benefits 
The  risks are very minor and include: 
 
Vascular function testing and 24-hour blood pressure monitor: Subject  may experience brief 
discomfort, pain or tingling sensations when the blood pressure cuff is inflated to reduce 
blood flow to the hand for a few minutes during the vascular function tests and/or 24-hour 
blood pressure monitor. 
 
Phlebotomy: Transient discomfort and minor bruising may occur when blood is drawn. 
 
Microbiome collection::  Collection will comprise of skin, saliva and stool samples utilizaing 
Norgen kits.  These tests carry very minor risks, with the transient mild skin irration at the sites 
where your skin is swabbed although this is very unlikely.  
 
Salt capsules: Sodium chloride is approved by the Food and Drug Administration (FDA) for a 
variety of clinical uses and is available over the counter (OTC). The capsules given as part of 
this study contain sodium chloride (salt) in powder form or placebo. The amount of salt added 
to subject’s usual daily diet as part of this study is equivalent to about half a teaspoon of salt 
or a packet of Ramen chicken noodle soup. The salt capsules are generally safe and well 
tolerated but side effects can occur including discomfort while swallowing, increased thirst, 
indigestion, nausea, vomiting, headaches, mild elevations in blood pressure, mild changes in 
blood chemicals (electrolytes), lightheadedness, tiredness, mild swelling of arms/legs, and/or 
diarrhea. Although it would be very rare to have an allergic reaction to the capsules in this 
study, any time a new medication is taken there is a small chance (less than 1 person out of 
1000 treated) that you could have an allergic reaction which would most commonly manifest 
itself as a rash.  
The GENE-FORECAST-SIT staff will monitor subjects during study participation to detect any 
potential serious adverse reactions to the salt capsules. 
 
24 Hour food intake recall: Subjects may be asked a series of questions by staff from the Clinical 
Center Nutrition Department to recall any foods or beverages they may have eaten or drank over 
the past 24 hours. There may be a minor psychological discomfort of not being able to recall any 
food or beverage item questions over the past 24 hours.  

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Mouth: At each clinical center visit, saliva samples from subjects mouth will be collected by 
passive drool or spit using a sterile collection tube. Providing study samples of these specimens 
that the body normally excretes will not cause any additional pain or discomfort. 
 
Stool: Subjects will be provided with a stool specimen collection kit prior to their visit. There 
may be some physical discomfort of inability to obtain sample (e.g., constipation). Providing 
study samples of these specimens that the body normally excretes will not cause any additional 
pain or discomfort. 
 
Urine: Subjects will be asked to give a urine sample in a sterile cup provided  at the NIH Clinical 
Center to test for urine chemistries (e.g., potassium, chloride, sodium). There may be  physical 
limitations in inability to provide sample (e.g. dehydration). Providing study samples of these 
specimens that the body normally excretes will not cause any additional pain or discomfort. 
 
 Psychophysical Taste Task with sucrose and salt detection thresholds and preference: Sucrose 
and salt detection thresholds and preference will be assessed to get subject responses to 
sweetness or saltiness. There may be a discomfort of tasting solutions and repeated spitting 
action to discard in waste receptacle.  
 
Physical activity and sleep questionnaire: Subjects will be asked a series of questions related 
to their physical activity and sleep. They may experience minor psychological discomfort of 
answering personal questions regarding their physical activity and sleeping habits.  
 
 
GENE-FORECAST-SIT staff will closely monitor the study participant in order to detect any 
potential severe adverse reactions to study medication. Specifically, GENE-FORECAST-SIT staff 
will contact individual study participant +/-2 days a week to anticipate missed calls and 
occasional inability to reach participant during the treatment phase of the trial to inquire about any 
severe adverse reactions.  Participant will also be given the direct mobile telephone number of 
GENE-FORECAST-SIT staff to discuss any adverse reactions.  
 
The risk of minimal potential risks outweighs the benefit of scientific knowledge gained from the 
effects of sodium on cardiovascular health, taste genotype and microbiome in African 
Americans.  
 
3 
OBJECTIVES AND ENDPOINTS 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Primary 
 
 
What effect does increase dietary 
sodium intake have on vascular 
function and microbiome? 
Vascular function 
Microbiome 
 
A sub-set of ‘salt-sensitive’ 
individuals exhibit changes 
in vascular function and 
modest elevations in blood 
pressure in response to 
relatively modest changes in 

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dietary sodium intake. 
Studies have suggested an 
apparent increased 
prevalence of ‘salt-sensitive’ 
blood pressure among 
African Americans 
The various patterns of gut 
microbiome composition 
have been implicated in the 
regulation of the immune 
system as well as the 
pathogenesis of obesity, 
autoimmune disorders and 
cardiovascular disease. 
Secondary 
 
 
What effect does increase dietary 
sodium have on blood pressure? 
 
What effect does increase dietary 
sodium have on whole blood 
epigenome? 
 
 
What effect does increase dietary 
sodium have on whole blood and urine 
transcriptome 
 
Blood pressure 
 
DNA methylation 
 
RNAseq 
 
 
It is well established that 
increased dietary sodium 
can predispose to an 
increase in blood pressure. 
DNA methylation is a well-
established measure of the 
epigenome. 
RNAseq is a well-
established measure of 
genetic transcriptome. 
 
4 
STUDY DESIGN 
4.1 
OVERALL DESIGN 
This study involves a call-back and recruitment of 40 normotensive participants from the GENE-
FORECAST protocol.  Those in the upper tertile of vascular stiffness (as assessed by pulse wave 
velocity measures) will be oversampled.  Participants will be randomized to a placebo or salt 
treatment arm for two weeks (Period 1), followed by a wash-out period for three-weeks, ending 
with a cross-over to the alternative treatment arm for two weeks (Period 2).  The total length of 
participation is 7 weeks (Figure 1).  
 
Study Site: 
All study activities will take place at the NIH Clinical Center. The NIH Clinical Center Pharmacy 
will dispense the treatment and placebo capsules.  
 
Statement of hypotheses: 
* Based on population history, it is postulated that the genomic architecture of individuals of 
African ancestry has been shaped by environmental pressures and natural selection in the context 
of high ambient temperatures in salt-poor conditions that may predispose to an increased 
sensitivity and avidity for sodium retention in African Americans. 

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* Increased dietary sodium intake is associated with vascular dysfunction and increased blood 
pressure in certain sub-sets of normotensive African Americans; and this effect is augmented in 
individuals with baseline evidence of vascular dysfunction. 
* Increased dietary sodium intake is associated with changes in the whole blood epigenome as 
assessed by DNA methylation. 
* Increased dietary sodium intake is associated with changes in the whole blood and urine 
transcriptome as assessed by RNA-seq. 
* Increased dietary sodium intake is associated with changes in the skin, oral and gut microbiome. 
* Increased dietary sodium take is associated with whole blood epigenome/transcriptome 
molecular signature that predisposes to a chronic “pro-inflammatory state.” 
 
Type/design of trial: 
A randomized, double-blind, placebo-controlled, cross-over design will be implemented. 
 
Method to minimize bias: 
Participants will be randomly assigned by a computer-generated code.  
 
Number of study arms and intervention duration: 
Participants will be randomized to a placebo or salt treatment arm for two weeks (Period 1), 
followed by a wash-out period for three-weeks, ending with a cross-over to the alternative 
treatment arm for two weeks (Period 2).  The total length of participation is 7 weeks (Figure 1).  
 
Name of study intervention: 
Sodium Chloride 
 
4.2 
SCIENTIFIC RATIONALE FOR STUDY DESIGN 
The study is testing the hypothesis that a modest increase in dietary sodium intake is sufficient to 
change the gut microbiome and trigger alterations in the blood transcriptome that may mediate 
the resultant in the well described salt-sensitive changes in vascular function, epigenome, 
transcriptome and blood pressure observed in African Americans. The study design as a 
randomized, double-blind, placebo-control cross-over design enables each participant to serve as 
her/his own control in response to the dietary salt intervention. The cross-over design allows for 
an intervening ‘wash-out’ period to ensure that the participant’s diet and sodium homeostasis 
returns to baseline. The design’s use of each participant as her/his own control reduces the 
impact of inter-individual variability in the basal dietary habits and microbiome; and optimizes 
statistical power to detect differences in baseline blood transcriptome gene expression profiles 
and gut microbiome in response to the dietary intervention.  Our previous studies of the human 
transcriptome indicate that the current design is very robust to detect changes in gene expression 
by DNA methylation, RNA-seq and vascular function. The randomization ensures that the 
sequence order of the intervention (placebo vs salt tablets) is due to chance and avoids a 
systematic ‘contamination’ or carryover effect of the active treatment. The double-blind 
administration of tasteless capsules ensures that neither the participant or research staff are aware 
that there could be an increased ingestion of dietary salt. The dose of sodium chloride in the 
capsule has previously been shown to affect biomarkers sensitive to salt balance such as 
measures of the renin-angiotensin-aldosterone system and blood pressure. The daily dose of 
sodium chloride tablets given in the protocol falls well within the wide variance of ‘normal’, 

GENE-FORECAST SIT 
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16 
 
safe, intake in various cultures around the world. The increment in salt intake beyond the 
participant’s usual diet is equivalent to eating 1-2 bowls of soup and is a dose commonly 
administered to treat orthostatic hypotension in patients who suffer from this disorder. It should 
be noted that the protocol focuses on normotensive volunteers and excludes patients with 
hypertension or heart failure for whom a transient increase in dietary sodium could pose a risk. 
We will assess, gut, skin and saliva microbiome. The oral cavity is an excellent source of easy-
to-access biological material for studies of oral microbiome 26. This is due to the quick, non-
invasive and low-cost collection 27. The most used source of oral samples is saliva (collected by 
passive drool or spit). The collection of the skin microbiome samples is commonly performed 
using the swabbing method 28.  The sampling for skin microbiome was guided by the aim to 
consider two of the three skin microenvironments types 29: Sebaceous and moist 
microenvironments. For the first, the Retroarticular Crease was chosen and for the latter, the 
Antecubital Fossa was selected. These sites provide also the advantage of being relatively less 
exposed than dry sites such as the forearm and hence less likely to have the external environment 
microbiome over-represented. 
The oral samples are not collected to investigate gut microbiome but rather to evaluate changes 
in oral microbiome 26 with respect to changes in sodium intake. The aim is to find out if changes 
in sodium intake are correlated with microbiome changes in the oral cavity, in the gut and on the 
skin. Each site is considered separately first and then patterns of changes between sites are 
considered. 
 
4.3 
JUSTIFICATION FOR DOSE 
Sodium chloride (1gm/capsule) and placebo will be dispensed by the NIH Pharmacy. Participant 
would be instructed to take 3 pills per day for a total of 3 grams. The dosing of three grams of 
sodium chloride per day was selected because it is sufficiently higher than normal dietary intake 
necessary to affect the outcome parameters and doesn’t pose any health dangers.  This dosage is 
the standard amount used in such protocol.8-10 
 
5 
STUDY POPULATION  
5.1 
INCLUSION CRITERIA 
African American men and women who are former GENE-FORECAST participants between 21 
and 65 years of age. I This criterion is inclusive of self-identified AA of both Hispanic, Latino 
and non-Hispanic, Latino ethnicities. Normotensive participants with systolic blood pressure 
(SBP) <140 mm Hg and diastolic blood pressure (DBP) <90 mm Hg and the absence of a history 
of prior diagnosis of hypertension. 
• 
Willingness and ability to participate in study procedures. 
 
5.2 
EXCLUSION CRITERIA 
• 
Individuals who are pregnant or breast-feeding.  
• 
Individuals with high blood pressure or a history of hypertension.  
• 
Individuals with a history of myocardial infarction, stroke, heart failure, diabetes, chronic 
liver or kidney diseases. 
• 
Individuals who are taking antihypertensive, antidepressants, antidiabetic and antibiotic 
medications. 
• 
Individuals currently participating in another NIH protocol. 
• 
Individuals unable to provide informed consent. 

GENE-FORECAST SIT 
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17 
 
 
5.3 
INCLUSION OF VULNERABLE PARTICIPANTS 
The study will not involve vulnerable groups other than NIH employees who volunteer for 
participation in study. NIH employees will be provided requisite information according to 
OHSRP Policy 404 prior to agreeing to participate in study. 
 
5.4 
LIFESTYLE CONSIDERATIONS 
During the study, participants are asked to: 
• 
Reframe from taking antibiotics. Taking such mediation will result in early withdrawal. 
 
5.5 
SCREEN FAILURES 
All Participants will go through screening activites after the consent has been signed. Females will 
undergo a pregnancy test. Participants who consent to participate in the trial and subsequently 
have a positive pregnancy test prior to randomization will be considered a screen failure and not 
enrolled in the study.  Participants who consent to participate but present with systolic blood 
pressure (SBP) >140 mm Hg and diastolic blood pressure (DBP) >90 mm Hg at physical 
examination will also be considered a screen failure. 
 
Screen failures are defined as participants who consent to participate in the study but are not 
subsequently assigned to the study intervention or enter the study.  A minimal set of screen failure 
information is required to ensure transparent reporting of screen failure participants to meet the 
Consolidated Standards of Reporting Trials (CONSORT), publishing requirements and respond to 
queries from regulatory authorities.  Minimum information will include demography, screen 
failure details, eligibility criteria, and any serious adverse event (SAE). 
 
5.6 
STRATEGIES FOR RECRUITMENT AND RETENTION 
• Target study sample: 
Participants will be recruited from those who previously participated in the GENE-FORECAST 
protocol. A total of 40 normotensive African American men and 21-65 years old will comprise 
the total sample size. We anticipate screening 150 potential participants. 
 
• Anticipated accrual rate: 
40 
 
• Source of participants: 
Former GENE-FORECAST participants. 
 
• Recruitment venues: 
The  GENE-FORECAST-SIT staff will contact individuals to determine interest in participating 
in GENE-FORECAST-SIT protocol and screen for eligibility over the telephone based on self-
report information. 
 
• How potential participants will be identified: 
A listing of former normotensive GENE-FORECAST participants will be generated, including 
those in the upper tertile with vascular stiffness, by the data base manager that consented for re-
contact via the GENE-FORECAST protocol.   

GENE-FORECAST SIT 
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18 
 
•  
5.6.1 Costs 
There will be no out-of-pocket cost to participants. 
 
5.6.2 Compensation 
Participants will receive $50 per visit disbursed through the Research Volunteer System (RVS) 
for Visits 1,2, 3, and 4.  A final payment for $300 will be mailed  or sent via direct deposit to the 
participant by the RVS at the completion of the study and recovery of the study equipment (24hr 
BP monitor; Smart-Bottles).  The total possible compensation for participating in the completed 
study will be $500.  Participant will also receive a $25 voucher for lunch each visit and be 
afforded a one-way pre-paid taxi trip to the NIH Clinical Center. 
 
6 
STUDY INTERVENTION 
6.1 
STUDY INTERVENTIONS(S) ADMINISTRATION 
6.1.1 Study Intervention Description 
There are no unapproved drugs or devices being used in this study.  
Sodium Chloride is Food and Drug Administration (FDA) approved for a variety of clinical 
conditions and is included in a wide variety of commonly consumed processed foods.  
 
6.1.2 Dosing and Administration 
Sodium Chloride and Placebo: Sodium Chloride (1gm/capsule) and corresponding placebo will 
be provided by the NIH Pharmacy. Participant will be instructed to take 3 pills per day for a total 
of 3 grams. The dosing of three grams of sodium chloride per day was selected because it is 
sufficiently higher than normal dietary intake necessary to affect the outcome parameters and 
doesn’t pose any health dangers.  This dosage is the standard amount used in such protocol.8-10 
 
6.1.2.1 Dose Escalation 
There will be no dose escalation.  Participants will be instructed to take 3 assigned salt or 
placebo pills per day for a total of 3 grams. 
 
6.1.2.2 Dose Limiting Toxicity 
Not applicable 
 
6.1.2.3 Dose Modifications 
Not applicable 
 
6.1.2.4 Drug Administration 
Participant will be instructed to take 3 pills per day for a total of 3 grams. If one capsule is 
missed per day, participant should not make up the next day by taking an additional capsule. 
 
6.2 
PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY  
6.2.1 Acquisition and Accountability 
The sodium chloride and placebo capsules will be manufactured by Pine Pharmaceuticals in 
Tonawanda, New York and sent directly to the NIH Clinical Center Investigational Drug & 
Research Section Outsourcing Unit (IDOU).  The sodium chloride and placebo capsules will be 
subsequently sent to the NIH Clinical Center pharmacy who will be dispense directly to 

GENE-FORECAST SIT 
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19 
 
participants based on randomized double-blind ID list previously provided by PI.  Expired and 
unused product will be discarded by the IDOU and not sent back to the manufacturer. 
 
6.2.2 Formulation, Appearance, Packaging, and Labeling 
The sodium chloride and placebo capsules will be formulated by Pine Pharmaceuticals in 
Tonawanda, New York based on FDA guidelines. There will be no distinguishing characteristics 
between both capsules. The sodium chloride and placebo capsules will be put in a separate 
‘smart bottle’ by Clinical Center pharmacy and labelled based on randomized double-blind 
participant ID list previously provided by PI with instruction to take two times a day. The smart 
bottle technology will electronically monitor adherence to daily treatment and is designed to 
remind participant to take treatment and to alert when treatment has been missed.  Smart bottles 
will be purchased from a reputable vender with a history of collaboration in such trials. 
 
6.2.3 Product Storage and Stability 
The product will be stored in the Clinical Center pharmacy based on protocol designed to protect 
from light, temperature, and humidity.  The smart bottle will also be designed to protect from 
such elements. 
 
6.2.4 Preparation 
The Clinical Center pharmacy will dispense the sodium chloride and placebo capsules in 
separate smart bottles. Capsules do not involve thawing, diluting, mixing and 
reconstitution/preparation. 
 
6.3 
MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING 
A randomized, double-blind, placebo-controlled, cross-over design will be implemented (Figure 
1).  Participants will be randomly assigned by a computer-generated code based on study ID.  All 
study staff will be blinded to randomization throughout the trial period.  The randomized list will 
be provided to the Clinical Center pharmacy who will have the key to specific participant 
randomization. 
 
6.4 
STUDY INTERVENTION COMPLIANCE 
The sodium chloride and placebo capsules will be put in separate smart bottle by Clinical Center 
pharmacy and labelled based on randomized double-blind ID previously provided by PI 
instructing participant to take three times a day. The smart bottle technology will electronically 
monitor adherence to daily treatment and is designed to remind participant to take treatment and 
to alert when treatment has been missed.  Smart bottles will be purchased from a reputable 
vender with a history of collaboration in such trials. 
The smart bottle includes an electronic interface and database that allows clinical study staff to 
monitor, document, and calculate compliance.  We will also collect spot urine test for 
sodium/potassium at baseline and at the end of each treatment test as an additional measure of 
adherence to the dietary sodium intervention. 
 
6.5 
CONCOMITANT THERAPY 
Antibiotics are excluded from this trial.  However, any other prescription medication is defined as 
a medication that can be prescribed by a properly authorized/licensed clinician is allowable.  

GENE-FORECAST SIT 
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20 
 
Medications will be reported in the Case Report Form (CRF) as concomitant prescription 
medication, including over-the-counter medications and supplements.  
 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL  
7.1 
DISCONTINUATION OF STUDY INTERVENTION 
Participant will be discontinued from participation in study if: 
• Completion of study trial 
• If any clinical adverse event (AE), laboratory abnormality, or other medical condition or 
situation occurs such that continued participation in the study would not be in the best 
interest of the participant 
• Positive pregnancy test 
• Participant unable to receive study intervention for two consecutive days. 
Any clinically relevant finding will be reported as an AE. All study related indices will be 
recorded and maintained up to the time of discontinuation.  Clinical staff will also follow-up with 
participant to capture AE, serious adverse events (SAE), and unanticipated problems (UPs) 
 
7.2 
PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
Participants are free to withdraw from participation in the study at any time upon request, or upon 
the development of an adverse reaction due to exposure to intervention. 
 
PI may discontinue or withdraw participant from the study for the following reasons: 
• Significant study intervention non-compliance 
• If the participant meets an exclusion criterion (either newly developed or not previously 
recognized) that precludes further study participation 
• Death 
• Screen failure 
 
The reason for participant discontinuation or withdrawal from the study will be recorded on the 
‘Current Status” CRF. Subjects who sign the informed consent form and are randomized and 
receive the study intervention and subsequently withdraw or are withdrawn or discontinued from 
the study will be replaced.  Prior to removal from study, efforts will be made to have participant 
complete a safety visit approximately one week following the last dose of study product. 
 
7.3 
LOST TO FOLLOW-UP 
 
A participant will be considered lost to follow-up if he or she fails to return for the three 
subsequent scheduled visits following baseline visit and is unable to be contacted by the study 
GENE-FORECAST-SIT staff .  
The following actions will be taken if a participant fails to return to the clinic for a required study 
visit: 
• The GENE-FORECAST-SIT staff will attempt to contact the participant and reschedule 
the missed visit one week and counsel the participant on the importance of maintaining 
the assigned visit schedule and ascertain if the participant wishes to and/or should 
continue in the study. 

GENE-FORECAST SIT 
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21 
 
• Before a participant is deemed lost to follow-up, the GENE-FORECAST-SIT staff will 
make every effort to regain contact with the participant (where possible, 3 telephone calls 
and, if necessary, a certified letter to the participant’s last known mailing address or local 
equivalent methods). These contact attempts will be documented in the participant’s 
medical record or study file.  
• Should the participant continue to be unreachable, he or she will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up. 
 
8 
STUDY ASSESSMENTS AND PROCEDURES 
8.1 
SCREENING PROCEDURES 
A listing of former normotensive GENE-FORECAST participants will be generated, including 
those in the upper tertile with vascular stiffness, by the data base manager that consented for re-
contact via the ‘Call Back’ protocol.  The Research Nurse will contact individual to determine 
interest in participating in GENE-FORECAST-SIT and screen via the telephone for eligibility 
based on self-reported health information.  Participant who agree to participate in study will be 
scheduled for baseline clinical visit based on availability and subsequently screened again at the 
Clinical Center. All participants will undergo screening activities upon coming to the NIH. 
Female participants will undergo a pregnancy test after consent signature. Participants who 
consent to participate in the trial and subsequently have a positive pregnancy test prior to 
randomization will be considered a screen failure and not enrolled in the study.    
 
8.2 
EFFICACY ASSESSMENTS 
1.  
Salt/Placebo Intervention: Capsules will be dispensed by Clinical Center Pharmacy. 
2.  
Flow Activating Cell Sorting (FACS) Analysis: Characterization of Leukocytes 
Populations :  As part of assessing the whole blood epigenome and transcriptome, the protocol 
will perform an in-depth characterization of the heterogeneous mixture of leukocytes circulating 
in the blood and how this profile might be altered in response to the dietary sodium intervention.   
We will utilize the NHLBI’s Flow Cytometry Core in collaboration with Dr. Phil McCoy. We 
will collect peripheral blood samples at the 4 time points (visit 1, 2 3 and 4). The FACS analysis 
will define changes in immune cell-type profiles as well as facilitate the isolation of DNA/RNA 
from specific immune cell sub-types (e.g. CD4 T-cells; CD8 T-cells, Monocytes etc.)  for 
epigenome and transcriptome analysis.  
3. 
Vascular function testing:  These tests will be done using specialized non-invasive 
machines, such as SphygmoCor and EndoPAT, to assess vascular health. 
4. 
 24-hour ambulatory blood pressure monitoring: Participants will be given an ambulatory 
blood pressure monitoring to device that will be worn for 24 hours within two days of NIH 
baseline visit and again for 24 hours within two days prior to the return visits.  
5  
A 24-hour dietary recall will be administered at baseline and at each subsequent visit to 
assess differential diet and salt intake among each participant.  Nutrition staff from the Clinical 
Center will meet with each participant and ask them to recall any foods or beverages they have 
eaten over the past 24 hours using an automated dietary analysis software application developed 
by the US Department of Agriculture for use in the National Health and Nutrition Examination 
Survey. The intakes for 108 nutrients (including salt) will be calculated for each participant 
based on individual diet. 
6.   
Psychophysical Taste Task: Sucrose and Salt Detection Thresholds: Sucrose and salt 
detection thresholds will be assessed using a two-alternative forced-choice staircase procedure 

GENE-FORECAST SIT 
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22 
 
was developed at the Monell Center for Adults.14-16 The two-alternative forced choice is a 
psychophysical method developed to elicit responses about an individual’s experience regarding 
a stimulus. It focuses on the evaluation of a single attribute (e.g., sweetness or saltiness), and the 
stimulus is adjusted based on the individual’s responses.17-18 For this study, all testing will take 
place in a private, comfortable room. Subjects will be fasted for at least one hour before the task 
and acclimate to the testing room and to the researcher for approximately 15 minutes before 
testing.  For the first trial and each subsequent trial, subjects will be presented with pairs of 
solutions in random order; within each pair, one solution will be distilled water, and the other 
will be the taste stimulus. Subjects will be instructed to taste the first solution presented within 
the pair, swish the solution in their mouth for 5 seconds, and expectorate. This will be repeated 
for the second solution within the pair. Between solutions, subjects will rinse their mouth with 
water; they will rinse once within a pair, and twice between successive pairs. After tasting both 
solutions within a pair, subjects will be asked to point to the solution that has a non-neutral taste. 
A tracking grid will be used to record subjects’ response.19-21  This method eliminates the need 
for a verbal response and has been shown to be an effective method for assessing both taste and 
olfaction in children.19,22 The task takes approximately 30 minutes to complete. 
7.  
Psychophysical Taste Task: Sucrose and Salt Preference: Sucrose and salt preference will 
be assessed using a two-series paired comparison-tracking method developed at the Monell 
Center for Adults.14-16 Subjects will be presented with pairs of solutions differing in sucrose 
concentration (3, 6, 12, 24, and 36 g per 100 mL) and salt (0.92–6.14% wt/vol NaCl). They will 
be asked to taste the solutions without swallowing and point to which of the pair they liked 
better. Subsequently, each pair presented will be determined by the subject’s preceding 
preference choice. The entire task is then repeated with the stimulus pairs presented in reverse 
order. After completion of the taste task, the geometric mean of the sucrose concentrations 
chosen will be determined. This serves as an estimate of the participant's most preferred level of 
sucrose.22-23 The task takes approximately 15 minutes to complete. 
8. 
Baecke’s Physical Activity Questionnaire: Physical activity will be assessed using 16 
questions involving three habitual physical activities (occupational physical activity, physical 
exercise, and leisure activities) during the previous 12 months.24 
9. 
Pittsburgh Sleep Quality Index (PSQI):  This is a 19-item self-rated questionnaire for 
evaluating subjective sleep quality over the previous month.25 
10. Microbiome: Skin, saliva and stool samples will be collected by the GENE-FORECAST-
SIT staff based on the utilization of Norgen kits.  Participant will be swabbed in three locations 
of the skin and will spit in a prepared value. Subjects will be required to not shower within 12 
hours of sampling based on the guidelines from the NIH common fund Human Microbiome 
Project, an initiative to generate resources and facilitate the characterization of the human 
microbiota (Human Microbiome Project, Nature, 2012). Stool kits will be previously mailed to 
participant for collection and participant will give stool sample to GENE-FORECAST-SIT staff 
at the clinic appointment. 
 
It is estimated that participants will spend approximately 5 hours at the Clinical Center during 
baseline and approximately 3 hours during each of the subsequent visit. Obtaining and explaining 
the informed consent will take about 20-30 minutes. Female prospective participant who signs 
consent and subsequently test positive for pregnancy prior to randomization will not be enrolled 
in the study. Administering the baseline questionnaires will take about 1.45 hours and 75 minutes 
for the 24-hour diet recall and taste assessments for each subsequent visit. The clinic visit 

GENE-FORECAST SIT 
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23 
 
assessment, plus vascular function test and microbiome collection, will take about 1.30 hours.  A 
member of the clinical study staff will follow-up with participant one week after visit to assess 
any adverse reaction to consuming intervention product.  All procedures and details for each can 
be found in the study’s Manual of Operation (MOP). 
 
Table 1: Estimated length of time for study procedures 
 NIH Clinic Visit 
Estimated 
time 4 hours 
Total 
Participants  
Duration of 
Study  
a) Informed consent 
(baseline only) 
20-30 
minutes  
40 
7 wks 
b) Phlebotomy 
30 minutes 
40 
7 wks 
 c)Physical 
examination 
20 minutes 
40 
7 wks 
d) Baseline 
Questionnaires:  
Baecke’s Physical 
Activity 
Pittsburgh Sleep 
Quality Index 
1.45 hours 
40 
Baseline 
 
e) 24 diet recall 
30 minutes 
40  
7wks 
f) Psychophysical 
Taste Task: Sucrose 
and Salt Detection 
Thresholds 
30 minutes  
40  
7 wks 
g) Psychophysical 
Taste Task: Sucrose 
and Salt Preference 
15 minutes  
40 
7 wks 
h) Microbiome 
15 minutes 
40 
7 weeks 
i) Vascular Function  
1.0 hrs 
40 
7 wks 
 
 
 
 
 
8.2.1 Clinical Evaluations 
Physical examination: A physical examination will be performed by the study Physician 
Assistant to include health/medication history and measurements of height and weight. Vital signs 
including temperature, pulse, respirations, and blood pressure, will be also be collected. 
 
Radiographic or other imaging assessments:  
Radiology or other imaging will not be conducted in this study. 
 
8.2.2 Biospecimen Evaluations 
Participants will undergo standardized blood collection for chemistry panel, blood pressure 
measurement and vascular function test.  Biological specimens will include peripheral venous 
blood collected by phlebotomy, urine, and stool. All biospecimens will be collected at the NIH 
Clinical Center which is in compliance with Clinical Laboratory Improvement Amendments 

GENE-FORECAST SIT 
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24 
 
(CLIA).  Blood, urine and stool samples will be collected from the Clinical Center by the 
GENE-FORECAST-SIT staff. The samples are  brought to PI’s lab by research study lab 
assistants where Laboratory Manager will prepare and store samples in freezers.  Shipment of 
blood and microbiome samples for each visit per participant will be sent overnight for assay in 
dry ice in compliance with standard procedures. Each sample will be labelled with participant 
study ID. 
 
Urine sample: (small quantity, 1 cup 90 ml) 
1. Urine chemistries (e.g. sodium , chloride, potassium ) 
2. Urine creatinine and microalbumin (ACR) 
 
3. Urine protein to creatinine ratio (PCR)  
1. Osmolality 
2. Urea Nitrogen 
3. Urine exosomes (RNA/microRNA)  
 
 
Microbiome samples: 
Stool/At home collection kit (quantity = 2 grams): 
Skin: swab of inner elbow and behind ears 
Oral: saliva  
 
Blood samples: 
 
Fasting samples for clinical chemistries: 
  
 
Blood samples: 
 
  
  
  
  
  
  
Clinical Center 
Research Lab 
Test 
Lab test 
Volume 
Tube (ml) 
Number of 
Tubes 
Total 
Volume 
(ml) 
Total 
Volume 
(tablespoon) 
Tube type 
CBC: CBC with 
differential count 
4 
2 
8 
0.54 
Lavender 
HbA1C: A1C 
 
Homocysteine 
Fasting lipid 
panel (serum):  
3.5 
2 
7 
0.47 
green/yellow 
Mineral panel  
Acute Care Panel 

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25 
 
 Total Protein 
ApoA1 and 
ApoB: APOAB 
Pro-BNP: BNP1 
C-reactive 
protein: CRPHS 
(high sensitivity 
CRP) 
Hepatic Panel 
3.5 
1 
3.5 
0.24 
Gold 
Serum hCG 
Serum Cortisol  
(CORT) 
8.5 
1 
8.5 
0.57 
Red SST 
Serum 
Aldosterone  
(ALD03,ADS01, 
ADS02) 
Clinical Center 
Research Labs 
Total  
19.5 
6 
27 
1.83 
  
 
Non-Clinical 
Center Research 
Lab Test 
  
  
  
  
  
Lab test 
Volume 
Tube 
(ml) 
Number 
of 
Tubes 
Total 
Volume 
(ml) 
Total 
Volume 
(tablespoon 
Tube type 
 
4 
1 
4 
0.27 
Lavender/EDTA  
DNA isolation 
(research, whole 
blood) 
RNA/miRNA 
isolation (whole 
blood) 
2.5 
2 
5 
0.34 
PaxGene 
Plasma for research 
archiving  
4 
1 
4 
0.27 
Green/ Lithium Heparin 
 
3.5 
1 
3.5 
0.24 
Red SST  
 
Research tube for 
specialized 
endothelial function 
markers (e.g. 
VCAM-1, VEGF) 

GENE-FORECAST SIT 
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26 
 
Research tube for 
specialized 
inflammatory 
markers (e.g. IL6, 
TNF alpha) 
Plasma Renin 
Activity (PRA, 
PRAK1) 
4 
1 
1 
0.20 
Lavendar/EDTA 
Serum for research 
archiving 
3.5 
1 
3.5 
0.24 
Red SST 
FACS Analysis 
10 
4 
40 
2.71 
Green/ Sodium Heparin 
Non-Clinical 
Center Research 
Labs Total  
27.5 
10 
60 
4.06 
  
Total per visit        
(4 visits)  
47 
16 
87 
5.88 
  
 
Microbiome: 
 
BODY SITE 
SPECIMEN 
COLLECTION TUBE 
 
Oral Cavity 
 
 
Saliva 
 
 
 
Saliva DNA Collection, 
Preservation, and Isolation 
Kit  
 
 
Skin  
Retroauricular Crease (Left) 
 
Swab Collection and DNA 
System  
Retroauricular Crease (Right) 
Antecubital Fossa (Left) 
Antecubital Fossa (Right) 
 
GI Tract 
 
Stool 
 
Norgen Stool Nucleic Acid 
Collection and Preservation 
System 
 
Total volume of blood collected per visit = 87 ml, which is equivalent to about 5.8-6 tablespoons 
of blood. 
 
Biological specimens collected from individuals will include peripheral venous blood and 
urine samples collected by phlebotomy.  Oral microbiome samples will be acquired using a 
spit collection kit. Skin swabs will be used to obtain skin microbiome samples from the left and 

GENE-FORECAST SIT 
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27 
 
right retroarticular crease, as well as, the left and right antecubital fossa. Stool microbiome 
samples will be collected in a stool kit mailed to participants prior to clinic visit.  Detailed 
instructions for the preparation, handling, storage, and shipment of specimens will be explained 
in the study’s MOP. 
 
8.2.3 Correlative Studies for Research/Pharmacokinetic Studies 
The protocol will not include correlative studies for research/pharmacokinetic studies. 
 
8.2.4 Samples for Genetic/Genomic Analysis 
8.2.4.1 Description of the scope of genetic/genomic analysis 
• Analyses will include whole blood epigenetics based on DNA methylation and 
transcriptomics analysis will use whole blood and urine based on RNAseq.  
• Germline and/or somatic analysis will not be conducted.  
• Whole blood and urine will be the biospecimens used for analyses. Privacy and 
confidentiality of medical information/biological specimens will be maximized. 
• No personal identifier will be maintained – including protected health information (PHI).  
Rather, participant will be assigned a unique numerical personal study identifier resulting 
in the unlikely occurrence of potential identification. 
• Clinical and demographic information will be collected on each participant.  Participant 
nor family members can be identified due to the use of a numerical personal study 
identifier.   
• All coded participant, demographic, survey, medical and biological information will be 
stored in a medical file and a secured NIH database with appropriate IT security measures 
(e.g. de- identified codes; encryption; password access protection) to maximize the 
protection of privacy. Only the database manager will have access to the password key 
for code.   
• Pedigree information will not be published. 
 
• The personal identifier will not be released to any third party. 
• No personal identifier containing data/sample information will be shared with other 
investigators.  Information will only be deposited into a relevant controlled access 
database as mandated by NIH policies. 
 
• For additional protection of confidentiality, all hard-copy information will be maintained 
in a locked file-cabinet with access only by the Research Nurse and Data-Base manager.  
Hard-copy information will be maintained and destroyed based on the duration 
mandated by NIH policies. In addition, we will use a custom designed NIH database -
Clinical Trial Data Base (CTDB) designed for the collection and storage of study indices 
from study participants.  Access to study participants’ information is limited to only a 
few of the investigators and support staff.  The database has password protection, 
stratified access, and firewall protection. 
 

GENE-FORECAST SIT 
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28 
 
• Furthermore, to help us protect participant’s privacy, this study has obtained a 
Certificate of Confidentiality from the National Institutes ofHealth.  Researchers 
cannot release identifiable information to any federal, state, or local civil, criminal, 
administrative, legislative, or other proceedings. Researchers will utilize the 
Certificate to minimize risks by adding a level of protection to study participants. 
 
• The Certificate cannot be used to resist a demand for information from personnel of the 
United States Government that is used for auditing or evaluation of federally funded 
projects or for information that must be disclosed in order to meet the requirements of 
the federal Food and Drug Administration (FDA). 
 
• The Certificate of Confidentiality does not prevent any participant or a member of their 
family from voluntarily releasing information about a participant involvement in this 
research. In the event that an employer, insurer or other person obtains a participant’s 
written consent to obtain research information, then the Certificate of Confidentiality 
cannot be used to withhold information. 
8.2.4.2 Management of Results 
Participants will be mailed a letter following baseline visit at the NIH Clinical Center (which is a 
CLIA certified lab) with results from their clinical visit, including Body Mass Index with health 
definitions, glucose, blood pressure.  
8.2.4.3 Genetic counseling 
Genetic counseling will not be provided.  We are not doing gene sequencing as a part of the 
study, therefore reporting the possibility of incidental findings is not applicable. 
 
8.2.4.4 Safety and  Other Assessments 
Table 3: List and description for study procedures 
 NIH Clinic Visit 
Description 
a) Physical 
examination 
Height, weight, temperature, pulse, respirations, blood 
pressure conducted by NIH Clinical Center Nursing Staff, 
Physician Assistant will perform physical examination. 
b) Phlebotomy 
Standard clinical laboratory chemistry- including whole 
blood for DNA methylation epigenome and RNAseq 
transcriptome collected by Clinical Center phlebotomy 
c) Urine 
Urine sample for RNAseq transcriptome collected by Clinical 
Center phlebotomy 
d) Microbiome 
     Skin, saliva, stool 
Swab, saliva and stool kits will be used to assess microbiome 
collected by study GENE-FORECAST-SIT staff 
e) Baseline 
Questionnaires  
Pittsburgh Sleep Quality Index (PSQI):  This is a 19-item 
self-rated questionnaire for evaluating subjective sleep 
quality over the previous month. Assessment will be 
completed in about 20 minutes. This will only be 
administered at baseline. 
Baecke’s Physical Activity Questionnaire: Physical activity 
will be assessed using 16 questions involving three habitual 
physical activities (occupational physical activity, physical 

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29 
 
exercise, and leisure activities) during the previous 12 
months. Assessment will be completed in about 15 minutes. 
This will only be administered at baseline. 
Questionnaires conducted by study GENE-FORECAST-SIT  
staff 
f) 24 diet recall 
Nutrition staff from the Clinical Center will meet with each 
participant and ask them to recall any foods or beverages they 
have eaten over the past 24 hours using an automated dietary 
analysis software application developed by the US 
Department of Agriculture for use in the National Health and 
Nutrition Examination Survey. The intakes for 108 nutrients 
(including salt) will be calculated for each participant based 
on individual diet. 
 
g) Psychophysical 
Taste Task: Sucrose 
and Salt Detection 
Thresholds 
Sucrose and salt detection thresholds will be assessed using a 
two-alternative forced-choice staircase procedure was 
developed at the Monell Center for Adults. The two-
alternative forced choice is a psychophysical method 
developed to elicit responses about an individual’s experience 
regarding a stimulus. It focuses on the evaluation of a single 
attribute (e.g., sweetness or saltiness), and the stimulus is 
adjusted based on the individual’s responses. After tasting 
both solutions within a pair, subjects will be asked to point to 
the solution that has a non-neutral taste. A tracking grid will 
be used to record subjects’ response.19-21  Assessment 
conducted by Associate Investigator Dr. Paule Joseph. 
h) Psychophysical 
Taste Task: Sucrose 
and Salt Preference 
Sucrose and salt preference will be assessed using a two-
series paired comparison-tracking method developed at the 
Monell Center for Adults.14-16 Subjects will be presented with 
pairs of solutions differing in sucrose concentration (3, 6, 12, 
24, and 36 g per 100 mL) and salt (0.92–6.14% wt/vol NaCl). 
They will be asked to taste the solutions without swallowing 
and point to which of the pair they liked better. Subsequently, 
each pair presented will be determined by the subject’s 
preceding preference choice. The entire task is then repeated 
with the stimulus pairs presented in reverse order. After 
completion of the taste task, the geometric mean of the 
sucrose concentrations chosen will be determined. This 
serves as an estimate of the participant's most preferred level 
of sucrose.22-23 The task takes approximately 15 minutes to 
complete. Assessment conducted by Associate Investigator 
Dr. Paule Joseph. 
 
i) Vascular Function  These tests will be done using specialized non-invasive 
machines, such as SphygmoCor and EndoPAT, to assess 
vascular health conducted by study Physician Assistant. 

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Biological specimen collection and laboratory evaluations: 
Biological specimen 
  
  
  
  
  
  
Clinical Center  
Research  Lab 
Test 
Lab test 
Volume  
Tube (ml) 
Number of 
Tubes 
Total 
Volume 
(ml) 
Total 
Volume 
(tablespoon ) 
Tube type 
CBC: CBC with 
differential count 
4 
2 
8 
0.54 
Lavender 
HbA1C: A1C 
 
Homocysteine 
Fasting lipid 
panel (serum):  
3.5 
2 
7 
0.47 
green/yellow 
Mineral panel  
Acute Care Panel 
 Total Protein 
ApoA1 and 
ApoB: APOAB 
Pro-BNP: BNP1 
C-reactive 
protein: CRPHS 
(high sensitivity 
CRP) 
Hepatic Panel 
3.5 
1 
3.5 
0.24 
Gold 
Serum hCG 
Serum Cortisol  
(CORT) 
8.5 
1 
8.5 
0.57 
Red SST 
Serum 
Aldosterone  
(ALD03,ADS01, 
ADS02) 

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Clinical Center 
Research Labs 
Total  
19.5 
6 
27 
1.83 
  
 
Non-Clinical 
Center Research 
Lab Test 
  
  
  
  
  
Lab test 
Volume 
Tube 
(ml) 
Number 
of 
Tubes 
Total 
Volume 
(ml) 
Total 
Volume 
(tablespoon 
) 
Tube type 
 
4 
1 
4 
0.27 
Lavender/EDTA  
DNA isolation 
(research, whole 
blood) 
RNA/miRNA 
isolation (whole 
blood) 
2.5 
2 
5 
0.34 
PaxGene 
Plasma for research 
archiving  
4 
1 
4 
0.27 
Green/ Lithium Heparin 
 
3.5 
1 
3.5 
0.24 
Red SST  
 
Research tube for 
specialized 
endothelial function 
markers (e.g. 
VCAM-1, VEGF) 
Research tube for 
specialized 
inflammatory 
markers (e.g. IL6, 
TNF alpha) 
Plasma Renin 
Activity (PRA, 
PRAK1) 
4 
1 
1 
0.20 
Lavendar/EDTA 
Serum for research 
archiving 
3.5 
1 
3.5 
0.24 
Red SST 
FACS Analysis 
10 
4 
40 
2.71 
Green/ Sodium Heparin 
Non-Clinical 
Center Research  
Labs Total  
27.5 
10 
60 
4.06 
  

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Total per visit        
(4 visits)  
47 
16 
87 
5.88 
  
 
Microbiome: 
 
BODY SITE 
SPECIMEN 
COLLECTION TUBE 
 
Oral Cavity 
 
 
Saliva 
 
 
 
Saliva DNA Collection, 
Preservation, and Isolation 
Kit  
 
 
Skin  
Retroauricular Crease (Left) 
 
Swab Collection and DNA 
System  
Retroauricular Crease (Right) 
Antecubital Fossa (Left) 
Antecubital Fossa (Right) 
 
GI Tract 
 
Stool 
 
Norgen Stool Nucleic Acid 
Collection and Preservation 
System 
 
Total volume of blood collected per visit = 87 ml, which is equivalent to about 5.8-6 tablespoons 
of blood.  More detailed description of all procedures can be found in the study MOP. 
 
Laboratory evaluations: 
DNA and RNA extractions will be done by ReproCELL USA, Inc. DNA and RNA sequencing 
will be done by the NIH Intramural Sequencing Center (NISC). Microbiome assay will be done 
by University of Pennsylvania Microbiome Center.  Flow Activating Flow Cell Sorting (FACS) 
Analysis will be conducted by the NHLBI’s Flow Cytometry Core in collaboration with Dr. Phil 
McCoy.  
 
Adverse Events and Serious Adverse Events 
8.2.5 Definition of Adverse Event 
We will define an adverse event (AE) as any untoward medical occurrence associated with the 
use of an intervention in humans, whether considered intervention relation based on 21 CFR 
312.32(a). 
 
8.2.6 Definition of Serious Adverse Events (SAE) 
8.2.7 Classification of an Adverse Event 
We will define a SAE or suspected SAE as “serious” if, in the view of either the investigator or 
sponsor, it results in any of the following outcomes: death, a life-threatening adverse event, 
inpatient hospitalization or prolongation of existing hospitalization, a persistent or significant 
incapacity of substantial disruption of the ability to conduct normal life functions, or a congenital 

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anomaly/birth defect.  Important medical events that may not result in death, life threatening, or 
require hospitalization may be considered serious when, based upon appropriate medical 
judgment, they may jeopardize the participant and may require medical or surgical intervention 
to prevent one of the outcomes listed in the definition.  Examples of such medical events include 
allergic bronchospasm requiring intensive treatment in an emergency room or at home, blood 
dyscrasias or convulsions that do not result in inpatient hospitalization or the development of 
drug dependency or drug abuse. 
 
8.2.7.1 Severity of Event 
All Adverse Events (AEs) will be assessed by the Staff Clinician. For AEs not included in the 
protocol defined grading system, the following guidelines will be used to describe severity. 
Mild: Events require minimal or no treatment and do not interfere with the participant’s daily 
activities. 
 
Moderate: 
Events result in a low level of inconvenience or concern with the therapeutic 
measures.  Moderate events may cause some interferences with functioning. 
 
Severe: 
Events interrupt a participant’s usual daily activities and may require systemic 
drug therapy or other treatment.  Severe events are usually potentially life-threatening or 
incapacitating.  Of note, the term “severe” does not necessarily equate to “serious”. 
 
8.2.7.2 Relationship to Study Intervention 
All adverse events (AEs) must have their relationship to study intervention assessed by the PI, in 
consultation with clinical study staff, who will examine and evaluate the participant based on 
temporal relationship and his clinical judgement.  The degree of certainty about causality will be 
graded using the categories below.  In a clinical trial, the study product must always be suspect. 
 
Related: 
The AE is known to occur with the study intervention, when there is a reasonable 
possibility that the study intervention caused the AE, or there is a temporal relationship between 
the study intervention and event.  Reasonable possibility means that there is evidence to suggest 
a causal relationship between the study intervention and the AE. 
 
Not Related: There is not a reasonable possibility that the administration of the study 
intervention caused the event, there is no temporal relationship between the study intervention 
and event onset, or an alternate etiology has been established. 
 
OR 
 
Definitely Related: There is clear evidence to suggest a causal relationship, and other possible 
contributing factors can be ruled out.  The clinical event, including an abnormal laboratory test 
result, occurs in a plausible time relationship to study intervention administration and cannot be 
explained by concurrent disease or other drugs or chemicals.  The response to withdrawal of the 
study intervention should be clinically plausible.  The event must be pharmacologically or 
phenomenologically definitive, with use of a satisfactory re-challenge procedure if necessary. 
 

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Probably Related: 
There is evidence to suggest a causal relationship, and the influence of 
other factors is unlikely.  The clinical event, including an abnormal laboratory test result, occurs 
within a reasonable time after administration of the study intervention, is unlikely to be attributed 
to concurrent disease or other drugs or chemicals, and follows a clinically reasonable response on 
withdrawal (de-challenge).  Re-challenge information is not required to fulfill this definition. 
 
Potentially Related: There is some evidence to suggest a causal relationship (e.g., the event 
occurred within a reasonable time after administration of the trial medication). However, other 
factors may have contributed to the event (e.g., the participant’s clinical condition, other 
concomitant events). Although an AE may rate only as “possibly related” soon after discovery, it 
can be flagged as requiring more information and later be upgraded to “probably related” or 
“definitely related”, as appropriate. 
 
Unlikely to be Related: 
A clinical event, including an abnormal laboratory test result, 
whose temporal relationship to study intervention administration makes a causal relationship 
improbable (e.g., the event did not occur within a reasonable time after administration of the 
study intervention and in which other drugs or chemicals or underlying disease provides 
plausible explanations (e.g., the participant’s clinical condition, other concomitant treatments). 
 
Not Related: The AE is completely independent of study intervention administration, and/or 
evidence exists that the event is definitely related to another etiology.  There must be an 
alternative, definitive etiology documented by the clinician. 
 
8.2.7.3 Expectedness  
The Staff Clinician will be responsible for determining whether and AE is expected or 
unexpected.  An AE will be considered unexpected if the nature, severity, or frequency of the 
event is not consistent with the risk information previously described for the study intervention. 
 
8.2.8 Time Period and Frequency for Event Assessment and Follow-Up 
The occurrence of an AE or SAE may come to the attention of clinical study staff during study 
visits and follow-up after study visit to assess reaction(s) to intervention product. 
 
All AEs including local and systemic reactions not meeting the criteria for SAEs will be capture 
on the appropriate Case Report Form (CRF).  Information to be collected includes events 
description, time of onset, Staff Clinician’s assessment of severity, relationship to study product 
and time of resolution/stabilization of the event.  All AEs occurring while on study will be 
documented appropriately regardless of relationship.  All AEs will be followed to adequate 
resolution. 
 
Any medical condition that is present at the time that the participant is screened will be 
considered as baseline and not reported as an AE.  However, if the study participant’s condition 
deteriorates at any time during the study, it will be recorded as an AE. 
 
Changes in the severity of an AE will be documented to allow an assessment of the duration of 
the event at each level of severity to be performed.  AEs characterized as intermittent require 
documentation of onset and duration of each episode. 

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35 
 
 
The study’s Physician Assistant will record all reportable events with start dates occurring any 
time after informed consent is obtained until 7 days (for non-serious AEs) or 30 days (for SAEs) 
after the last day of study participation.  At each study visit, the PI will inquire about the 
occurrence of AE/SAEs since the last visit.  Events will be followed for outcome information 
until resolution or stabilization. 
 
8.2.8.1Adverse Event Reporting  
The study PI will report all nonserious AEs to the Independent Safety Monitor and to the NIH 
IRB at the time of the continuing review.  The PI will be responsible for signing off of all AE 
reports.  Full details of procedures, including flow chart, will be included in the study’s Manual 
of Operating Procedures (MOP).  Potential side effects associated with the intervention product 
(i.e. sodium chloride) may include discomfort while swallowing, increased thirst, indigestion, 
nausea, vomiting, headaches, mild elevations in blood pressure, mild changes in blood chemicals 
(electrolytes), lightheadedness, tiredness, mild swelling of arms/legs, and/or diarrhea, and rash. 
There are no disease related conditions related to the intervention product.  
8.2.8 Serious Adverse Event Reporting  
The study PI will immediately report to the NIH IRB any serious adverse event, whether or not 
considered study intervention related, including those listed in the protocol and will include an 
assessment of whether there is a reasonable possibility that the study intervention caused the 
event.  Study endpoints that are SAEs (e.g., all-cause mortality) must be reported in accordance 
with the protocol unless there is evidence suggesting a causal relationship between the study 
intervention and the event (e.g., death from anaphylaxis).   
 
All SAEs will be followed until satisfactory resolution or until the PI deems the event to be 
chronic or the participant is stable.  Other supporting documentation of the event will be made 
available to the Independent Safety Monitor or Intramural Program as soon as possible upon 
request.  
 
8.2.9 Events of Special Interest  
Not applicable 
8.2.10 Reporting of Pregnancy  
If a participant test positive for pregnancy during baseline visit, she will not be enrolled.  If the 
participant test positive at any subsequent visit or becomes pregnant in between visits, she will be 
discontinued, and this will be reported as an AE to the NIH IRB during the time of continuing 
review.  
8.3 
UNANTICIPATED PROBLEMS 
8.3.1 Definition of Unanticipated Problems (UP) 
Any incident, experience, or outcome that meets all of the following criteria shall be considered 
an UP: 
• 
Unexpected in terms of nature, severity, or frequency given (a) the research procedures 
that are described in the protocol related documents, such as the IRB-approved research protocol 
and informed consent documents; and (b) the characteristics of the participant population being 
studied; and 

GENE-FORECAST SIT 
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36 
 
• 
Related or possibly related to participation in the research (“possibly related” means there 
is a reasonable possibility that the incident, experience, or outcome may have been caused by the 
procedures involved in the research); and 
• 
Suggests that the research places participants or others (which many include research 
staff, family members or other individuals not directly participating in the research) at a greater 
risk of harm (including physical, psychological, economic, or social harm) than was previously 
known or expected. 
8.3.2 Unanticipated Problem Reporting  
The investigator will report unanticipated problems (UPs) to the NIH Institutional Review Board 
(IRB) as per Policy 801. 
8.3.3 NIH Intramural IRB Reporting of IND Safety Reports 
Only IND Safety Reports that meet the definition of an unanticipated problem will be reported to 
the NIH Intramural IRB. 
 
9 
STATISTICAL CONSIDERATIONS 
The protocol will not have a formal Statistical Analytical Plan (SAP).  
9.1 
STATISTICAL HYPOTHESIS 
1) Increased dietary sodium intake will be associated with vascular dysfunction and increased 
blood pressure in the treatment group compared to the placebo group. 
2) Increased dietary sodium intake will be associated with changes in the whole blood epigenome 
as assessed by DNA methylation in the treatment group compared to the placebo group. 
3) Increased dietary sodium intake will be associated with changes in the whole blood and urine 
transcriptome as assessed by RNA-seq in the treatment group compared to the placebo group. 
4) Increased dietary sodium intake will be associated with changes in the skin, oral and gut 
microbiome in the treatment group compared to the placebo group. 
5) Increased dietary sodium take will be associated with blood epigenome/transcriptome 
molecular signature in the treatment group compared to the placebo group.   
 
Endpoints: 
Primary: 
Vascular function 
Microbiome 
      Secondary: 
      Blood pressure 
      Whole blood epigenome 
      Whole blood and urine transcriptome 
9.2 
SAMPLE SIZE DETERMINATION 
Data will be tested for normality using the Shapiro-Wilk test. Continuous variables will be 
described as mean ± standard deviation (SD) if normally distribute. Variables with skewed 
distribution (i.e.  aldosterone, BNP, etc.) were logarithmically transformed prior to analyses and 
are presented as median and interquartile range (IQR).  Categorical variables will be expressed 
as numbers (percentages).  
When data are normally distributed, comparisons between the treatment (sodium intake) and 
control (placebo) groups will be made using paired Student’s t-tests, and when data are non-

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normally distributed, the Mann-Whitney U test will be employed. Chi-square test will be used 
for categorical variables.  Tests for sodium intake effect on the primary outcomes without 
correction for period effect will be done using Mann-Whitney test on the differences. Tests for 
period effect will be assessed using two-sample Wilcoxon tests on the differences between 
treatment groups, and tests for carry-over effect will be assessed by a two-sample Mann-Whitney 
test on the averages in the two treatment periods. Correlations were determined with Pearson's 
correlation coefficient. 
Additionally, we will evaluate the effects of sodium intake on changes in blood pressure, 24-hr 
ambulatory blood pressure monitoring (ABPM), biochemical markers, vascular functions, and 
specific transcriptome and epigenome by using a linear mixed model, with subject as a random 
effect, thereby accounting for the period, sequence, and carryover effects and to model the 
various sources of intra-individual and inter-individual variability.  All statistical tests will be 
two-sided and P values <0·05 will be regarded as significant. Statistical analyses will be carried 
out using SAS software (SAS Institute, Inc., Cary, NC, USA).  
Sample size and statistical power: We expect to enroll and randomize a total of 40 eligible 
subjects for this protocol. The sample size is assessed to detect meaningful effects of sodium 
intake on the study’s primary outcomes and secondary outcomes, including changes in vascular 
function (PWV m/s), sitting SBP and DBP (mm Hg), ABPM (mm Hg), biochemical markers 
(plasma aldosterone, pg/mL, brain natriuretic peptide (pg/mL), as well as epigenome, 
transcriptome, and microbiome under an assumption about the effect size, standard deviation 
outcomes, correlation of measurements within individual. We estimated that a sample size of 28 
participants will be required in a cross-over study to detect changes in PWV between high salt 
intake and control groups of 0.6 m/s (estimates based on Pimenta et al. 47), with 80% power and 
P < 0.05, assuming common SD of 0.9 m/s for high sodium intake and control groups, and 
correlation of 0.3 within subjects (Figure 2).  Accounting for a potential of 20% dropout during 
the study, the sample size would be 34.  
In addition, the estimated sample size will provide us with more than 80 percent power to detect 
a difference of 6 mm Hg (SD=9 mm Hg) in sitting SBP between sodium intake and the placebo 
treatment at 0.05 significance level (estimates based on Tzemos et al. 48).  Similarly, the 
proposed sample size would give more than 90% power to detect significant changes in plasma 
aldosterone (pg/ml) and brain natriuretic peptide (pg/mL) between high sodium intake and 
control at 0.05 significance level 47,48. The sample size calculation was performed using a paired 
t test assuming no carryover effect and no interactions between subjects, treatments, and periods.   
Figure 2. 

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38 
 
 
 
9.3 
POPULATIONS FOR ANALYSES 
We will use the per-protocol analysis dataset for our study.  We are confident that participants 
will comply with the protocol sufficiently to ensure that the data will be likely to represent the 
effects of exposure to treatment and placebo arms based on the underlying scientific model. 
 
9.3.1 Evaluable for toxicity 
9.3.2 Evaluable for objective response  
Not applicable 
9.3.3 Evaluable Non-Target Disease Response 
Not applicable 
9.4 
STATISTICAL ANALYSES 
9.4.1 General Approach 
For descriptive statistics, categorical variables will be assessed via chi-square and presented as 
percentages.  Continuous variables will be assessed via t-test and will be presented as means with 
standard deviations. We will check assumptions underlying statistical procedure and correct 
procedure if necessary. 
9.4.2 Analysis of the Primary Endpoints 
The two primary endpoints are vascular function and microbiome. 
First, vascular function difference between placebo and treatment (salt intake) will be estimated. 
Then, microbiome differential between treatment and placebo will be identified and estimate for 
each of the microbiome collection sides (skin, oral and gut). Details of the vascular function and 
microbiome analyses with respect to treatment and placebo are provided below. 
 
Vascular function (e.g. pulse wave velocity, PWV) will be first analyzed as a continuous 
variable. Its relationship with salt intake will be investigated through multiple linear regression 
with vascular function as outcome and salt intake (a binary variable with two categories: 

GENE-FORECAST SIT 
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39 
 
treatment and placebo) as exposure variable. For this analysis only samples with complete data 
for vascular function and salt intake will be considered. The outcome variable will be log 
transformed if it departs markedly from the Gaussian distribution. The aim of this first analysis is 
to estimate the effect of salt intake on vascular function. The effect size, p-value and 95% 
confidence interval will be provided as results.  
 
The second endpoint is microbiome; the microbiome data of each of the all the collection sites 
(oral, gut and skins) will be analyzed separately. Prior to investigating the relationship between 
microbiome and salt intake, the raw microbiome data requires extensive processing and quality 
controls (QC) because of challenges posed by this type of data 30,31 and the potential of spurious 
results if appropriate QC are not applied. The raw data will be processed in mainly 5 steps: (a) 
reads preparation (assembly of paired reads, filtering of low quality reads and identification of 
unique sequences), (b) Operational Taxonomic Units - OTU clustering (selection of unique OTU 
sequences), (c) quality controls (identification and removal of artifacts from amplicon reads, in 
OUT sequences). These tasks will be carried out using pipelines included in the standards 
operating protocols (SOPs) developed and used by the NIH Human Microbiome Project 31,32. For 
the metagenomic microbiome data, the HUMAnN2 33 pipeline will be used and for the 16s 
microbiome data the DADA2 34 pipeline will be used.  
 
The output of the pipelines are count data which are discrete, not continuous, and include only 
positive values 30,33,34. The counts, Cij, are the observed number of microbes for the ith sample 
and jth feature where the features are the OTUs or microbial taxa. The data are normalized 
because library sizes (total counts for each sample, across all features) often have a large 
magnitude difference, which can cause a bias 35. The data are right skewed with, usually, a large 
number of data points having the value 0. Such data follows a Poisson distribution and therefore 
a Poisson regression model type (e.g. negative binomial model) will be used to investigate the 
relationship between microbiome and salt intake. For each feature, a model will be fitted with the 
feature as outcome and salt intake (a binary variable with two categories: treatment and placebo) 
as covariate. The differential count (fold change difference) between treatment and placebo will 
be estimated for each feature (OTU). To adjust for the multiple testing of several OTUs, a false 
discovery rate penalty will be applied and only fold changes with an adjusted p-value 
(Benjamini-Hochberg multiple test correction) smaller or equal to 0.05 will be considered 
statistically significant.  
 
The analysis describes above using a Poisson regression model assumes the features (OTUs) are 
independent but it has been shown that microbial communities do interact in such way that 
changes in one feature affect another feature 36-38. However, the number of interactions can be 
large due to the number of features, and not easily fitted in a conventional regression model.  
Therefore, a machine learning algorithm (Random Forest or Neural Network) will be used to 
investigate the combined response of the features to salt intake. These algorithms can incorporate 
multiple and complex interactions between features without making an assumption about their 
distribution. The features will be used as predictor variables to predict salt intake (a binary 
variable with two categories: treatment and placebo). The results will be presented as the area 
under the curve (AUC) of a receiver operating curve (ROC) that reflect how well the features can 
predict treatment and placebo. Because these ensemble techniques do not assume the predictors 

GENE-FORECAST SIT 
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40 
 
variable (OTUs) are independent, no multiple testing penalty is required but rather cross-
validation will be undertaken. 
 
9.4.3 Analysis of the Secondary Endpoint(s) 
The secondary endpoints will be assessed independent of the results associated with the primary 
endpoints; secondary endpoints include blood pressure, whole blood epigenome, and whole blood 
and urine transcriptome. The effect of treatment and placebo will be investigated will be 
investigated for each secondary endpoint, as detailed below. 
 
The association between blood pressure (systolic and diastolic) and salt intake will be assessed 
using multiple linear regression with salt intake (two categories: treatment and placebo) as the 
exposure variable and blood pressure outcome variable. The effect size, p-value and 95% 
confidence interval of the regression model will be provided as results. 
 
The effect of salt intake on gene expression will be investigated in a differential expression 
analysis to identify genes whose expression differs significantly between treatment and placebo. 
The gene expression data will be normalized, using the weighted trimmed mean of M-values 
(TMM) method, an optimal method for the normalization of mRNA count data 39. Genes with an 
expression less than 1 Count Per Million (CPM = count/sum [counts] x 1million) in at least 2 
samples will be excluded because results from genes with extremely low expression are not 
reliable. Gene expression is generated as count data which follows a Poisson distribution; 
therefore, a negative binomial model will be fitted using the R library edgeR 40 developed to 
handle the specificities of mRNA sequencing data. The model computes likelihood ratio tests for 
the coefficients whilst taking into account dispersion. A deviance of goodness of fit test will be 
carried out to identify genes where the model fit was poor indicating that the dispersion estimate 
is away from the average dispersion. Dispersion outliers will not be considered for the results 
because the deviance indicates low quality or marked expression difference for the gene. 
Finally, genes with a log fold change not equal to 0 and a false discovery rate adjusted p-value ≤ 
0.05 will be reported as significantly differentially expressed between treatment and placebo. 
Multiple testing correction, for the total number of genes considered, will be carried out using 
the Benjamini-Hochberg method. 
 
The epigenetic effect of salt intake will be investigated trough a two-sample t–test to identify 
methylation sites with significant methylation level difference between treatment and placebo. At 
a minimum of 5X coverage and an average coverage of 30X, the SureSelect-MethSeq (SS-
Methseq) platform captures between 2.6 and 2.3 million methylation sites 41. After thorough 
processing and QC of the methylation data, using the Bismark pipeline 42, samples and 
methylation sites that passed all QC filters will be considered for the analysis.  Each methylation 
site will be treated as a continuous variable and salt intake will be treated as a nominal variable 
with two categories (treatment and placebo), in the t-test. In such analysis, the methylation sites 
are assumed to be independent and there is, hence, a high burden of multiple testing due to the 
millions of sites being tested. Therefore, a significance threshold of p-value < 9 × 10− 8 will be 
applied to control for false positive findings; this threshold was derived from empirical work 43 
based on the Illimunia EPIC array which has a lower density than the SS-Methseq platform but 
includes carefully selected sites that span the whole genome. The estimated mean difference, p-
value and 95% confidence interval of the t-test will be presented as the results. 

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After the transcriptome, epigenome and microbiome data have been considered separately in the 
analyses described above and to understand the links between gene expression and methylation, 
an ensemble technique such as Random Forests will be used to leverage the high dimensionality 
of the data (number of features >> number of observations) 44,45 and identify the best combination 
of predictors of salt intake in the transcriptome and epigenome data. Subsequently, the OTUs 
identified in the microbiome data will be integrated to find out if microbiome information 
improves the prediction accuracy of the omics data 46. This will provide further insights into the 
interplay between the three domains (microbiome, transcriptome and epigenome). 
 
 
9.4.4 Safety Analyses 
Not applicable. 
 
9.4.5 Baseline Descriptive Statistics 
Not applicable. 
Baseline analysis stratified by treatment and placebo arms will be assessed based on clinical and 
demographic characteristics. Chi-square will be used to assess categorical variables and t-test 
will be used to assess continuous variables.  
9.4.6 Planned Interim Analyses 
Not applicable 
 
9.4.7 Sub-Group Analyses 
Primary and secondary endpoints will be compared based on age and sex differences stratified by 
treatment and placebo groups.). 
 
9.4.8 Tabulation of individual Participant Data 
Individual participant data will not be presented by time point.  
9.4.9 Exploratory Analyses 
Exploratory analyses will not be conducted. 
10 REGULATORY AND OPERATIONAL CONSIDERATIONS 
10.1 INFORMED CONSENT PROCESS 
 
10.1.1 Consent/Assent Procedures and Documentation 
Consent will be obtained at the NIH Clinical Center during baseline visit. The GENE-
FORECAST-SIT staff is experienced in administering consent and will escort potential 
participant to a private area of the Clinical Center to discuss the consent form. 
 
The GENE-FORECAST-SIT staff will provide the individual a copy of the consent forms, read 
all sections of the consent form and solicit for any questions.  The consent process will take 
between 20-30 minutes.  The GENE-FORECAST-SIT staff is experienced in obtaining informed 
consent for NIH trials and will not coerce or influence potential participant to enroll in the trial.  
Potential participant will be given the opportunity to consult with others prior to providing 
consent.   Consent for minors when they reach the age of majority  
Not applicable. Our study population will be 21-65 years of age. 
 

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10.1.2 Considerations for Consent of NIH staff, or family members of study team members  
Consent from staff members will be obtained by an individual independent of the staff member’s 
team whenever possible.  Otherwise, the consent procedure will be independently monitored by 
the CC Department of Bioethics Consultation Service in order to minimize the risk of undue 
pressure on the staff member. 
10.1.3 Participation of Subjects who are/become Decisionally Impaired 
Adults unable to provide consent are excluded from enrolling in the protocol. However, it is 
possible that subjects enrolled in the protocol may permanently lose the capacity to consent for 
themselves during the course of this study.  In the event this occurs, the subjects will  be 
withdrawn from the study. .  
 
10.2 STUDY DISCONTINUATION AND CLOSURE 
Study participant can discontinuation participation in protocol if any potential side effects occur 
due to consumption of study product (e.g., nausea, dizziness, rash).  The possibility of a 
temporary suspension of the study is highly unlikely. 
10.3 CONFIDENTIALITY AND PRIVACY 
Participant confidentiality and privacy is strictly held in trust by the PI, participating 
investigators, and study staff.  This confidentiality is extended to cover testing of biological 
samples and genetic tests in addition to the clinical information relating to participants.  
Therefore, the study protocol, documentation, data, and all other information generated will be 
held in strict confidence. No information concerning the study or the data will be released to any 
unauthorized third party without prior written approval of study participant. 
 
All research activities will be conducted in as private a setting as possible. 
 
Representatives of the NIH IRB and/or regulatory agencies may inspect all documents and 
records required to be maintained by the PI, including but not limited to, medical records (office, 
clinic) and pharmacy records for the participants in this study.  The NIH Clinical Center will 
permit access to such records. 
 
The study participant’s contact information will be securely stored for internal use during the 
study.  At the end of the study, all records will continue to be kept in a secure location for as long 
a period as dictated by the reviewing IRB. 
 
Study participant research data, which is for purposes of statistical analysis and scientific 
reporting, will be transmitted to and stored on dedicated and secured NIH servers and the NIH 
electronic Clinical Trial Database (CTDB).  This will not include the participant’s contact or, 
identified by a unique study identification number. 
  
The study data entry and study management systems used by the research staff will be secured 
and password protected.  At the end of the study, all study databases will be identified and 
archived on PI’s dedicated NIH server. 
 
To further protect the privacy of study participants, a Certificate of Confidentiality has been 
issued by NIH.  This certificate protects identifiable research information from forced disclosure.  
It allows the investigator and others who have access to research records to refuse to disclose 

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identifying information on research participation in any civil, criminal, administrative, legislative, 
or other proceedings, whether at the federal, state, or local level.  By protecting researchers and 
institutions from being compelled to disclose information that would identify research 
participants. Certificates of Confidentiality help achieve the research objectives and promote 
participation in studies by helping assure confidentiality and privacy to participants. 
10.4 FUTURE USE OF STORED SPECIMENS AND DATA 
No unintended specimens or residual specimens will be retained after the study is completed. 
10.5 SAFETY OVERSIGHT 
We will have an Independent Safety Monitor (ISM) responsible for safety oversight.  The ISM 
will be composed of individuals with appropriate expertise (e.g., physician, nurse, 
epidemiologist), will be independent from the study and free of conflict of interest.  The ISM 
will meet at least semiannually to assess safety and efficacy data on each arm of the study.  The 
ISM will provide input to the study PI and study staff. 
10.6 CLINICAL MONITORING 
Clinical site monitoring will be conducted to ensure the rights and well-being of trial participants 
are protected, that the reported trial data are accurate, complete, and verifiable, and that the 
conduct of the trial is in compliance with International Conference on Harmonisation Good 
Clinical Practice (ICH GCP), and with applicable regulatory requirements. 
10.7 QUALITY ASSURANCE AND QUALITY CONTROL 
The database manager will perform internal quality management of study conduct, data and 
biological specimen collection, documentation and completion.  An individualized quality 
management plan will be developed to describe quality management. 
 
Quality control (QC) procedures will be implemented beginning with the data entry system and 
data QC checks that will be run on the database will be generated.  Any missing data or data 
anomalies will be communicated to the PI and other relevant study staff for 
clarification/resolution. 
Following written (Manual of Operating Procedures (MOP), the database manager will verify 
that the study is conducted and data are generated and biological specimens are collected, 
documented (recorded), and reported in compliance with the protocol, ICH GCP, and applicable 
regulatory requirements (e.g., Good Laboratory Practices (GLP), Good Manufacturing Practices 
(GMP). 
 
The study will provide direct access to all source data/documents, and reports for the purpose of 
monitoring and auditing and inspection by local and regulatory authorities. 
 
10.8 DATA HANDLING AND RECORD KEEPING 
10.8.1 Data Collection and Management Responsibilities 
Data collection will be the responsibility of the clinical staff (i.e., Research Nurse and Physician 
Assistant) under the supervision of the PI.  The PI is ultimately responsible for ensuring the 
accuracy, completeness, legibility, and timeliness of the data reported. 
 
All source documents will be completed in a neat, legible manner and recorded in electronic 
CTDB to ensure accurate interpretation of data. 
 

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Hardcopies of the study visit worksheets will also be used as source document worksheets for 
recording data for each participant enrolled in the study.  Data recorded in the electronic case 
report form (eCRF) derived from source documents will be consistent with the data recorded on 
the source documents. 
 
All study related data (including AEs, concomitant medications, and expected adverse reaction 
data) and clinical laboratory data will be entered into the CTDB on the PIs server.  The CTDB 
will be password protected and internal quality checks conducted by the database manager, 
such as automatic range checks, to identify data that appear inconsistent, incomplete, or 
inaccurate.  Data will be entered directly from the source documents. 
10.8.2 Study Records Retention 
Study documents will be retained for a minimum of 2 years or until at least 2 years have 
elapsed since the formal discontinuation of the study or as per the NIH Intramural Records 
Retention Schedule.   
 
10.9 PROTOCOL DEVIATIONS 
It is the responsibility of the PI to use continuous vigilance to identify and report deviations to 
the NIH Institutional Review Board as per Policy 801.  The PI responsible for knowing and 
adhering to the reviewing IRB requirements. 
10.9.1 NIH Definition of Protocol Deviation 
A protocol deviation will be any change, divergence, or departure from the IRB approved 
research protocol and defined as: 
• Major deviations: Deviations from the IRB approved protocol that have, or may have 
the potential to, negatively impact the rights, welfare or safety of the subject, or to 
substantially negatively impact the scientific integrity or validity of the study. 
• Minor deviations:  Deviations that do not have the potential to negatively impact the 
rights, safety or welfare of subjects or others, or the scientific integrity or validity of the 
study. 
 
10.10 PUBLICATION AND DATA SHARING POLICY 
10.10.1Human Data Sharing Plan 
This study will be conducted in accordance with the following publication and data sharing 
policies and regulations: 
NIH Public Access Policy, which ensures that the public has access to the published results of 
NIH funded research.  This requires scientists to submit final peer-reviewed journal manuscripts 
that arise from NIH funds to the digital archive PubMed Central upon acceptance for 
publication. 
 
This study will comply with the NIH Data Sharing Policy on the Dissemination of NIH-Funded 
Clinical Trial Information and the Clinical Trials Registration and Results Information 
Submission rule.  As such, this trial will be registered at the ClinicalTrials.gov, and results from 
this trial will be submitted to ClinicalTrials.gov.  In addition, every attempt will be made to 
publish results in peer reviewed journals.   
10.10.2Genomic Data Sharing Plan 
Not applicable. 
. 

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10.11 COLLABORATIVE AGREEMENTS  
Not applicable. 
10.11.1Agreement Type 
Not applicable.  
 
10.12 CONFLICT OF INTEREST POLICY 
The independence of this study from any actual or perceived influence, such as by a 
pharmaceutical industry, is critical.  Therefore, any actual conflict of interest of persons who 
have a role in the design, conduct, analysis, publication, or any aspect of this trial will be 
disclosed and managed. Furthermore, persons who have a perceived conflict of interest will be 
required to have such conflicts managed in a way that is appropriate to their participation on the 
design and conduct of the study.  The study leadership in conjunction with the NHLBI has 
established policies and procedures for all study group members to disclose all conflicts of 
interest and will establish a mechanism for the management of all reported dualities of interest. 
 
11 ABBREVIATIONS 
AE 
Adverse Event 
AI 
Associate Investigator 
CFR 
Code of Federal Regulations 
CLIA 
Clinical Laboratory Improvement Amendments 
CMP 
Clinical Monitoring Plan 
COC 
Certificate of Confidentiality 
CRF 
Case Report Form 
CONSORT 
Consolidated Standards of Reporting Trials 
CTDB 
Clinical Trial DataBase 
DCC 
Data Coordinating Center 
DHHS 
Department of Health and Human Services 
DSMB 
Data Safety Monitoring Board 
DRE 
Disease-Related Event 
EC 
Ethics Committee 
eCRF 
Electronic Case Report Forms 
FDA 
Food and Drug Administration 
FDAAA 
Food and Drug Administration Amendments Act of 2007 
FFR 
Federal Financial Report 
FACS 
Flow Activated Cell Sorting 
GCP 
Good Clinical Practice 
GENE-
FORECAST:SIT 
Genomics, Environmental Factors and Social Determinants of 
Cardiovascular Disease In African Americans: Sodium Intervention 
Trial 
GLP 
Good Laboratory Practices 
GMP 
Good Manufacturing Practices 
GWAS 
Genome-Wide Association Studies 
HIPAA 
Health Insurance Portability and Accountability Act  
ICH 
International Conference on Harmonisation  

GENE-FORECAST SIT 
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46 
 
ICMJE 
International Committee of Medical Journal Editors 
IDE 
Investigational Device Exemption 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
ISO 
International Organization for Standardization 
LSMEANS 
Least-squares Means 
MOP 
Manual of Procedures 
NCT 
National Clinical Trial 
NIH  
National Institutes of Health 
NIH IC 
NIH Institute or Center 
NIH IDOU 
NIH Investigational Drug & Research Section Outsourcing Unit 
NHGRI 
National Human Genome Research Institute 
NHLBI 
National Heart, Lung, and Blood Institute 
OHRP 
Office for Human Research Protections 
PI 
Principal Investigator 
QA 
Quality Assurance 
QC 
Quality Control 
RVS 
Research Volunteer System 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SMC 
Safety Monitoring Committee 
SOA 
Schedule of Activities 
SOP 
Standard Operating Procedure 
TMAO 
Trimetylamine-oxidase 
UP 
Unanticipated Problem 
 
12 ADDENDUM FOR TRANSFER OF GENEFORECAST DATA 
 
 
GENE-FORECAST (GENomics, Environmental FactORs and the Social DEterminants of 
Cardiovascular Disease in African Americans  Study) was established to explore the intricate 
relationship between the unique genomic variations characteristic of African-Americans (AA) and 
the broader array of social determinants and environmental factors, collectively known as the 
'exposome', that impact the development of cardiovascular disease (CVD) in this population. 
The Sodium Intervention Trial (SIT) is a clinical trial protocol (000070) aimed at investigating the 
impact of dietary salt on the cardiovascular system, leveraging the existing pool of participants of 
the GENE-FORECAST protocol (18-HG-0048). Initially embedded within GENE-FORECAST, 
SIT sought to enroll participants who had previously consented to be re-contacted. However, as 
guidelines prohibited the inclusion of a sub-study within an ongoing natural history protocol, SIT 
was separated from GENE-FORECAST after recruiting an initial cohort of 20 participants. 
Following the division of the protocols, an additional 8 participants were enrolled in SIT, resulting 
in a total of 28 participants. To conduct comprehensive analyses of the SIT clinical trial data, it is 
imperative to have access to the baseline characteristics of all 28 participants recruited from the 

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GENE-FORECAST protocol. Therefore, we kindly request the transfer of data pertaining to these 
28 participants from the GENE-FORECAST protocol to the SIT protocol. 
 
 
 
 
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