The Genetic, Protein, and Lipid Basis of Variation in Cholesterol Efflux
Conditions: Lipid Metabolism Disorders
Sponsor: University of Texas Southwestern Medical Center

STUDY PROTOCOL 
 
 
 
Official title:  The Genetic, Protein, and Lipid Basis of 
Variation in Cholesterol Efflux 
 
NCT number: NCT04061018 
IRB Approved date: 06-21-21 

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Protocol for Family Pedigree Study on Cholesterol Efflux in the Dallas Heart Study 
 
 
 
“The Genetic, Protein, and Lipid Basis of Variation in Cholesterol Efflux” 
 
Anand Rohatgi, MD, MSCS 
5323 Harry Hines Blvd Dallas, TX 75390-8830 
214/645-7500 Fax 214/645-2480 
anand.rohatgi@utsouthwestern.edu 
 
 
 
 
Objective 
 
 
 
 
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Background 
 
 
 
 
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Study Design                                                 Pg 2 
Eligibility Criteria 
 
 
 
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Recruitment 
 
 
 
 
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Training 
 
 
 
 
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Research Information  
 
 
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Policies for Repository 
 
 
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Removal from Study  
 
 
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Possible Risks 
 
 
 
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Adverse Events 
 
 
 
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Possible Benefits 
 
 
 
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Costs to subjects 
 
 
 
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Biostatistics 
 
 
 
 
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Optional Sub Study 
 
 
 
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OBJECTIVE 
To create a family pedigree and biobank repository of blood and DNA from participants from the 
Dallas Heart Study with extreme low or high cholesterol efflux.    
 
BACKGROUND  
Low high-density lipoprotein cholesterol (HDL-C) is a major risk factor for atherosclerotic 
cardiovascular disease (ASCVD), the leading cause of death.  However, strategies to improve 
atheroprotection by raising HDL-C levels have failed to improve outcomes.   Cholesterol efflux 
from the macrophage to the circulation is the first critical step of reverse cholesterol transport, a 
key atheroprotective pathway.  We directly measured the cholesterol efflux capacity of plasma 
in over 3,000 participants in the population-based Dallas Heart Study and were the first to show 
that cholesterol efflux at baseline has a graded, inverse association with incident ASCVD events 
and improves risk prediction of ASCVD beyond cardio-metabolic factors and lipid measures.  
Our findings established that measurement of cholesterol efflux as a direct reflection of the anti-
atherosclerotic pathway is clinically relevant.  Intriguingly, we found only weak correlations 
between cholesterol efflux and HDL-C, which may explain the failure of strategies targeting 
HDL-C levels to promote atheroprotection.  The mechanisms that underlie variation in 
cholesterol efflux are unknown.  In this study will try to identify genetic, protein, and lipid factors 
that govern variation in cholesterol efflux at a population level.   
 
STUDY DESIGN 
To establish the heritability of extreme cholesterol efflux with a prospective family pedigree 
cohort, participants and their relatives (parents, siblings, adult children, grandparents, 
aunts/uncles, cousins) of extreme low or high cholesterol efflux will be recruited by a study 
coordinator under my direction (the original consent for participation in the Dallas Heart Study 
also includes separate consent to be contacted for future studies, of which 99% assented to be 
contacted). The Dallas Heart Study maintains an active contact list of all participants and has 
successfully enrolled participants in multiple prospective family studies. 
 
At enrollment, which will occur at UTSouthwestern Medical Center or at proband’s home if the 
participant is not able or readily willing to come to UTSW for a study visit, Dr. Rohatgi or a study 
coordinator will perform a survey to collect information including: demographics health history, 
lifestyle measures, and medications. Blood will then be collected on-site by venipuncture and 
plasma, serum, and cells will be stored at -80° Celcius. All efflux measurements will be 
completed in the PI’s laboratory. Plasma stores at -80° celcius will be depleted of apolipoprotein 
B lipoproteins using polyethylene glycol precipitation. Efflux will be determined as previously 
described from J774 macrophages to apolipoprotein B-depleted plasma. All efflux assays will be 
performed in duplicate in blinded fashion. Results will be normalized to the measured efflux by a 
pooled reference sample evaluated on every plate. All samples will be run in duplicate and the 
average value will be reported. In our own prior studies, intra-plate CV was 3.3% and inter-plate 
CV was 7.4%. Efflux measurements on each participant and their family members will be run on 
the same plate and repeated in each quadrant to minimize potential sources of analytical 
variability. The PI’s laboratory is well-versed in high-throughput assessment of cholesterol 
efflux, having performed measurements in over 3000 participants in the Dallas Heart Study and 

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selected as a core laboratory to measure cholesterol efflux by an external investigator for over 
1500 participants. 
 
The expected outcome is the determination of degree of heritability of the extreme low or high 
cholesterol efflux phenotype, specific for sex and ethnicity. This will establish for the first time to 
what degree inherited verses environmental factors associate with variation in cholesterol efflux. 
 
ELIGIBILTY CRITERIA 
There are around 500 male and female participants who enrolled in the initial Dallas Heart 
Study (DHS1), who provided blood and had risk factor assessment are eligible.  Those with 
existing cholesterol efflux measurements from our prior study will be included. Specific inclusion 
for this study will include those below or above the sex- and ethnicity-specific 10th and 90th% of 
cholesterol efflux. Family members of the DHS participants may also be invited to participate.  
All ethnic and racial categories will be included.  Participants or their legal guardian must be 
able to give informed written consent prior to participating. This study includes no prospective 
direct benefit to individual subjects.  
 
Exclusion criteria: Pregnant women or those with existing HIV, malignancy, or autoimmune 
disease will be excluded from the study.    
 
RECRUITMENT 
Family pedigree studies have been successfully completed from the Dallas Heart Study. 
Participants enrolled in the Dallas Heart Study (DHS1) who provided consent to be re-contacted 
for future studies and meet the inclusion/exclusion criteria will be recruited by phone, hard mail, 
and email.  Two-five family members of these participants may also be included.  Recruitment 
and study procedures will take place at UTSW in a research clinic.  All participants who agree to 
participate will give informed written consent.  A study staff member will explain the study and 
review the consent with each participant.  A copy of each signed form will be kept in the 
participant’s study file.  Each participant will also receive signed copies. The total needed to 
complete the study would be around 180 participants.   
 
TRAINING 
All study staff have completed the appropriate training. 
 
SOURCES OF RESEARCH MATERIAL 
1)  Blood samples for storage of plasma and serum, extraction of DNA, and isolation of 
monocytes and lymphocytes.  The amount of blood drawn will be at most 75ml. Blood samples 
obtained will be for research purposes only, 2) demographic information, 3) family history and 
pedigree and 4) other self-reported pertinent information recorded on questionnaire. 
  
 
 
POLICIES OF THE REPOSITORY 

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1) Collection of samples - Subjects will be identified on the collection container subject ID 
and/or accession number. When samples are distributed to the laboratory the only identifiers will 
be subject ID and/or accession number.  
2) Storage of samples – Samples will be stored indefinitely in the Rohatgi Lab (K4.120) on 
South Campus or in the Human Genetics Core Laboratory, NA2-118 at UT Southwestern 
Medical Center.  The primary contact will be Anand Rohatgi 214/645-7500, 
anand.rohatgi@utsouthwestern.edu.  Storage containers will be labeled with the study subject 
ID number and/or accession number.  These numbers can only be traced to subjects via a 
password protected, secure computer database in the Core Laboratory or via the subject’s chart 
kept in a locked records cabinet in study coordinator’s office. 
3) Linkage of sample to the subject - When samples are distributed to the laboratory the only 
identifiers will be subject ID and/or accession number.  All subsequent laboratory procedures 
will refer to the subject ID and/or accession number.  Linkage of the subject’s PHI (name, etc.) 
to subject IDs will be limited to the study staff.  
4) Use of samples – Samples will be used for the study of cholesterol efflux.  Plasma and 
serum aliquots will be stored for testing of biomarkers. DNA will be extracted from blood 
samples. Single nucleotide polymorphism (SNP) and sequence analysis will be performed on 
the DNA samples.  Monocytes and lymphocytes will be isolated and transformed for 
immortalized cell lines.  Samples will not be used by investigators outside of UT Southwestern 
except as part of an academic collaboration or a commercial agreement approved by the Office 
of Contracts management.  Samples may be shared as part of such agreement, but in all cases, 
copies of these materials will be kept at UT Southwestern.  Identifying information will not be 
shared with investigators outside of UT Southwestern.   
5) Use of samples by investigators not affiliated with UT Southwestern Medical Center – A 
current record of Multiple Assurance for an IRB at another medical center will be maintained if 
data and specimens are sent from the repository at UT Southwestern to another medical center.  
All samples will be de-identified prior to being shared with any investigator and if an investigator 
at another medical center receives data and specimens from investigators at UT Southwestern, 
documentation will be maintained that a) the recipient of the samples from the UT Southwestern 
repository acknowledges that the conditions for use of the research material are governed by 
the IRB at UT Southwestern in accordance with 45 CFR 46, b) the recipient agrees to comply 
fully with all such conditions and to report promptly to the repository at UT Southwestern any 
proposed changes in the research project and any unanticipated problems involving risks to 
subjects or others, c) the recipient remains subject to applicable State or local laws or 
regulations and institutional policies which provide additional protections for human subjects, 
and d) the research material may be utilized only in accordance with the conditions stipulated by 
the IRB at UT Southwestern.  Any additional use of this material requires prior review and 
approval by the IRB at UT Southwestern and, where appropriate, by an IRB at the recipient site. 
6)  Approval of sample use – Samples will be available to investigators and their collaborators 
after approval by the principal investigator.  All research utilizing these samples will require 
approval by the IRB at UT Southwestern. 
7) Computer security – All data pertaining to subjects’ identities will be kept in a password 
protected file and will not be connected to the internet.  
8) Reports of DNA tests results - The results of DNA tests are not intended to be used to 
influence decisions about a subject’s medical care.  The results of the genetic research tests will 
not be given to the study participants or their physicians.  Results will not be given to private 
physicians, medical insurance providers, life insurance providers, or employers.  
9) Publication of private information – Any information that could be linked to a subject will be 
published only with the subject’s consent. 
10) Certificate of Confidentiality – A Certificate of Confidentiality will be obtained. 

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11) Release of subject’s sample to a third party for purposes other than research – Samples will 
not be released to a third party for purposes other than research. 
12) Future contact with subjects – Subjects may be contacted to participate in future research, 
to invite a subject’s family members to participate in research, or to obtain health information. 
REMOVAL FROM STUDY 
A subject may request to discontinue study participation.  The subject will not be asked for 
further information or samples.  The identity of the subject will be removed from the research 
records.  However, neither the data generated by the subject’s samples nor any samples will be 
destroyed because they cannot be linked to the subject in any way. 
 
POSSIBLE RISKS TO SUBJECTS 
There is minimal risk to subjects participating in this study. Risks from blood draws include, 1) 
discomfort, bleeding, and/or bruising, 2) dizziness or feeling faint, 3) infection may develop on 
rare occasions at the site where blood was collected. Blood will be drawn by trained, 
experienced personnel.   There is also a risk of psychological, social, or legal harm to subjects 
in the event of loss of confidentiality.  All research staff have completed appropriate Human 
Subjects and HIPAA training.  Results of DNA tests will not be released for purposes other than 
research and a certificate of confidentiality will be obtained. 
 
ADVERSE EVENTS 
The study coordinator will be responsible for reporting any adverse events. 
 
POSSIBLE BENEFITS 
There is not prospect of direct benefit to the individual subject.  Findings may benefit others in 
the treatment and prevention of atherosclerotic heart disease.  
 
COSTS TO SUBJECTS 
There are no costs to the subjects to participate in this research. After successful completion of 
the study visit, a participant will receive a $35 gift card and additional amount of $35 to cover for 
their transportation expenses. 
 
BIOSTATISTICS 
NA 
OPTIONAL SUBSTUDY 
Title – Understanding the Effect of Food Challenge on Lipid Metabolism and Cholesterol Efflux 
Capacity 
Background-  
Elevated plasma lipids and decreased high density cholesterol levels are associated with 
increased risk of atherosclerotic cardiovascular diseases. Plasma lipids are usually measured 
after 9hrs of fast even though most of the time in human life is spent in post prandial state (PP). 
It is known that lipid containing lipoprotein particles change in PP state and effect of such 
particle remodeling on cholesterol efflux is unknown. Our preliminary findings from current study 
show clear difference in cholesterol efflux pattern in fasting vs post prandial state. Therefore we 
plan to test this phenomenon in a controlled intervention.  

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Aim- The purpose of this optional sub study is to evaluate the effect of lipoprotein remodeling in 
post prandial state on cholesterol efflux.  
After completion of the primary visit, we will determine eligibility of study participants to 
participate in the metabolic remodeling sub study based on their cholesterol efflux profiles. A 
total of 100 participants will be recruited from two groups - 10th and 90th% of cholesterol efflux 
(high and low). CEFS study participants who provided consent to be re-contacted for future 
studies and meet the inclusion-exclusion criteria will be recruited by phone, hard mail or email.  
All participants who agree to participate will give informed written consent.  A study staff 
member will explain the study and review the consent with each participant.  A copy of each 
signed form will be kept in the participant’s study file.  Each participant will also receive signed 
copies. 
 
At enrollment, which will occur at UT Southwestern Medical Center, up to 50ml of fasting blood 
sample will be collected by venipuncture after 9hr overnight fast to isolate plasma, serum and 
cells. A standardized meal replacement shake, Ensure Plus (8oz) consisting of 13 g protein, 52 
g carbohydrate and 11 g fat will be provided to the study participant and up to 25ml of post 
prandial blood sample will be obtained 2hrs after ingestion of the shake. Both, fasting and post 
prandial samples will be analyzed to measure the cholesterol efflux capacity, plasma lipids etc. 
During the visit, study staff will also perform a survey to collect demographics, health history, 
lifestyle measures and medications of the study participant.   
After successful completion of the study visit, a participant will receive a $100 gift card and a 
parking voucher to cover parking expenses. There are no funds available to pay for 
transportation to and from UTSW, lost time away from work and other activities, lost wages or 
child care expenses.