Association of Sickle Cell Trait With Incidence of Coronary Heart Disease Among African American Individuals

Key Points Question Is sickle cell trait associated with increased risk of myocardial infarction and coronary heart disease among African American individuals? Findings In this cohort study of 23 197 African American individuals in 5 cardiovascular epidemiologic studies, sickle cell trait was not associated with increased risk of myocardial infarction or coronary heart disease among African American individuals. Meaning In this study, sickle cell trait was not associated with increased risk of fatal and nonfatal myocardial infarction or coronary heart disease, suggesting that these disorders may not be associated with sickle cell trait–related sudden death.

© 2021 Hyacinth HI et al. JAMA Network Open. sampled a total of 30,239 community-dwelling adult individuals aged ≥45 years, who self-reported as Non-Hispanic Black or White. Sampling was started in January, 2003 and was completed by October, 2007. Potential enrollees where determined from a well characterized commercially available list from the GENESYS database/sampling system. The exclusion criteria included any self-reported medical conditions (e.g. cancer) that would prevent long-term participation, or being on a waiting list for a nursing home. The sample design was such that participants were balanced on race and sex, and across the stroke buckle and stroke belt, and the rest of the contiguous United States. The resulting sample was such that, 21% were from the buckle of the stroke belt, 35% from the rest of the stroke belt area (i.e. minus the buckle) and 44% from the other 40 contiguous states of the United States. Further, 42% of the total sample were Blacks, and 55% were women.
Initial study contact between participants and interviewers was via a mailed questionnaire. This was subsequently followed by an initial in-home visit where informed consent was obtained and blood samples collected for various laboratory measures. Participants or their proxies were contacted every six months to obtain medical history and identify hospitalizations, emergency department visits, overnight stays in nursing homes or rehabilitation centers, or death within the last 6 months. For those who reported an event such as CHD or MI, medical records were sought and events were adjudicated by an expert panel of trained adjudicators, based on pre-specified adjudication criteria (see below).
A total of 10,731 participants had direct Taqman® genotyping for rs334 of which 10,714 have the relevant phenotype and covariate data, and were included in this study.

Multi-Ethnic Study of Atherosclerosis (MESA)
The MESA study was designed to investigate the correlates of subclinical cardiovascular disease (CVD) progression in a longitudinal multi-ethnic cohort free of CVD at baseline. The details of the methods and design of the MESA study have been previously reported. 4 Participants were excluded if they had physician-diagnosed CVD prior to enrollment, including angina, myocardial infarction, heart failure, stroke or TIA, resuscitated cardiac arrest or a cardiovascular intervention (e.g., CABG, angioplasty, valve replacement, or pacemaker/defibrillator placement). Pre- For this study, participant characteristics were obtained from data collected at the enrollment visit from physical measures, standardized questionnaires, and laboratory tests. These data include demographic information (age, sex, race/ethnicity), medical history, medications, and alcohol and tobacco use. Resting blood pressure was determined by taking three measurements with the participant in the seated position. Systolic and diastolic blood pressures were recorded as the average value of the last two measurements from both the first and second study examinations.  For this study, a total of 1,556 subjects (who had complete genotype, phenotype and covariate data) were included; out of which 152 were directly genotyped for rs334 via sequencing and the rest were imputed.

Jackson Heart Study (JHS)
The JHS is a single-site, prospective, population-based study designed to explore the environmental, behavioral, and genetic factors that influence the development of CVD among African Americans. A total of 5,301 women and men between the ages of 21 and 94 were recruited between 2000 and 2004 from a tri-county area of Mississippi: Hinds, Madison, and Rankin Counties. Participants were recruited from four sources, including (1) randomly sampled households from a commercial listing; (2) ARIC participants; (3) a structured volunteer sample that was designed to mirror the eligible population; and (4) a nested family cohort. Overviews of the JHS including the sampling and recruitment, sociocultural data, and laboratory methods have been described and published previously. [5][6][7][8] The institutional review boards of the following participating institutions approved the study: the University of Mississippi Medical Center, Jackson State University, and Tougaloo College. All participants provided written informed consent for the parent JHS and subsequent ancillary studies including this one. Unrelated participants were between 35 and 84 years old, and members of the family cohort were ≥ 21 years old when consent for genetic testing was obtained and blood was drawn for DNA extraction.
The baseline examination consisted of a home interview, self-administered questionnaires, and a clinic visit. Medications taken in the prior 2 weeks were brought to clinic and transcribed verbatim with subsequent coding by a pharmacist. After an overnight fast, anthropometric and seated blood pressure measurements were obtained and venipuncture/urine collection was performed in accordance with the National Committee for Clinical Laboratory Standards. Blood pressure was © 2021 Hyacinth HI et al. JAMA Network Open. measured by trained technicians using a Hawksley random zero manometer and determined by the arithmetic average of two readings taken 1 minute apart after a five-minute rest. 9 A total of 2,175 subjects (having the relevant phenotype and covariate data) were included in the current study, of which 1,827 were directly genotyped for rs334 via sequencing, the rest were imputed.

Atherosclerosis Risk in Communities (ARIC) study
The Atherosclerosis Risk in Communities (ARIC) Study, sponsored by the National Heart, Lung and Blood Institute (NHLBI), is a prospective epidemiologic study conducted in four U.S. pressures (BP) were recorded as an average of two of the last three measurements performed with the participant comfortably seated. Hypertension was defined as systolic BP≥140 mmHg, diastolic BP≥90 mmHg, or self-reported use of antihypertensive medication. Body mass index (kg/m 2 ) was calculated from weight and height measurements. Blood glucose was determined from blood samples collected during clinic visits. Diabetes was defined as fasting glucose≥126 mg/dl, non-fasting glucose≥200 mg/dl, self-reported physician diagnosis of diabetes, or selfreported use of oral hypoglycemic medication or insulin. 11 The current study includes 2,848 African American subjects from ARIC who had provided consent for genetic studies and who had sufficient genetic material and data, all of whom were directly genotyped for rs334.

Exome Sequencing
Exome sequencing (N=2,052) was performed through the National Heart, Lung, and Blood

Genotyping and Imputation
Direct genotype data for the rs334 variant was obtained by custom genotyping from the REGARDS and ARIC study. Whole blood DNA was isolated from the buffy coat layer using the Gentra Puregene Blood Kit (Qiagen, Inc., Valencia, CA; www.qiagen.com). Carriers of HbS were identified from biallelic variation in the single nucleotide polymorphism, rs334. In addition, carriers information to include in this analysis. Standard quality control measures were performed. 14 Both target and reference panels were pre-phased using BEAGLE. 15 The reference panel was then imputed into the target using minimac. 16 The imputation quality score Rsq (which is equivalent to the squared correlation between proximal imputed and genotyped SNP) was 0.86. Individuals with the rs334 genotype derived via imputation were coded as having 0, 1, or 2 risk alleles using the most probable genotype. The imputation of genotype in these cohorts, have been done as part of multiple projects [17][18][19][20] and the similarity in description with this supplemental methods is due to the fact that the same set of data is being utilized for the current study. Difference in the number of participants between projects is due to phenotype and/or covariate missingness, which is purely random.
Using N=1,132 samples with both exome sequence and imputed genotype data from JHS, we validated our imputed genotypes (Table S1). The kappa correlation for sequenced and imputed values was 0.88 (95%CI 0.84 -0.92).

WHI
The details of the method for ascertainment and adjudication of cardiovascular disease, specifically coronary heart disease (CHD) phenotypes among WHI participants has already been published. 21 But briefly, WHI participants were contacted every 6 months or 12 months for those in clinical trials or observational study arm respectively; for medical history updates with regards to incident cardiovascular and specifically coronary heart disease outcomes. Any report of occurrence of coronary heart disease outcome in a participant was then investigated and adjudicated by a panel of independent physician adjudicators. If the coronary disease outcome is a myocardial infarction (MI), only the first one is adjudicated. Note that an MI following an angina or any other non-MI CHD outcome also required adjudication. 21 MI and CHD events were defined based on established criteria using a combination medical history, serum cardiac enzyme and electrocardiographic findings. 22

REGARDS
A full and detailed method for case ascertainment and adjudication has been published. 23  Composite CHD phenotype included fatal and non-fatal MI, coronary revascularization procedures, and non-MI fatal or non-fatal CHD.

MESA
In addition to the standard MESA examinations, participants or next of kin were contacted by history, serum cardiac enzyme levels and ECG findings. They are classified using the Minnesota code as "Definite", "Probable" or "No/Absent".

ARIC
In addition to the standard ARIC examinations, participants were contacted by telephone annually, and hospital surveillance in the ARIC communities was performed to obtain interim information about hospitalizations. A complete detail of the methodology for ascertainment of and adjudication of cardiovascular events has already been published. 30 Briefly, MI or CHD events were identified based on a review of the medical and death records by ARIC staff members. MI was diagnosed based on clinical symptoms, abnormal cardiac enzyme levels and ECG findings.
A computer algorithm was used to assign electrocardiographic (ECG) diagnosis based on the Minnesota code. An independent expert physician reviews cases for which there is a disagreement between the discharge diagnosis and the computer assigned diagnosis. MI were classified as "Definite", "Probable", "Suspect" or "No/Absent". Fatal CHD was classified on the basis of chest pain, prior history of CHD, and underlying cause of death.
In all cohorts, definite and probable MI were combined to define MI. For this study, we only analyzed incident MI or CHD. Participants with a prior history of MI or CHD were excluded from our analysis.