Design and Analysis of Replication Cohorts
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Supplementary Information
Design and Analysis of Replication Cohorts
Study Populations
The replication study populations consisted of The Nurses’ Health Study (NHS) and The
Health Professionals Follow-Up Study (HPFS) participants. In both replication cohorts, nested case-control studies were designed using incident coronary heart disease (CHD), with non-fatal MI as per the WHO criteria (14) and fatal CHD as outcomes. Among participants who provided blood samples and who were free of diagnosed cardiovascular disease or cancer at blood draw, 928 cases of incident CHD were identified. Using risk- set sampling, controls were selected randomly and matched in a 1:2 ratio on age, smoking, and month of blood return. Analyses were restricted to participants with available genotype data as well as measures of blood lipids and inflammatory markers, yielding final sample sizes of 1 200 for NHS and 1 295 for HPFS.
Biochemical Measurements
Blood samples was requested from all active participants in NHS and HPFS and collected from 32 826 women and 18 225 men. Plasma lipids and inflammatory markers were assessed using standard methods with reagents from Roche Diagnostics (Indianapolis,
IN) and Bayer Diagnostics (New York, NY).
Genotyping Genotyping was performed using the Affymetrix Genome-Wide Human SNP 6.0 array and the Birdseed calling algorithm. Genotypic data for a total of 1 330 HPFS samples
(98%) and 96% of the NHS samples passed laboratory technical quality control criteria and missing call <0.05. Population structure was investigated by principal component analysis and samples with evidence of non-European genetic ancestry were excluded
(n=24). Imputation of ~2.5 million SNPs was performed using MACH software (Version
1.0.16) with HapMap CEU phased II data (Release 22) as the reference panel. Of seven
SNPs used in the analyses, two (rs3734397 and rs2294867) were on the Affymetrix chip, while five (rs10498676, rs17239120, rs17544464, rs2115564, rs761179) were imputed with r-squared values > 0.9.
Statistical Analysis
Linear and conditional logistic regression analyses with individual SNPs as predictors and inflammation markers, blood lipids, and MI as outcomes as described in Materials and Methods were replicated in the NHS and HPFS. All analyses were adjusted for sex
(by restriction) and age. Least square means1 (+/- standard errors) of inflammatory markers, HDL-
cholesterol, and triglycerides by genotype among controls.
Homozygous Heterozygous Homozygous P value2
(major allele) (minor allele) rs10498676 (ELOVL2) hsCRP, mg/L
Costa Rica Study (n=1 493) 2.20±0.09 2.18±0.11 2.08±0.27 0.70
NHS (n=797) 1.78±0.09 1.75±0.13 2.08±0.61 0.77
HPFS (n=860) 1.03±0.05 1.10±0.08 1.15±0.27 0.69 VCAM-1, ng/mL
Costa Rica Study (n=1 500) 722.38±8.65 705.39±10.51 715.15±24.93 0.30
NHS (n=411) 669.84±8.75 670.62±13.02 809.67±52.76 0.45
HPFS (n=508) 1282.00±15.87 1258.69±22.36 1390.12±74.30 0.17 HDL cholesterol, mg/dL
Costa Rica Study (n=1 773) 41.97±0.30 42.00±0.37 41.39±0.88 0.73
NHS (n=797) 60.80±0.74 58.71±1.05 60.29±3.43 0.27
HPFS (n=860) 46.36±0.53 45.40±0.76 45.10±2.09 0.53 Triglycerides, mg/dL
Costa Rica Study (n=1 789) 189.91±3.07 188.19±3.78 192.66±9.55 0.72
NHS (n=540) 98.79±2.54 105.56±4.17 101.35±13.73 0.35
HPFS (n=492) 113.38±3.41 115.20±5.08 119.51±14.84 0.87 rs3734397 (ELOVL2) hsCRP, mg/L
Costa Rica Study (n=1 493) 2.16±0.08 2.17±0.12 2.85±0.50 0.29
NHS (n=797) 1.90±0.10 1.58±0.11 1.87±0.41 0.09
HPFS (n=860) 1.11±0.06 1.01±0.07 0.84±0.16 0.18 VCAM-1, ng/mL Costa Rica Study (n=1 500) 718.57±8.04 707.27±11.67 731.29±33.14 0.67
NHS (n=411) 653.09±9.44 647.85±11.87 663.26±35.40 0.88
HPFS (n=508) 1284.26±16.74 1260.14±20.83 1348.30±62.51 0.31 HDL cholesterol, mg/dL
Costa Rica Study (n=1 773) 42.08±0.28 41.65±0.40 42.01±1.50 0.46
NHS (n=797) 60.27±0.77 60.30±1.00 56.5±2.70 0.47
HPFS (n=860) 45.42±0.56 46.70±0.72 47.71±1.75 0.23 Triglycerides, mg/dL
Costa Rica Study (n=1 789) 188.41±2.89 188.58±4.16 183.35±11.90 0.83
NHS (n=540) 101.86±5.46 98.80±3.57 102.87±10.91 0.77
HPFS (n=492) 116.37±3.66 113.45±4.68 98.42±10.47 0.25 rs17239120 (ELOVL4) hsCRP, mg/L
Costa Rica Study (n=1 493) 2.16±0.08 2.31±0.16 2.42±0.99 0.31
NHS (n=797) 1.78±0.08 1.83±0.23 0.93±0.62 0.31
HPFS (n=860) 1.06±0.05 1.00±0.13 1.57±2.14 0.74 VCAM-1, ng/mL
Costa Rica Study (n=1 500) 716.23±7.49 711.64±14.47 768.70±65.51 0.96
NHS (n=411) 653.53±7.66 635.86±21.61 661.82±96.08 0.73
HPFS (n=508) 1280.82±13.61 1261.28±37.68 1256.29±231.2 0.88 HDL cholesterol, mg/dL
Costa Rica Study (n=1 773) 42.03±0.26 41.69±0.51 39.27±2.30 0.30
NHS (n=797) 59.63±0.64 62.48±1.66 73.65±6.33 0.07
HPFS (n=860) 46.10±0.46 45.30±1.26 45.23±7.24 0.83 Triglycerides, mg/dL
Costa Rica Study (n=1 789) 189.60±2.68 183.13±5.18 168.04±23.60 0.15
NHS (n=540) 102.34±2.31 91.80±5.70 82.75±25.38 0.16 HPFS (n=492) 113.97±2.96 115.85±8.01 111.45±60.25 0.97 rs17544464 (ELOVL5) hsCRP, mg/L
Costa Rica Study (n=1 493) 2.23±0.09 2.10±0.12 2.15±0.32 0.42
NHS (n=797) 1.79±0.08 1.65±0.23 4.74±9.13 0.39
HPFS (n=860) 1.06±0.05 1.01±0.13 2.20±4.40 0.62 VCAM-1, ng/mL
Costa Rica Study (n=1 500) 709.51±8.08 726.98±11.89 737.65±28.68 0.14
NHS (n=411) 651.48±7.56 654.55±23.65 590.83±163.26 0.90
HPFS (n=508) 1278.38±13.45 1266.51±39.74 1654.48±303.7 0.25 HDL cholesterol, mg/dL
Costa Rica Study (n=1 773) 41.87±0.28 42.33±0.41 40.65±1.03 0.99
NHS (n=797) 60.19±0.63 59.78±1.81 47.86±11.89 0.58
HPFS (n=860) 46.08±0.46 45.63±1.25 35.84±8.87 0.49 Triglycerides, mg/dL
Costa Rica Study (n=1 789) 188.12±2.89 188.02±4.23 192.51±11.08 0.81
NHS (n=540) 100.12±2.24 106.36±7.22 115.23±55.63 0.62
HPFS (n=492) 114.30±2.94 112.77±8.04 174.23±158.67 0.71 rs2115564 (ELOVL5) hsCRP, mg/L
Costa Rica Study (n=1 493) 2.11±0.13 2.18±0.10 2.28±0.14 0.20
NHS (n=797) 1.64±0.12 1.83±0.11 1.87±0.17 0.38
HPFS (n=860) 1.16±0.09 0.97±0.05 1.19±0.11 0.05 VCAM-1, ng/mL
Costa Rica Study (n=1 500) 702.09±12.55 716.42±9.10 720.02±12.67 0.75
NHS (n=411) 643.61±13.21 659.83±10.52 644.74±15.17 0.54
HPFS (n=508) 1287.43±24.69 1262.33±17.50 1307.20±28.20 0.36 HDL cholesterol, mg/dL Costa Rica Study (n=1 773) 41.30±0.44 42.05±0.32 42.37±0.44 0.06
NHS (n=797) 60.49±1.10 59.53±0.85 60.92±1.27 0.61
HPFS (n=860) 45.87±0.82 46.36±0.59 45.26±0.95 0.60 Triglycerides, mg/dL
Costa Rica Study (n=1 789) 193.15±4.66 187.88±3.24 184.43±4.44 0.13
NHS (n=540) 96.31±3.75 101.78±3.11 105.31±4.93 0.28
HPFS (n=492) 119.10±5.64 110.34±3.69 118.04±6.40 0.30 rs2294867 (ELOVL5) hsCRP, mg/L
Costa Rica Study (n=1 493) 2.15±0.11 2.22±0.10 2.17±0.15 0.82
NHS (n=797) 1.74±0.12 1.81±0.11 1.82±0.23 0.87
HPFS (n=860) 1.10±0.07 1.00±0.06 1.18±0.15 0.33 VCAM-1, ng/mL
Costa Rica Study (n=1 500) 706.43±10.95 715.76±9.32 732.46±14.44 0.14
NHS (n=411) 649.57±11.27 659.03±10.60 630.29±20.26 0.43
HPFS (n=508) 1277.69±20.34 1274.05±18.34 1298.36±37.67 0.83 HDL cholesterol, mg/dL
Costa Rica Study (n=1 773) 41.27±0.38 42.23±0.32 42.40±0.50 0.04
NHS (n=797) 59.80±0.93 60.58±0.88 59.42±1.66 0.75
HPFS (n=860) 46.23±0.69 45.60±0.61 46.93±1.23 0.57 Triglycerides, mg/dL
Costa Rica Study (n=1 789) 186.84±3.93 191.54±3.39 182.64±5.00 0.68
NHS (n=540) 94.80±3.07 104.50±3.34 109.32±6.55 0.03
HPFS (n=492) 119.56±4.73 109.93±3.79 115.59±8.08 0.25 rs761179 (ELOVL5) hsCRP, mg/L
Costa Rica Study (n=1 493) 2.17±0.09 2.14±0.11 2.63±0.32 0.33 NHS (n=797) 1.73±0.11 1.81±0.12 1.85±0.24 0.83
HPFS (n=860) 1.07±0.07 1.05±0.06 1.09±0.15 0.95 VCAM-1, ng/mL
Costa Rica Study (n=1 500) 718.91±8.73 704.17±10.41 752.19±23.82 0.89
NHS (n=411) 647.56±11.12 659.22±10.73 637.05±20.49 0.55
HPFS (n=508) 1275.29±19.71 1277.60±18.75 1293.22±38.54 0.91 HDL cholesterol, mg/dL
Costa Rica Study (n=1 773) 41.51±0.30 42.44±0.36 42.78±0.82 0.02
NHS (n=797) 59.73±0.90 60.46±0.90 60.22±1.69 0.85
HPFS (n=860) 46.10±0.66 45.49±0.62 47.87±1.30 0.25 Triglycerides, mg/dL
Costa Rica Study (n=1 789) 187.72±3.13 189.72±3.74 184.99±8.56 0.94
NHS (n=540) 97.08±3.09 103.38±3.36 105.99±6.65 0.24
HPFS (n=492) 117.05±4.40 112.63±3.98 110.36±8.49 0.65