Online Appendix for the following JACC article
TITLE: Remnant Cholesterol as a Causal Risk Factor for Ischemic Heart Disease
AUTHORS: Anette Varbo, MD, Marianne Benn, MD, PhD, DMSc, Anne Tybjærg-
Hansen, MD, DMSc, Anders B. Jørgensen, MD, Ruth Frikke-Schmidt, MD, PhD, DMSc,
Børge G. Nordestgaard, MD, DMSc
APPENDIX

Supplementary Table 1. Characteristics of individuals in the Copenhagen City Heart Study and Copenhagen General Population Study combined by genotypes.
Alleles No.
Age years
Women
%
Body mass Alcohol consumption index kg/m 2 %
Diabetes mellitus
%
Menopause, women only
%
Hypertension Smoking Statin use
% % %
Remnant cholesterol
TRIB1 rs2954029
GCKR rs1260326
APOA5 rs651821
Uncorrected P-trend
Bonferroni corrected P-trend
0-1
2
3
22,018
22,236
17,119
Remnant cholesterol and HDL cholesterol
57(47-67)
57(47-67)
57(46-67)
0.1
NS
LPL rs1801177
LPL G188E
LPL rs268
LPL rs328
Uncorrected P-trend
Bonferroni corrected P-trend
0-1
2
3-4
11,304
45,832
4,237
57(47-67)
57(47-67)
57(47-67)
0.3
NS
55
55
56
0.8
NS
55
56
55
0.6
NS
26(23-28)
26(23-28)
25(23-28)
0.2
NS
26(23-28)
26(23-28)
25(23-28)
0.02
NS
65
65
65
0.4
NS
65
65
64
0.7
NS
65
66
64
0.1
NS
65
66
65
0.8
NS
4
4
4
0.4
NS
4
4
4
0.8
NS
19
18
17
2 x 10 -4
0.07
18
18
18
0.4
NS
8
9
9
5 x 10 -7
2 x 10-4
9
8
8
0.3
NS
26
26
26
0.2
NS
26
26
26
0.7
NS

HDL cholesterol
LIPC -480C>T
ABCA1 N1800H
ABCA1 R2144X
Uncorrected P-trend
Bonferroni corrected P-trend
0
1
2-3
2,766
20,768
37,839
57(47-67)
57(47-67)
57(47-67)
0.7
NS
55
55
56
0.4
NS
26(23-28)
25(23-28)
26(23-28)
0.3
NS
17
18
18
0.3
NS
4
4
4
0.5
NS
64
65
66
0.2
NS
66
65
65
0.6
NS
25
27
26
0.5
NS
LDL cholesterol
APOB rs5742904
LDLR W23X
LDLR W66G
LDLR W556S
PCSK9 rs11591147
Uncorrected P-trend
Bonferroni corrected P-trend
0-1
2
3
1,587
59,707
79
57(47-67)
57(47-67)
54(42-67)
1.0
NS
0.6
NS
55
55
52
26(23-29)
26(23-28)
26(23-28)
0.2
NS
0.9
NS
18
18
13
0.1
NS
3
3
4
0.5
NS
64
65
66
0.6
NS
65
65
59
0.6
NS
25
26
17
Continuous values are summarized as median and interquartile range and categorical values are summarized in percent. Diabetes is self-
5
8
55
7 x 10 -17
3 x 10 -14 reported disease, nonfasting plasma glucose >11.0 mmol/L and/or hospitalization or death due to diabetes (ICD8:249,250;
9
8
8
0.2
NS

ICD10:E10,E11,E13,E14). Alcohol consumption is percent individuals with intake >14 units per week for women and >21 units per week for men (1 unit=12g). P-values for trend are by Cuzick’s extension of a Wilcoxon rank-sum test. The Bonferroni corrected P-trend is uncorrected P-trend multiplied with the number of tests (=36) to accommodate multiple testing. NS=not significant.

Remnant cholesterol
N
12,668
TRIB1 rs2954029
27,405
14,851
GCKR rs1260326
22,080
25,294
7,550
APOA5 rs651821
48,360
6,368
196
1 x 10
-31
7 x 10
-37
1 x 10
-84
1 x 10
-29
3 x 10
-23
2 x 10
-71
4 x 10
-13 p=0.007
2 x 10
-19
2 x 10
-19 p=0.1
9 x 10
-7
Remnant cholesterol and HDL cholesterol
LPL rs1801177
53,338
1,576
10
6 x 10
-15
LPL
G188E
LPL rs268
54,882
42
52,230
2,665
29 p=0.001
4 x 10
-30
LPL rs328
597
10,032
44,295
3 x 10
-49
7 x 10
-18 p=0.001
1 x 10
-40
6 x 10
-60
2 x 10
-12 p=0.005
5 x 10
-37
2 x 10
-48 p=0.1
p=0.9
p=0.04
p=0.05
HDL cholesterol
LIPC
-480C>T
2,478
18,538
33,908
ABCA1
N1800H
54,843
81
ABCA1
R2144X
54,918
6
2 x 10
-6 p=0.8
p=0.5
p=0.002
2 x 10
-4 p=0.4
1 x 10
-56
9 x 10
-19 p=0.002
LDL cholesterol
APOB rs5742904
54,902
22
LDLR
W23X
LDLR
W66G
LDLR
W556S
54,923
1
54,913
11
54,922
2
PCSK9 rs11591147
9
1,469
53,446 p=0.9
p=0.2
p=0.1
p=0.1
p=0.8
0.4
0.8
1.2
Remnant cholesterol
(mmol/L) p=0.9
p=0.4
p=0.03
p=0.2
p=0.5
p=0.9
p=0.6
p=0.02
p=0.9
p=0.1
0.2
0.6
1.0
Remnant cholesterol to HDL cholesterol
(ratio)
1.0
1.5
2.0
HDL cholesterol
(mmol/L) p=0.6
p=0.02
p=0.1
5 x 10
-12 p=0.09
3 x 10
-6 p=0.03
6 x 10
-64
3.0
5.0
7.0
LDL cholesterol
(mmol/L)

Supplementary Figure 1.
Lipoprotein levels as a function of single site genotypes in the
Copenhagen General Population Study, Copenhagen City Heart Study, and Copenhagen
Ischemic Heart Disease Study controls combined. Columns show mean lipoprotein levels with standard error bars and P-values for trend across genotypes. Participants using lipid lowering therapy were excluded. To convert cholesterol values to mg/dL, multiply values in mmol/L by 38.6. HDL=high density lipoprotein. LDL=low density lipoprotein.

Risk of ischemic heart disease
Remnant cholesterol
Observational: Elevated plasma levels
Causal: Genetically elevated levels
Hazard/Odds ratio P-value
P for comparison
1.15 (1.12-1.19)
1.61 (1.35-1.93)
5 x 10
-17
1 x 10
-7
3 x 10
-4
Remnant cholesterol to HDL cholesterol ratio
Observational: Elevated plasma levels
Causal: Genetically elevated levels
1.12 (1.10-1.14)
1.80 (1.42-2.28)
9 x 10
-33
1 x 10
-6
1 x 10
-4
HDL cholesterol
Observational: Elevated plasma levels
Causal: Genetically elevated levels
LDL cholesterol
Observational: Elevated plasma levels
Causal: Genetically elevated levels
1.25 (1.20-1.31)
0.85 (0.62-1.18)
9 x 10
-27
0.3
0.02
1.14 (1.11-1.18)
1.47 (1.33-1.63)
2 x 10
-14
4 x 10
-13
7 x 10
-6
1 2 3
Hazard/Odds ratio for a 1 standard deviation increase/decrease (95%CI)
Supplementary Figure 2.
Observational and causal risk estimates for ischemic heart disease. Observational risk estimates as hazard ratios for a 1 standard deviation increase or decrease in plasma lipoprotein levels are from the prospective Copenhagen General
Population Study, Copenhagen City Heart Study, and Copenhagen Ischemic Heart
Disease Study controls combined, and adjusted for age, sex, smoking, hypertension, time since last meal, time of day for blood sampling, and lipid lowering therapy. Causal risk estimates for a 1 standard deviation increase or decrease in genetic lipoprotein levels in odds ratios are from the Copenhagen General Population Study, the Copenhagen City
Heart Study, and the Copenhagen Ischemic Heart Disease Study combined. P-values are

for significance of risk estimates and P-values for comparison are for differences between observational and causal estimates. CI=confidence interval. HDL=high-density lipoprotein. LDL=low-density lipoprotein.

R
2
(%)
Remnant cholesterol
TRIB1 rs 2954029
GCKR rs1260326
ApoA5 rs651821
Combined
0.3
0.3
0.9
1.5
F
93
111
296
161
Odds ratio for ischemic heart disease P-value
--> 17
0.002
0.1
8 x 10
-5
1 x 10
-7
Remnant cholesterol to HDL cholesterol ratio
LPL D9N
LPL G188E
LPL N291S
0.1
0.0
0.3
0.4
37
20
93
133 LPL S 447X
Combined 0.8
74
HDL cholesterol
LIPC 480C/T
ABCA1 N1800H
ABCA1 R2144X
Combined
0.5
0.2
0.0
0.7
163
87
11
71
--> 13
--> 9
--> 18
0.1
0.4
1 x 10
-4
0.003
1 x 10
-6
0.6
0.3
0.1
0.3
LDL cholesterol
ApoB R3500Q
LDLR W23X
LDLR W66G
LDLR W556S
PCSK9 R46L
Combined
0.4
0.1
0.4
0.2
0.6
1.7
178
96
247
134
186
134
1 x 10
-10
1 x 10
-7
2 x 10
-8
0.007
0.04
4 x 10
-13
0 1 2 3 4 5 6
Odds ratio for a 1 mmol/L or ratio increase/decrease (95%CI)
Supplementary Figure 3.
Causal risk estimates for ischemic heart disease for the variants separately and in variant combinations in the Copenhagen General Population

Study, Copenhagen City Heart Study, and Copenhagen Ischemic Heart Disease Study combined. F-statistics indicates the strength of the instrument and R 2 is a measure of the contribution of genotypes to the variation in the different lipoprotein levels.
CI=confidence interval. HDL=high-density lipoprotein. LDL=low-density lipoprotein.

R
2
(%)
Remnant cholesterol
CGPS
CCHS
CIHDS
Combined
1.6
2.0
1.2
1.5
F
116
36
18
161
Odds ratio for ischemic heart disease P-value
0.02
0.1
1 x 10
-4
1 x 10
-7
Remnant cholesterol to HDL cholesterol ratio
CGPS 0.7
49
CCHS
CIHDS
Combined
1.1
1.1
0.8
17
15
74
HDL cholesterol
CGPS
CCHS
CIHDS
Combined
0.7
0.8
0.6
0.7
44
19
9
71
--> 105
0.1
0.05
1 x 10
-7
1 x 10
-6
0.6
0.5
0.6
0.3
LDL cholesterol
CGPS
CCHS
CIHDS
Combined
1.5
1.6
3.4
1.7
88
28
44
134
0.02
0.03
3 x 10
-11
4 x 10
-13
0 1 2 3 4 5 6 7 8 9 10 11
Odds ratio for a 1 mmol/L or ratio increase/decrease (95%CI)
Supplementary Figure 4.
Causal risk estimates for ischemic heart disease for the three studies separately and combined. F-statistics indicates the strength of the instrument and

R
2
is a measure of the contribution of genotypes to the variation in the different lipoprotein levels. CGPS=Copenhagen General Population Study. CCHS=Copenhagen
City Heart Study. CIHDS=Copenhagen Ischemic Heart Disease Study. CI=confidence interval. HDL=high-density lipoprotein. LDL=low-density lipoprotein.