Analysis of Circulating Cholesterol Levels as a Mediator of an Association Between ABO Blood Group and Coronary Heart Disease Yequn Chen, Chang Chen, Xiayi Ke, Longgen Xiong, Yongying Shi, Jiafu Li, Xuerui Tan and Shu Ye Circ Cardiovasc Genet. 2014;7:43-48; originally published online January 6, 2014; doi: 10.1161/CIRCGENETICS.113.000299 Circulation: Cardiovascular Genetics is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 1942-325X. Online ISSN: 1942-3268
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Original Article Analysis of Circulating Cholesterol Levels as a Mediator of an Association Between ABO Blood Group and Coronary Heart Disease Yequn Chen, PhD*; Chang Chen, MD*; Xiayi Ke, PhD*; Longgen Xiong, MD; Yongying Shi, MD; Jiafu Li, MD; Xuerui Tan, PhD; Shu Ye, MD, PhD Background—Non-O type of ABO blood group has been associated with a predisposition to coronary heart disease. It is thought that this association is partly mediated by increased cholesterol levels in non–O-type individuals. In this study, we sought to estimate the mediation effect size. Methods and Results—In a group of individuals (n=6476) undergoing coronary angiography, we detected associations of non-O type with significant coronary artery disease with >50% stenosis in ≥1 coronary arteries (odds ratio, 1.24; 95% confidence interval, 1.10–1.39; P=2.6×10−4) and with prevalent or incident myocardial infarction (odds ratio, 1.22; 95% confidence interval, 1.09–1.37; P=1.2×10−3). Subjects of non-O type had higher levels of total cholesterol, low-density lipoprotein cholesterol, and non–high-density lipoprotein cholesterol (mean [SEM] in mmol/L: 4.931[0.021], 3.041 [0.018], and 3.805 [0.020] in non-O type compared with 4.778 [0.026], 2.906 [0.021], and 3.669 [0.024] in O type; P=3.8×10−7, P=1.5×10−7, and P=3.1×10−7, respectively). Mediation analyses indicated that 10% of the effect of non-O type on coronary artery disease susceptibility was mediated by increased low-density lipoprotein cholesterol level (P=7.8×10−4) and that 11% of the effect of non-O type on myocardial infarction risk was mediated by raised low-density lipoprotein cholesterol level (P=2.0×10−3). Conclusions—In a model in which it is presumed that cholesterol is a mediator of the associations of ABO group with coronary artery disease and myocardial infarction, around 10% of the effect of non-O type on coronary artery disease and myocardial infarction susceptibility was mediated by its influence on low-density lipoprotein cholesterol level. (Circ Cardiovasc Genet. 2014;7:43-48.) Key Words: ABO blood-group system ◼ cholesterol ◼ cholesterol, LDL ◼ coronary disease ◼ myocardial infarction
S
everal epidemiological studies have shown a relation between the human ABO blood group and coronary heart disease (CHD), such that individuals of a non-O type (A, B, or AB type) are more likely to have CHD than O-type individuals.1–3 However, the mechanistic basis underlying this relationship requires further investigations.
Clinical Perspective on p 48 It is well established that low-density lipoprotein cholesterol (LDLc) plays an essential role in the pathogenesis of atherosclerosis, the pathology underling CHD, and that high plasma concentrations of cholesterol, in particular those of LDLc, are a major risk factor for the disease.4 An association of ABO group with total cholesterol (TC) and LDLc levels
has also long been recognized5,6 and has recently been reinforced in genetic studies of variation at the ABO locus.7–12 Specifically, the non-O types, which confer an increased risk of CHD,1–3 are associated with increased TC and LDLc levels.5,6,12 Furthermore, studies have shown that non–O-type individuals have higher cholesterol absorption rates12,13 and that the cholesterol absorption rate is positively associated with cardiovascular risk.12,14 Therefore, it is thought that the effect of ABO on CHD susceptibility is in part mediated by its influence on cholesterol levels.8,12 However, it has remained unknown as to how much of the effect of non-O type on CHD susceptibility is attributable to the mediation of cholesterol levels. In this study, we sought to estimate this mediation effect size. In a large group
Received July 24, 2013; accepted December 18, 2013. From the Department of Cardiology, First Affiliated Hospital of Shantou University Medical College, Shantou, China (Y.C., C.C., X.T., S.Y.); Department of Cardiology, Southern Medical University, Guangzhou, China (C.C.); MRC Centre of Epidemiology for Child Health, University College London, London, United Kingdom (X.K.); Department of Cardiology, Second Affiliated Hospital of Guangzhou Medical College, Guangzhou, China (L.X.); Department of Cardiology, First Affiliated Hospital of Guangzhou Medical College, Guangzhou, China (Y.S.); Department of Cardiology, First Affiliated Hospital of Luzhou Medical College, Luzhou, China (J.L.); Shantou University Medical College, Shantou, China (S.Y.); and Department of Clinical Pharmacology, William Harvey Research Institute, Queen Mary University of London, London, United Kingdom (S.Y.). *Drs Y. Chen, C. Chen, and Ke contributed equally to this work. The Data Supplement is available at http://circgenetics.ahajournals.org/lookup/suppl/doi:10.1161/CIRCGENETICS.113.000299/-/DC1. Correspondence to Shu Ye, MD, PhD, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, United Kingdom, or First Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong 515041, China. E-mail [email protected] or Xuerui Tan, PhD, Department of Cardiology, First Affiliated Hospital of Shantou University Medical College, Guangdong 515041, China. E-mail [email protected] © 2014 American Heart Association, Inc. Circ Cardiovasc Genet is available at http://circgenetics.ahajournals.org
DOI: 10.1161/CIRCGENETICS.113.000299
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44 Circ Cardiovasc Genet February 2014 Table 1. Characteristics of Study Subjects Characteristics
Mean (SD) or %
Age, y
64.11 (11.42)
Sex (% of men)
71.4
ABO group (% of AB, A, B, and O)
7.2, 27.1, 25.8, 39.9
Total cholesterol, mmol/L
4.88 (1.25)
LDL cholesterol, mmol/L
2.99 (1.04)
HDL cholesterol, mmol/L
1.12 (0.33)
NHDL cholesterol, mmol/L
3.76 (1.26)
Triglycerides, mmol/L
1.62 (1.30)
Systolic blood pressure, mm Hg
135.39 (23.88)
Diastolic blood pressure, mm Hg
80.16 (13.88)
Diabetes mellitus, %
22.4
Cases with >50% coronary stenosis, %
69.8
Myocardial infarction, %
32.2
HDL indicates high-density lipoprotein; LDL, low-density lipoprotein; and NHDL, non–high-density lipoprotein.
of individuals (n=6476) undergoing diagnostic or interventional coronary angiography, we first verified the associations among ABO group, cholesterol levels, and CHD phenotypes and then performed mediation analyses in a model with cholesterol levels as a mediator for the effect of ABO group on CHD phenotypes.
Methods Subjects We studied 6476 consecutive patients undergoing diagnostic or interventional coronary angiography in the First Affiliated Hospital of Shantou University Medical College, the First Affiliated Hospital of Guangzhou Medical College, and the Second Affiliated Hospital of Guangzhou Medical College in Southern China. Data required for this study were collected from hospital records for the patients. All subjects were Chinese. The study was approved by the appropriate research ethics committee. We collected demographic and clinical data including age, sex, ABO blood type, plasma levels of TC, LDLc, high-density lipoprotein cholesterol (HDLc) and triglycerides, coronary angiographic findings, prevalent or incident myocardial infarction (MI) diagnosed according to the World Health Organization criteria, systolic and diastolic blood pressure, and the presence or absence of diabetes mellitus. ABO blood typing and cholesterol and triglyceride measurements were conducted by the clinical chemistry department of the
respective hospitals. Blood samples for cholesterol and triglyceride measurements were collected after overnight fasting. The main demographic and clinical characteristics of the study subjects are summarized in Table 1 and in Table I in the Data Supplement. Of the 6476 subjects, 4518 had significant angiographically documented coronary artery disease (CAD) as having >50% diameter stenosis in ≥1 major epicardial coronary artery. A total of 2083 subjects had prevalent or incident MI.
Statistical Analysis Values of TC, LDLc, HDLc, and triglycerides were logarithm transformed to normalize their distributions. Non-HDL cholesterol (NHDLc) values were calculated as TC minus HDLc and then normalized by logarithmic transformation. χ2 test and logistic regression analysis were used to ascertain association of ABO blood types with CAD and MI, respectively, with adjustment for age, sex, and hospitals as covariates. t Test and general linear model analysis were used to examine differences in lipid levels between CAD and non-CAD groups, between MI and non-MI groups, and between O and non-O blood types, with adjustment for age, sex, and hospitals as covariates. The above analyses were then performed in male and female subjects separately, with age and hospitals as covariates. All P values were 2 sided. For causal mediation analysis, the R package Mediation was used.15 Briefly, the average causal mediation effect is estimated as the expected difference in the potential outcome (Y) when the mediator (M) takes the value that would realize under the treatment condition (T) as opposed to the control condition, while the treatment status is held constant, which is expressed in the following equation: δ(t)=E{Y(t,M(t1))–Y(t,M(t0))},where t, t1, t0 are specific values of T, with t1≠t0. Similarly, the average direct effect is defined as the expected difference in the potential outcome when the treatment is changed while the mediator is held constant at the value that would realize whether the treatment equals t, according to the following equation:ζ(t)=E{Y(t1,M(t))–Y(t0,M(t))}. The total effect of the treatment on the outcome is composed of the average causal mediation effect and the average direct effect. This model allows the mediation effect to be estimated for a variety of data types.15 In this study, the blood type was regarded as the treatment variable in either a 2-group classification setting (A/B/AB versus O) or a 3-group classification setting (AB versus A/B versus O). The level of TC, LDLc, or NHDLc was regarded as the mediator. First, in the mediator model, a linear regression model was used to estimate the association between the treatment variable and the mediator. Second, in the outcome model, a generalized linear model was used to estimate the association of the mediator and the treatment variable and the outcome (either CAD or MI), using binomial probit link function. Finally, a nonparametric bootstrapping method with ≤100 000 simulations was applied to obtain accurate P values of the estimates of the total effects, direct effects, as well as mediation effects. The analyses were performed first in the sample as a whole with adjustment for age, sex, and hospitals and then separately in men and women with adjustment for age and hospitals.
Table 2. Association of ABO Blood Group With CAD and MI CAD* ABO Type
MI‡
Cases
Controls
OR (95% CI)†
Yes
No
OR (95% CI)†
AB
323 (71.6%)
128 (28.4%)
1.27 (1.01–1.60)
A
1227 (70.9%)
465 (29.1%)
1.29 (1.12–1.49)
144 (31.9%)
307 (68.1%)
1.14 (0.91–1.43)
578 (34.2%)
1114 (65.8%)
B
1142 (70.9%)
469 (29.1%)
1.28 (1.12–1.47)
1.18 (1.02–1.36)
524 (32.5%)
1087 (67.5%)
1.18 (1.02–1.35)
O
1685 (67.7%)
803 (32.3%)
Non-O
2692 (71.7%)
728 (29.3%)
1760 (70.7%)
1062 (28.3%)
1.24 (1.10–1.39)
1246 (33.2%)
O
1685 (67.7%)
2508 (66.8%)
1.22 (1.09–1.37)
803 (32.3%)
P=2.6×10−4
728 (29.3%)
1760 (70.7%)
P=1.2×10−3
CAD indicates coronary artery disease; CI, confidence interval; MI, myocardial infarction; and OR, odds ratio. *Significant CAD indicated by >50% stenosis in ≥1 coronary artery in angiography. †OR (95% CI) for non-O type vs O type. ‡Prevalent or incident MI.
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Chen et al ABO Blood Group and CHD 45 Table 3. Lipid Levels by ABO Blood Types Total Cholesterol
LDL Cholesterol
HDL Cholesterol
Non-HDL Cholesterol
Triglycerides
AB
4.968 (0.061); n=418
3.037 (0.051); n=408
1.142 (0.018); n=417
3.826 (0.057); n=417
1.695 (0.062); n=418
A
4.928 (0.031); n=1530
3.049 (0.027); n=1504
1.120 (0.008); n=1530
3.812 (0.029); n=1527
1.598 (0.029); n=1534
B
4.923 (0.032); n=1495
3.034 (0.027); n=1472
1.139 (0.009); n=1496
3.792 (0.030); n=1493
1.637 (0.037); n=1500
O
4.778 (0.026); n=2284
2.906 (0.021); n=2245
1.112 (0.007); n=2285
3.669 (0.024); n=2279
1.603 (0.028); n=2297
P value
9.1×10
4.4×10
−6
−6
3.7×10
−2
6.8×10
−6
n.s.
Non-O
4.931 (0.021); n=3443
3.041 (0.018); n=3384
1.131 (0.006); n=3443
3.805 (0.020); n=3437
1.627 (0.022); n=3452
O
4.778 (0.026); n=2284
2.906 (0.021); n=2245
1.112 (0.007); n=2285
3.669 (0.024); n=2279
1.603 (0.028); n=2297
P value
3.8×10
1.5×10
−7
−7
2.1×10
−2
3.1×10
−7
n.s.
Lipid levels shown are mean (SEM) in mmol/L. HDL indicates high-density lipoprotein; LDL, low-density lipoprotein; and n.s., nonsignificant.
Results We observed an association between non-O type (AB, A, or B) and the presence of significant CAD indicated by >50% stenosis in ≥1 coronary artery in angiography. The odds ratio (OR) for significant CAD was 1.24 (95% confidence interval [CI], 1.10–1.39; P=3.0×10−4) for subjects of non-O type as compared with O-type individuals with adjustment for age, sex, and hospitals (Table 2) and was similar in men (OR, 1.25; 95% CI, 1.08–1.45; P=3.0×10−3) and women (OR, 1.23; 95% CI, 1.02–1.48; P=3.5×10−2; Table III in the Data Supplement). Among those subjects with significant CAD, there was no association between ABO group and the number (1 or 2 or 3) of coronary arteries with >50% stenosis (Table III in the Data Supplement). There was a significant association between non-O type and prevalent or incident MI in the sample as a whole (OR, 1.22; 95% CI, 1.09–1.37; P=1.2×10−3; Table 2) and in men (OR, 1.26; 95% CI, 1.02– 1.44; P=1.0×10−3), with a similar but nonsignificant trend in women (OR, 1.08; 95% CI, 0.86–1.36; P>0.05; Table II in the Data Supplement). Subjects of the AB, A, or B type had similar concentrations of TC in the blood, whereas subjects of the O type had lower levels (mean [SEM]: 4.931 [0.021] mmol/L in non-O type compared with 4.778 [0.026] mmol/L in O type; P=3.8×10−7; Table 3), and so was the case for LDLc (3.041 [0.018] mmol/L in non-O type compared with 2.906 [0.021] in O type; P=1.5×10−7; Table 3). HDLc levels were marginally higher (rather than lower) in non–O-type than in
O-type subjects (1.131 [0.006] mmol/L versus 1.112 [0.007] mmol/L; P=2.1×10−2; Table 3), the reason for which is unknown. However, the levels of NHDLc, previously shown to be an independent and superior predictor of CHD from the Framingham Heart Study,16 were higher in non–O-type than in O-type individuals (3.805 [0.020] mmol/L versus 3.669 [0.024]; P=3.1×10−7; Table 3). There was no significant association between ABO group and triglycerides level (Table 3), which is consistent with findings from several other studies.7–12 The results were generally similar when men and women were analyzed separately (Table IV in the Data Supplement). As expected, levels of TC, LDLc, and NHDLc were higher in subjects with significant CAD than in those without and similarly were higher in the MI group as compared with the non-MI group (Table 4), and these were also the case when men and women were analyzed separately (Table V in the Data Supplement). HDLc levels were lower in cases with significant CAD than in those without (Table 4), with a similar trend in both sexes (Table V in the Data Supplement), while no significant association was detected between HDLc and MI (Table 4 and Table V in the Data Supplement). In the sample as a whole and in women, triglyceride level was higher in CAD cases but did not significantly differ between the MI and non-MI groups (Table 4 and Table V in the Data Supplement). However, in men, it was lower in CAD and MI cases, the reason for which is unknown (Table V in the Data Supplement).
Table 4. Lipid Levels by CAD Status and MI Status Total Cholesterol
LDL Cholesterol
HDL Cholesterol
Non-HDL Cholesterol
Triglycerides
Cases
4.919 (0.020); n=4128
3.036 (0.017); n=4056
1.109 (0.005); n=4128
3.812 (0.019); n=4119
1.629 (0.020); n=4145
Controls
4.776 (0.027); n=1805
2.897 (0.023); n=1775
1.157 (0.008); n=1809
3.626 (0.025); n=1803
1.591 (0.030); n=1812
P value
1.3×10−12
9.2×10−11
3.4×10−2
6.4×10−16
Yes
4.922 (0.030); n=1920
3.078 (0.025); n=1884
1.120 (0.007); n=1920
3.807 (0.027); n=1915
1.597 (0.031); n=1927
No
4.853 (0.019); n=4012
2.954 (0.016); n=3946
1.126 (0.005); n=4015
3.731 (0.018); n=4006
1.632 (0.020); n=4029
1.5×10−4
2.2×10−7
n.s.
5.0×10−6
CAD*
5.1×10−7
MI†
P value
n.s.
Lipid levels shown are mean (SEM) in mmol/L. CAD indicates coronary artery disease; HDL, high-density lipoprotein; LDL, low-density lipoprotein; MI, myocardial infarction; and n.s., nonsignificant. *Significant CAD indicated by >50% stenosis in ≥1 coronary artery in angiography. †Prevalent or incident MI.
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46 Circ Cardiovasc Genet February 2014 Having detected that subjects of non-O type had higher ORs of significant CAD and MI as well as higher total TC, LDLc, and NHDLc levels, together with the observation that TC, LDLc, and NHDL levels were higher in subjects with significant CAD (than in those without) and in those with MI (than those without), we performed mediation analyses with TC, LDLc, and NHDLc as mediators for the associations of ABO group with CAD and MI. Mediation analysis assesses an intermediate variable as a mediator in the pathway between a risk factor and an outcome to estimate the extent to which the effect of the risk factor occurs through the mediator.17–20 This method has been used in various research fields including studies of effects of genetic variants.21–24 A mediation analysis in a model with CAD as the dependent variable, non-O versus O type as the independent variable, and LDLc level as the mediator variable, with age, sex,
and hospitals as covariates, indicated that there was a 4.9% difference in CAD susceptibility between non-O type and O type (P=4.0×10−5) and that 10% of this difference was mediated by increased LDLc level (0.5% of the difference in CAD susceptibility was mediated by LDLc level; P=7.8×10−4 for mediation effect; Table 5). In the above model, substituting LDLc with TC or NHDLc as the mediator variable showed that TC and NHDLc had a similar mediation effect size to that of LDLc (Table 5). As expected, the levels of TC, LDLc, and NHDLc were highly correlated (correction coefficient, 0.873 for TC and LDLc; 0.890 for LDLc and NHDLc; and 0.959 for TC and NHDLc; all P50% stenosis in ≥1 coronary artery in angiography. ‡Prevalent or incident MI.
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Chen et al ABO Blood Group and CHD 47 risk between non-O type and O type (P=8.0×10−3) and that 11% of this difference was mediated by increased LDLc level (0.4% of the difference in MI risk was mediated by LDLc level; P=2.0×10−3 for mediation effect; Table 5). Substituting LDLc with TC or NHDLc as the mediator variable in this model showed that TC and NHDLc had a somewhat smaller mediation effect size (6% and 7%, respectively) than that of LDLc (Table 5). In mediation analyses in which ABO group was coded as AB=2, A or B=1, and O=0, rather than non-O type=1 and O type=0 as in the models described above, the results were similar to those described above (Table 5). When the mediation analyses were performed in men and women separately, the mediation effect sizes of LDLc, TC, and NHDLc for CAD were similar; however, with regard to MI, mediation effects of LDLc, TC, and NHDLc were significant in men but not in women (Tables VI and VII in the Data Supplement). Some limitations to our study warrant mentioning. First, the usefulness of the results of the mediation analyses will be dependent on the correctness of the presumed model. Second, there were no available data for ABO subtypes (such as A1 and A2) or single-nucleotide polymorphisms at the ABO locus for analysis in this study. However, the A2 allele is very rare in the Chinese, and therefore it is likely that type A in this study was mostly the A1 type.25 Third, because this study was conducted in individuals of Chinese ancestry, the findings may not be generalizable to other races/ethnicities.
Discussion In summary, our study confirms the presence of associations of ABO blood group with cholesterol levels and CHD susceptibility, with non-O types associating with higher TC, LDLc, and NHDLc levels and greater risks of significant CAD and MI in an investigation of a large group of individuals undergoing coronary angiography. In a presumed model with cholesterol levels as mediators for the associations of ABO group with CHD, our mediation analyses indicated that ≈10% of the effect of non-O type on CAD and MI susceptibility was attributable to the mediation of LDLc level and that this 10% was significantly nonzero. These findings provide a useful insight for understanding the genetic mechanisms underlying CHD susceptibility.
Sources of Funding Work in the laboratory of author Dr Ye is supported by the British Heart Foundation and forms part of the research themes contributing to the translational research portfolio of Barts Cardiovascular Biomedical Research Unit, which is supported and funded by the National Institute of Health Research. The UK Medical Research Council has provided financial support for the MRC Centre of Epidemiology for Child Health, UCL Institute of Child Health, where Dr Ke works.
Disclosures None.
References 1. Wu O, Bayoumi N, Vickers MA, Clark P. ABO(H) blood groups and vascular disease: a systematic review and meta-analysis. J Thromb Haemost. 2008;6:62–69. 2. Reilly MP, Li M, He J, Ferguson JF, Stylianou IM, Mehta NN, et al; Myocardial Infarction Genetics Consortium; Wellcome Trust Case Control Consortium. Identification of ADAMTS7 as a novel locus for coronary atherosclerosis and association of ABO with myocardial infarction in the presence of coronary atherosclerosis: two genome-wide association studies. Lancet. 2011;377:383–392. 3. He M, Wolpin B, Rexrode K, Manson JE, Rimm E, Hu FB, et al. ABO blood group and risk of coronary heart disease in two prospective cohort studies. Arterioscler Thromb Vasc Biol. 2012;32:2314–2320. 4. Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med. 1999;340:115–126. 5. Langman MJ, Elwood PC, Foote J, Ryrie DR. ABO and Lewis blood-groups and serum-cholesterol. Lancet. 1969;2:607–609. 6. Oliver MF, Geizerova H, Cumming RA, Heady JA. Serum-cholesterol and ABO and rhesus blood-groups. Lancet. 1969;2:605–606. 7. Teslovich TM, Musunuru K, Smith AV, Edmondson AC, Stylianou IM, Koseki M, et al. Biological, clinical and population relevance of 95 loci for blood lipids. Nature. 2010;466:707–713. 8. Schunkert H, König IR, Kathiresan S, Reilly MP, Assimes TL, Holm H, et al; Cardiogenics; CARDIoGRAM Consortium. Large-scale association analysis identifies 13 new susceptibility loci for coronary artery disease. Nat Genet. 2011;43:333–338. 9. Wu Y, Waite LL, Jackson AU, Sheu WH, Buyske S, Absher D, et al. Trans-ethnic fine-mapping of lipid loci identifies population-specific signals and allelic heterogeneity that increases the trait variance explained. PLoS Genet. 2013;9:e1003379. 10. Deloukas P, Kanoni S, Willenborg C, Farrall M, Assimes TL, Thompson JR, et al. Large-scale association analysis identifies new risk loci for coronary artery disease. Nat Genet. 2013;45:25–33. 11. Willer CJ, Schmidt EM, Sengupta S, Peloso GM, Gustafsson S, Kanoni S, et al. Discovery and refinement of loci associated with lipid levels. Nat Genet. 2013;45:1274–1283. 12. Silbernagel G, Chapman MJ, Genser B, Kleber ME, Fauler G, Scharnagl H, et al. High intestinal cholesterol absorption is associated with cardiovascular disease and risk alleles in ABCG8 and ABO: evidence from the LURIC and YFS cohorts and from a meta-analysis. J Am Coll Cardiol. 2013;62:291–299. 13. Teupser D, Baber R, Ceglarek U, Scholz M, Illig T, Gieger C, et al. Genetic regulation of serum phytosterol levels and risk of coronary artery disease. Circ Cardiovasc Genet. 2010;3:331–339. 14. Strandberg TE, Tilvis RS, Pitkala KH, Miettinen TA. Cholesterol and glucose metabolism and recurrent cardiovascular events among the elderly: a prospective study. J Am Coll Cardiol. 2006;48:708–714. 15. Imai K, Kees L, Tingley D, Yamamoto T. Causal mediation analysis using R. In: Vinod HD, ed. Advances in Social Science Research Using R. New York: Springer; 2010:129–154. 16. Liu J, Sempos CT, Donahue RP, Dorn J, Trevisan M, Grundy SM. Non-high-density lipoprotein and very-low-density lipoprotein cholesterol and their risk predictive values in coronary heart disease. Am J Cardiol. 2006;98:1363–1368. 17. Baron RM, Kenny DA. The moderator-mediator variable distinction in social psychological research: conceptual, strategic, and statistical considerations. J Pers Soc Psychol. 1986;51:1173–1182. 18. Huang B, Sivaganesan S, Succop P, Goodman E. Statistical assess ment of mediational effects for logistic mediational models. Stat Med. 2004;23:2713–2728. 19. Li Y, Schneider JA, Bennett DA. Estimation of the mediation effect with a binary mediator. Stat Med. 2007;26:3398–3414. 20. Albert JM. Mediation analysis via potential outcomes models. Stat Med. 2008;27:1282–1304. 21. Stoltenberg SF, Twitchell GR, Hanna GL, Cook EH, Fitzgerald HE, Zucker RA, et al. Serotonin transporter promoter polymorphism, peripheral indexes of serotonin function, and personality measures in families with alcoholism. Am J Med Genet. 2002;114:230–234.
Downloaded from http://circgenetics.ahajournals.org/ at Queen's University--Kingston (CANADA) on August 18, 2014
48 Circ Cardiovasc Genet February 2014 22. VanderWeele TJ, Asomaning K, Tchetgen Tchetgen EJ, Han Y, Spitz MR, Shete S, et al. Genetic variants on 15q25.1, smoking, and lung cancer: an assessment of mediation and interaction. Am J Epidemiol. 2012;175:1013–1020. 23. Machiela MJ, Lindström S, Allen NE, Haiman CA, Albanes D, Barricarte A, et al. Association of type 2 diabetes susceptibility variants with advanced prostate cancer risk in the Breast and Prostate Cancer Cohort Consortium. Am J Epidemiol. 2012;176:1121–1129.
24. Siedlinski M, Tingley D, Lipman PJ, Cho MH, Litonjua AA, Sparrow D, et al; COPDGene and ECLIPSE Investigators. Dissecting direct and indirect genetic effects on chronic obstructive pulmonary disease (COPD) susceptibility. Hum Genet. 2013;132:431–441. 25. Zhu F, Tao S, Xu X, Ying Y, Hong X, Zhu H, et al. Distribution of ABO blood group allele and identification of three novel alleles in the Chinese Han population. Vox Sang. 2010;98:554–559.
CLINICAL PERSPECTIVE It has long been recognized that the ABO blood group has an influence on risk of coronary heart disease (CHD), such that individuals of a non–O-type (A, B, or AB type) are more likely to have CHD than O-type individuals. However, the mechanistic basis underlying this relationship is still unclear. Interestingly, it has also been shown that non–O-type individuals tend to have higher cholesterol absorption rates and higher levels of total cholesterol and low-density lipoprotein cholesterol in the blood. Therefore, it is thought that the effect of ABO blood group on CHD susceptibility is in part mediated by its influence on circulating cholesterol levels, a well-established risk factor that plays an important role in the development of CHD. However, it has remained unknown as to how much of the effect of non-O type on CHD susceptibility is attributable to the mediation of cholesterol levels. In this study, we sought to estimate this mediation effect size. In a large group of individuals (n=6476) undergoing diagnostic or interventional coronary angiography, we first verified the associations among ABO group, cholesterol levels, and CHD phenotypes and then performed mediation analyses in a model with cholesterol levels as a mediator for the effect of ABO group on CHD phenotypes. The mediation analyses indicated that ≈10% of the effect of non-O type on CHD susceptibility and risk of myocardial infarction was mediated by increased low-density lipoprotein cholesterol level.
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SUPPLEMENTAL MATERIAL
Table S1. Characteristics of study subjects by gender Mean (SD) or % Males (n=4296)
Females (n=2180)
Age (years)
62.95 (11.67)
66.41 (10.53)
ABO group (% of AB, A, B, and O)
7.2, 28.8, 25.9, 40.1 7.3, 27.6, 25.6, 39.5
Total cholesterol (mmol/L)
4.76 (1.24)
5.01 (1.24)
LDL-cholesterol (mmol/L)
2.95 (1.04)
3.07 (1.01)
HDL-cholesterol (mmol/L)
1.09 (0.31)
1.19 (0.33)
NHDL- cholesterol (mmol/L)
3.68 (1.16)
3.91 (1.15)
Triglycerides (mmol/L)
1.72 (1.17)
1.57 (1.50)
Systolic blood pressure (mmHg)
133.24 (23.39)
139.62 (24.27)
Diastolic blood pressure (mmHg)
79.99 (14.04)
80.49 (13.57)
Diabetes mellitus (%)
18.1
22.7
Cases with >50% coronary stenosis (%) 75.1
59.2
MI (%)
19.2
38.7
1
Table S2. Association of ABO blood group with CAD and MI, by gender Gender Males
Females
AB A B O
*CAD ‡OR (95% CI) cases controls 236 (79.5%) 61 (20.5%) 1.49 (1.10, 2.02) 858 (77.6%) 248 (22.4%) 1.28 (1.07, 1.54) 810 (75.8%) 258 (24.2%) 1.16 (0.97, 1.39) 1208 (73.2%) 443 (26.8%)
†MI ‡OR (95% CI) Yes No 119 (40.1%) 178 (59.9%) 1.25 (0.96, 1.62) 460 (41.6%) 646 (58.4%) 1.34 (1.14, 1.58) 412 (38.6%) 656 (61.4%) 1.18 (1.01, 1.39) 576 (34.9%) 1075 (65.1%)
non-O O
1904 (77.1%) 567 (22.9%) 1.25 (1.08, 1.45) 1208 (73.2%) 443 (26.8%) p=3.010-3
991 (40.1%) 1480 (59.9%) 1.26 (1.02, 1.44) 576 (34.9%) 1075 (65.1%) p=1.010-3
AB A B O
87 (56.5%) 369 (63.0%) 332 (61.1%) 477 (57.0%)
67 (43.5%) 1.00 (0.69, 1.45) 217 (37.0%) 1.31 (1.04, 1.66) 211 (38.9%) 1.21 (0.96, 1.54) 360 (43.0%)
25 (16.2%) 118 (20.1%) 112 (20.6%) 152 (18.2%)
non-O O
788 (61.4%) 477 (57.0%)
495 (38.6%) 1.23 (1.02, 1.48) 360 (43.0%) p=3.510-2
255 (19.9%) 1028 (80.1%) 1.08 (0.86, 1.36) 152 (18.2%) 685 (81.8%) n.s.
ABO type
129 (83.8%) 468 (79.9%) 431 (79.4%) 685 (81.8%)
0.85 (0.53, 1.37) 1.09 (0.82, 1.43) 1.15 (0.87, 1.52)
*significant CAD indicated by >50% stenosis in at least one coronary artery in angiography. †prevalent or incident MI. ‡odds ratio (95% confidence interval) for non-O type versus O type.
2
Table S3. Patients with one, two or three coronary arteries with >50% stenosis, by ABO types Gender
ABO type
Both
AB A B O
Males
Females
Number of coronary artery with >50% stenosis One Two Three 110 (6.8%) 104 (7.9%) 109 (7.5%) 439 (27.2%) 372 (28.4%) 416 (28.6%) 434 (26.9%) 339 (25.9%) 369 (25.4%) 631 (39.1%) 495 (37.8%) 559 (38.5%)
Non-O O
983 (60.9%) 631 (39.1%)
815 (62.2%) 495 (37.8%)
894 (61.5%) 559 (38.5%)
AB A B O
81 (7.5%) 286 (26.4%) 304 (28.1%) 412 (38.0%)
69 (7.2%) 272 (28.4%) 244 (25.5%) 372 (38.9%)
86 (8.0%) 300 (28.0%) 262 (24.4%) 424 (39.6%)
Non-O O
671 (62.0%) 412 (38.0%)
585 (61.1%) 372 (38.9%)
648 (60.4%) 424 (39.6%)
AB A B O
29 (5.5%) 153 (28.8%) 130 (24.5%) 219 (41.2%)
35 (9.9%) 100 (28.3%) 95 (26.9%) 123 (34.8%)
23 (6.0%) 116 (30.4%) 107 (28.1%) 135 (35.4%)
Non-O O
312 (58.8%) 219 (41.2%)
230 (65.2%) 123 (34.8%)
246 (64.6%) 135 (35.4%)
3
Table S4. Lipid levels according to ABO blood types, by gender Gender Males
ABO type AB A B O p-value
Total cholesterol 4.903 (0.075), n=276 4.802 (0.037), n=1015 4.792 (0.039), n=990 4.685 (0.033), n=1521 3.010-3
non-O O p-value
4.810 (0.025), n=2281 2.987 (0.022), n=2244 1.091 (0.007), n=2277 3.722 (0.024), n=2275 1.581 (0.025), n=2286 4.685 (0.033), n=1521 2.876 (0.027), n=1494 1.083 (0.008), n=1522 3.675 (0.031), n=1517 1.542 (0.031), n=1531 n.s. n.s. 4.010-4 2.210-4 1.110-4
Females AB A B O p-value non-O O p-value
5.094 (0.105), n=142 5.177 (0.055), n=515 5.181 (0.056), n=505 4.962 (0.043), n=763 2.010-3
LDL-cholesterol 3.016 (0.065), n=270 2.991 (0.032), n=999 2.976 (0.036), n=975 2.876 (0.027), n=1494 3.010-3
3.079 (0.081), n=138 3.164 (0.048), n=505 3.149 (0.046), n=497 2.965 (0.035), n=751 2.010-3
HDL-cholesterol Non-HDL-cholesterol 1.117 (0.023), n=275 3.787 (0.071), n=275 1.076 (0.009), n=1013 3.729 (0.035), n=1012 1.101 (0.010), n=989 3.697 (0.036), n=988 1.083 (0.008), n=1522 3.675 (0.031), n=1517 n.s. 1.010-3
1.192 (0.026), n=142 1.206 (0.014), n=517 1.213 (0.017), n=507 1.169 (0.012), n=764 n.s.
3.902 (0.095), n=142 3.974 (0.052), n=515 3.979 (0.051), n=505 3.801 (0.040), n=762 1.010-2
Triglycerides 1.694 (0.074), n=276 1.569 (0.035), n=1018 1.563 (0.039), n=992 1.542 (0.031), n=1531 n.s.
1.698 (0.114), n=142 1.655 (0.053), n=516 1.782 (0.078), n=508 1.725 (0.056), n=766 n.s.
5.168 (0.037), n=1162 3.147 (0.031), n=1140 1.207 (0.010), n=1166 3.967 (0.034), n=1162 1.716 (0.044), n=1166 4.962 (0.043), n=763 2.965 (0.035), n=751 1.169 (0.012), n=764 3.801 (0.040), n=762 1.725 (0.056), n=766 -4 -4 -3 n.s. 2.110 1.310 8.010 1.010-3
Lipid levels shown are mean (SEM) in mmol/L. n.s.: non-significant.
4
Table S5. Lipid levels by CAD status and MI status, respectively, in males and in females Gender Males
Females
*CAD
Cases Controls p-value
Total cholesterol 4.818 (0.023), n=2971 4.601 (0.036), n=982 2.210-9
LDL-cholesterol 2.998 (0.020), n=2925 2.794 (0.031), n=961 8.410-10
HDL-cholesterol 1.083 (0.006), n=2970 1.105 (0.011), n=982 n.s.
Non-HDL-cholesterol 3.738 (0.022), n=2963 3.500 (0.034), n=980 1.610-11
Triglycerides 1.561 (0.021), n=2986 1.584 (0.039), n=984 4.510-2
†MI
Yes No p-value
4.875 (0.033), n=1541 4.693 (0.025), n=2411 1.610-5
3.071 (0.027), n=1513 2.869 (0.021), n=2372 3.810-9
1.104 (0.008), n=1541 1.079 (0.006), n=2410 n.s.
3.776 (0.030), n=1537 3.616 (0.023), n=2405 1.710-6
1.533 (0.029), n=1547 1.588 (0.024), n=2422 9.010-3
*CAD
Cases Controls p-value
5.176 (0.038), n=1157 4.985 (0.040), n=823 2.010-3
3.135 (0.032), n=1131 3.018 (0.033), n=814 3.010-2
1.178 (0.012), n=1158 1.218 (0.010), n=827 n.s.
4.004 (0.036), n=1156 3.775 (0.036), n=823 3.110-4
1.807 (0.047), n=1159 1.598 (0.047), n=828 9.810-6
†MI
Yes No p-value
5.111 (0.070), n=379 5.093 (0.030), n=1601 n.s.
3.108 (0.056), n=371 3.081 (0.025), n=1574 n.s.
1.185 (0.016), n=379 1.197 (0.009), n=1606 n.s.
3.936 (0.063), n=378 3.902 (0.028), n=1601 n.s.
1.811 (0.097), n=380 1.698 (0.035), n=1607 n.s.
Lipid levels shown are mean (SEM) in mmol/L. *significant CAD indicated by >50% stenosis in at least one coronary artery in angiography. †prevalent or incident MI. n.s.: non-significant.
5
Table S6. TC, LDLc or NHDLc level as a mediator for association of ABO with CAD and MI, respectively, in males (n=3802) Effect size Mediator Model Parameter *CAD †MI -3 Total cholesterol (TC) Model 1 Total effect of ABO group 0.048, p=1.110 0.048, p=2.610-3 -3 (non-O versus O type) Effect not mediated by TC 0.043, p=2.310 0.046, p=3.810-3 Effect mediated by TC 0.005, p=8.710-3 0.003, p=5.310-2 Proportion of ABO effect mediated by TC 0.09 0.06
LDL-cholesterol (LDLc)
Non-HDL-cholesterol (NHDLc)
Model 2 Total effect of ABO group (AB=2, A or B=1, O=0) Effect not mediated by TC Effect mediated by TC Proportion of ABO effect mediated by TC
0.042, p=2.010-4 0.038, p=4.010-4 0.004, p=7.410-3 0.09
0.034, p=5.410-3 0.032, p=8.610-3 0.002, p=3.610-2 0.07
Model 1 Total effect of ABO group (non-O versus O type) Not mediated by LDLc Mediated by LDLc Proportion of ABO effect mediated by LDLc
0.046, p=1.010-3 0.042, p=3.210-3 0.005, p=7.810-3 0.10
0.045, p=5.310-3 0.041, p=1.210-2 0.005, p=1.510-2 0.10
Model 2 Total effect of ABO group (AB=2, A or B=1, O=0) Effect not mediated by LDLc Effect mediated by LDLc Proportion of ABO effect mediated by LDLc
0.039, p=2.010-4 0.036, p=1.210-3 0.004, p=1.210-2 0.09
0.029, p=1.710-2 0.026, p=3.810-2 0.004, p=1.010-2 0.12
Model 1 Total effect of ABO group (non-O versus O type) Effect not mediated by TC Effect mediated by TC Proportion of ABO effect mediated by TC
0.049, p=5.210-4 0.043, p=2.010-3 0.006, p=5.210-3 0.12
0.048, p=2.010-3 0.045, p=4.710-3 0.003, p=2.810-2 0.07
Model 2 Total effect of ABO group (AB=2, A or B=1, O=0) Effect not mediated by TC Effect mediated by TC Proportion of ABO effect mediated by TC
0.043, p=2.010-3 0.038, p=2.010-3 0.005, p=1.210-2 0.11
0.033, p=8.710-3 0.031, p=1.510-2 0.003, p=2.310-2 0.08
*the difference in disease risk. †significant CAD indicated by >50% stenosis in at least one coronary artery in angiography. ‡prevalent or incident MI.
6
Table S7. TC, LDLc or NHDLc level as a mediator for association of ABO with CAD and MI, respectively, in females (n=1925) *Effect size Mediator Model Parameter †CAD ‡MI -2 Total cholesterol (TC) Model 1 Total effect of ABO group 0.013, p=0.45 0.052, p=1.210 (non-O versus O type) Effect not mediated by TC 0.047, p=2.510-2 0.014, p=0.44 Effect mediated by TC 0.006, p=4.510-2 -0.0008, p=0.55 Proportion of ABO effect mediated by TC 0.11 -0.06
LDL-cholesterol (LDLc)
Non-HDL-cholesterol (NHDLc)
Model 2 Total effect of ABO group (AB=2, A or B=1, O=0) Effect not mediated by TC Effect mediated by TC Proportion of ABO effect mediated by TC
0.029, p=0.11 0.025, p=0.16 0.004, p=0.06 0.13
0.004, p=0.76 0.004, p=0.73 -0.0005, p=0.57 -0.13
Model 1 Total effect of ABO group (non-O versus O type) Not mediated by LDLc Mediated by LDLc Proportion of ABO effect mediated by LDLc
0.054, p=8.010-3 0.050, p=1.7×10-2 0.004, p=7.110-2 0.08
0.013, p=0.48 0.013, p=0.48 -0.0001, p=0.92 -0.01
Model 2 Total effect of ABO group (AB=2, A or B=1, O=0) Effect not mediated by LDLc Effect mediated by LDLc Proportion of ABO effect mediated by LDLc
0.030, p=0.08 0.027, p=0.10 0.003, p=0.11 0.10
0.003, p=0.83 0.003, p=0.81 -0.0001, p=0.95 -0.01
Model 1 Total effect of ABO group (non-O versus O type) Effect not mediated by TC Effect mediated by TC Proportion of ABO effect mediated by TC
0.053, p=2.010-2 0.047, p=2.6×10-2 0.006, p=3.410-2 0.12
0.01, p=0.47 0.01, p=0.47 0.000004, p=0.97 0.00001
Model 2 Total effect of ABO group (AB=2, A or B=1, O=0) Effect not mediated by TC Effect mediated by TC Proportion of ABO effect mediated by TC
0.030, p=0.08 0.025, p=0.14 0.004, p=0.06 0.15
0.004, p=0.79 0.004, p=0.80 0.00003, p=0.98 0.008
*the difference in disease risk. †significant CAD indicated by >50% stenosis in at least one coronary artery in angiography. ‡prevalent or incident MI.
7
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Original Article Analysis of Circulating Cholesterol Levels as a Mediator of an Association Between ABO Blood Group and Coronary Heart Disease Yequn Chen, PhD*; Chang Chen, MD*; Xiayi Ke, PhD*; Longgen Xiong, MD; Yongying Shi, MD; Jiafu Li, MD; Xuerui Tan, PhD; Shu Ye, MD, PhD Background—Non-O type of ABO blood group has been associated with a predisposition to coronary heart disease. It is thought that this association is partly mediated by increased cholesterol levels in non–O-type individuals. In this study, we sought to estimate the mediation effect size. Methods and Results—In a group of individuals (n=6476) undergoing coronary angiography, we detected associations of non-O type with significant coronary artery disease with >50% stenosis in ≥1 coronary arteries (odds ratio, 1.24; 95% confidence interval, 1.10–1.39; P=2.6×10−4) and with prevalent or incident myocardial infarction (odds ratio, 1.22; 95% confidence interval, 1.09–1.37; P=1.2×10−3). Subjects of non-O type had higher levels of total cholesterol, low-density lipoprotein cholesterol, and non–high-density lipoprotein cholesterol (mean [SEM] in mmol/L: 4.931[0.021], 3.041 [0.018], and 3.805 [0.020] in non-O type compared with 4.778 [0.026], 2.906 [0.021], and 3.669 [0.024] in O type; P=3.8×10−7, P=1.5×10−7, and P=3.1×10−7, respectively). Mediation analyses indicated that 10% of the effect of non-O type on coronary artery disease susceptibility was mediated by increased low-density lipoprotein cholesterol level (P=7.8×10−4) and that 11% of the effect of non-O type on myocardial infarction risk was mediated by raised low-density lipoprotein cholesterol level (P=2.0×10−3). Conclusions—In a model in which it is presumed that cholesterol is a mediator of the associations of ABO group with coronary artery disease and myocardial infarction, around 10% of the effect of non-O type on coronary artery disease and myocardial infarction susceptibility was mediated by its influence on low-density lipoprotein cholesterol level. (Circ Cardiovasc Genet. 2014;7:43-48.) Key Words: ABO blood-group system ◼ cholesterol ◼ cholesterol, LDL ◼ coronary disease ◼ myocardial infarction
S
everal epidemiological studies have shown a relation between the human ABO blood group and coronary heart disease (CHD), such that individuals of a non-O type (A, B, or AB type) are more likely to have CHD than O-type individuals.1–3 However, the mechanistic basis underlying this relationship requires further investigations.
Clinical Perspective on p 48 It is well established that low-density lipoprotein cholesterol (LDLc) plays an essential role in the pathogenesis of atherosclerosis, the pathology underling CHD, and that high plasma concentrations of cholesterol, in particular those of LDLc, are a major risk factor for the disease.4 An association of ABO group with total cholesterol (TC) and LDLc levels
has also long been recognized5,6 and has recently been reinforced in genetic studies of variation at the ABO locus.7–12 Specifically, the non-O types, which confer an increased risk of CHD,1–3 are associated with increased TC and LDLc levels.5,6,12 Furthermore, studies have shown that non–O-type individuals have higher cholesterol absorption rates12,13 and that the cholesterol absorption rate is positively associated with cardiovascular risk.12,14 Therefore, it is thought that the effect of ABO on CHD susceptibility is in part mediated by its influence on cholesterol levels.8,12 However, it has remained unknown as to how much of the effect of non-O type on CHD susceptibility is attributable to the mediation of cholesterol levels. In this study, we sought to estimate this mediation effect size. In a large group
Received July 24, 2013; accepted December 18, 2013. From the Department of Cardiology, First Affiliated Hospital of Shantou University Medical College, Shantou, China (Y.C., C.C., X.T., S.Y.); Department of Cardiology, Southern Medical University, Guangzhou, China (C.C.); MRC Centre of Epidemiology for Child Health, University College London, London, United Kingdom (X.K.); Department of Cardiology, Second Affiliated Hospital of Guangzhou Medical College, Guangzhou, China (L.X.); Department of Cardiology, First Affiliated Hospital of Guangzhou Medical College, Guangzhou, China (Y.S.); Department of Cardiology, First Affiliated Hospital of Luzhou Medical College, Luzhou, China (J.L.); Shantou University Medical College, Shantou, China (S.Y.); and Department of Clinical Pharmacology, William Harvey Research Institute, Queen Mary University of London, London, United Kingdom (S.Y.). *Drs Y. Chen, C. Chen, and Ke contributed equally to this work. The Data Supplement is available at http://circgenetics.ahajournals.org/lookup/suppl/doi:10.1161/CIRCGENETICS.113.000299/-/DC1. Correspondence to Shu Ye, MD, PhD, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, United Kingdom, or First Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong 515041, China. E-mail [email protected] or Xuerui Tan, PhD, Department of Cardiology, First Affiliated Hospital of Shantou University Medical College, Guangdong 515041, China. E-mail [email protected] © 2014 American Heart Association, Inc. Circ Cardiovasc Genet is available at http://circgenetics.ahajournals.org
DOI: 10.1161/CIRCGENETICS.113.000299
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44 Circ Cardiovasc Genet February 2014 Table 1. Characteristics of Study Subjects Characteristics
Mean (SD) or %
Age, y
64.11 (11.42)
Sex (% of men)
71.4
ABO group (% of AB, A, B, and O)
7.2, 27.1, 25.8, 39.9
Total cholesterol, mmol/L
4.88 (1.25)
LDL cholesterol, mmol/L
2.99 (1.04)
HDL cholesterol, mmol/L
1.12 (0.33)
NHDL cholesterol, mmol/L
3.76 (1.26)
Triglycerides, mmol/L
1.62 (1.30)
Systolic blood pressure, mm Hg
135.39 (23.88)
Diastolic blood pressure, mm Hg
80.16 (13.88)
Diabetes mellitus, %
22.4
Cases with >50% coronary stenosis, %
69.8
Myocardial infarction, %
32.2
HDL indicates high-density lipoprotein; LDL, low-density lipoprotein; and NHDL, non–high-density lipoprotein.
of individuals (n=6476) undergoing diagnostic or interventional coronary angiography, we first verified the associations among ABO group, cholesterol levels, and CHD phenotypes and then performed mediation analyses in a model with cholesterol levels as a mediator for the effect of ABO group on CHD phenotypes.
Methods Subjects We studied 6476 consecutive patients undergoing diagnostic or interventional coronary angiography in the First Affiliated Hospital of Shantou University Medical College, the First Affiliated Hospital of Guangzhou Medical College, and the Second Affiliated Hospital of Guangzhou Medical College in Southern China. Data required for this study were collected from hospital records for the patients. All subjects were Chinese. The study was approved by the appropriate research ethics committee. We collected demographic and clinical data including age, sex, ABO blood type, plasma levels of TC, LDLc, high-density lipoprotein cholesterol (HDLc) and triglycerides, coronary angiographic findings, prevalent or incident myocardial infarction (MI) diagnosed according to the World Health Organization criteria, systolic and diastolic blood pressure, and the presence or absence of diabetes mellitus. ABO blood typing and cholesterol and triglyceride measurements were conducted by the clinical chemistry department of the
respective hospitals. Blood samples for cholesterol and triglyceride measurements were collected after overnight fasting. The main demographic and clinical characteristics of the study subjects are summarized in Table 1 and in Table I in the Data Supplement. Of the 6476 subjects, 4518 had significant angiographically documented coronary artery disease (CAD) as having >50% diameter stenosis in ≥1 major epicardial coronary artery. A total of 2083 subjects had prevalent or incident MI.
Statistical Analysis Values of TC, LDLc, HDLc, and triglycerides were logarithm transformed to normalize their distributions. Non-HDL cholesterol (NHDLc) values were calculated as TC minus HDLc and then normalized by logarithmic transformation. χ2 test and logistic regression analysis were used to ascertain association of ABO blood types with CAD and MI, respectively, with adjustment for age, sex, and hospitals as covariates. t Test and general linear model analysis were used to examine differences in lipid levels between CAD and non-CAD groups, between MI and non-MI groups, and between O and non-O blood types, with adjustment for age, sex, and hospitals as covariates. The above analyses were then performed in male and female subjects separately, with age and hospitals as covariates. All P values were 2 sided. For causal mediation analysis, the R package Mediation was used.15 Briefly, the average causal mediation effect is estimated as the expected difference in the potential outcome (Y) when the mediator (M) takes the value that would realize under the treatment condition (T) as opposed to the control condition, while the treatment status is held constant, which is expressed in the following equation: δ(t)=E{Y(t,M(t1))–Y(t,M(t0))},where t, t1, t0 are specific values of T, with t1≠t0. Similarly, the average direct effect is defined as the expected difference in the potential outcome when the treatment is changed while the mediator is held constant at the value that would realize whether the treatment equals t, according to the following equation:ζ(t)=E{Y(t1,M(t))–Y(t0,M(t))}. The total effect of the treatment on the outcome is composed of the average causal mediation effect and the average direct effect. This model allows the mediation effect to be estimated for a variety of data types.15 In this study, the blood type was regarded as the treatment variable in either a 2-group classification setting (A/B/AB versus O) or a 3-group classification setting (AB versus A/B versus O). The level of TC, LDLc, or NHDLc was regarded as the mediator. First, in the mediator model, a linear regression model was used to estimate the association between the treatment variable and the mediator. Second, in the outcome model, a generalized linear model was used to estimate the association of the mediator and the treatment variable and the outcome (either CAD or MI), using binomial probit link function. Finally, a nonparametric bootstrapping method with ≤100 000 simulations was applied to obtain accurate P values of the estimates of the total effects, direct effects, as well as mediation effects. The analyses were performed first in the sample as a whole with adjustment for age, sex, and hospitals and then separately in men and women with adjustment for age and hospitals.
Table 2. Association of ABO Blood Group With CAD and MI CAD* ABO Type
MI‡
Cases
Controls
OR (95% CI)†
Yes
No
OR (95% CI)†
AB
323 (71.6%)
128 (28.4%)
1.27 (1.01–1.60)
A
1227 (70.9%)
465 (29.1%)
1.29 (1.12–1.49)
144 (31.9%)
307 (68.1%)
1.14 (0.91–1.43)
578 (34.2%)
1114 (65.8%)
B
1142 (70.9%)
469 (29.1%)
1.28 (1.12–1.47)
1.18 (1.02–1.36)
524 (32.5%)
1087 (67.5%)
1.18 (1.02–1.35)
O
1685 (67.7%)
803 (32.3%)
Non-O
2692 (71.7%)
728 (29.3%)
1760 (70.7%)
1062 (28.3%)
1.24 (1.10–1.39)
1246 (33.2%)
O
1685 (67.7%)
2508 (66.8%)
1.22 (1.09–1.37)
803 (32.3%)
P=2.6×10−4
728 (29.3%)
1760 (70.7%)
P=1.2×10−3
CAD indicates coronary artery disease; CI, confidence interval; MI, myocardial infarction; and OR, odds ratio. *Significant CAD indicated by >50% stenosis in ≥1 coronary artery in angiography. †OR (95% CI) for non-O type vs O type. ‡Prevalent or incident MI.
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Chen et al ABO Blood Group and CHD 45 Table 3. Lipid Levels by ABO Blood Types Total Cholesterol
LDL Cholesterol
HDL Cholesterol
Non-HDL Cholesterol
Triglycerides
AB
4.968 (0.061); n=418
3.037 (0.051); n=408
1.142 (0.018); n=417
3.826 (0.057); n=417
1.695 (0.062); n=418
A
4.928 (0.031); n=1530
3.049 (0.027); n=1504
1.120 (0.008); n=1530
3.812 (0.029); n=1527
1.598 (0.029); n=1534
B
4.923 (0.032); n=1495
3.034 (0.027); n=1472
1.139 (0.009); n=1496
3.792 (0.030); n=1493
1.637 (0.037); n=1500
O
4.778 (0.026); n=2284
2.906 (0.021); n=2245
1.112 (0.007); n=2285
3.669 (0.024); n=2279
1.603 (0.028); n=2297
P value
9.1×10
4.4×10
−6
−6
3.7×10
−2
6.8×10
−6
n.s.
Non-O
4.931 (0.021); n=3443
3.041 (0.018); n=3384
1.131 (0.006); n=3443
3.805 (0.020); n=3437
1.627 (0.022); n=3452
O
4.778 (0.026); n=2284
2.906 (0.021); n=2245
1.112 (0.007); n=2285
3.669 (0.024); n=2279
1.603 (0.028); n=2297
P value
3.8×10
1.5×10
−7
−7
2.1×10
−2
3.1×10
−7
n.s.
Lipid levels shown are mean (SEM) in mmol/L. HDL indicates high-density lipoprotein; LDL, low-density lipoprotein; and n.s., nonsignificant.
Results We observed an association between non-O type (AB, A, or B) and the presence of significant CAD indicated by >50% stenosis in ≥1 coronary artery in angiography. The odds ratio (OR) for significant CAD was 1.24 (95% confidence interval [CI], 1.10–1.39; P=3.0×10−4) for subjects of non-O type as compared with O-type individuals with adjustment for age, sex, and hospitals (Table 2) and was similar in men (OR, 1.25; 95% CI, 1.08–1.45; P=3.0×10−3) and women (OR, 1.23; 95% CI, 1.02–1.48; P=3.5×10−2; Table III in the Data Supplement). Among those subjects with significant CAD, there was no association between ABO group and the number (1 or 2 or 3) of coronary arteries with >50% stenosis (Table III in the Data Supplement). There was a significant association between non-O type and prevalent or incident MI in the sample as a whole (OR, 1.22; 95% CI, 1.09–1.37; P=1.2×10−3; Table 2) and in men (OR, 1.26; 95% CI, 1.02– 1.44; P=1.0×10−3), with a similar but nonsignificant trend in women (OR, 1.08; 95% CI, 0.86–1.36; P>0.05; Table II in the Data Supplement). Subjects of the AB, A, or B type had similar concentrations of TC in the blood, whereas subjects of the O type had lower levels (mean [SEM]: 4.931 [0.021] mmol/L in non-O type compared with 4.778 [0.026] mmol/L in O type; P=3.8×10−7; Table 3), and so was the case for LDLc (3.041 [0.018] mmol/L in non-O type compared with 2.906 [0.021] in O type; P=1.5×10−7; Table 3). HDLc levels were marginally higher (rather than lower) in non–O-type than in
O-type subjects (1.131 [0.006] mmol/L versus 1.112 [0.007] mmol/L; P=2.1×10−2; Table 3), the reason for which is unknown. However, the levels of NHDLc, previously shown to be an independent and superior predictor of CHD from the Framingham Heart Study,16 were higher in non–O-type than in O-type individuals (3.805 [0.020] mmol/L versus 3.669 [0.024]; P=3.1×10−7; Table 3). There was no significant association between ABO group and triglycerides level (Table 3), which is consistent with findings from several other studies.7–12 The results were generally similar when men and women were analyzed separately (Table IV in the Data Supplement). As expected, levels of TC, LDLc, and NHDLc were higher in subjects with significant CAD than in those without and similarly were higher in the MI group as compared with the non-MI group (Table 4), and these were also the case when men and women were analyzed separately (Table V in the Data Supplement). HDLc levels were lower in cases with significant CAD than in those without (Table 4), with a similar trend in both sexes (Table V in the Data Supplement), while no significant association was detected between HDLc and MI (Table 4 and Table V in the Data Supplement). In the sample as a whole and in women, triglyceride level was higher in CAD cases but did not significantly differ between the MI and non-MI groups (Table 4 and Table V in the Data Supplement). However, in men, it was lower in CAD and MI cases, the reason for which is unknown (Table V in the Data Supplement).
Table 4. Lipid Levels by CAD Status and MI Status Total Cholesterol
LDL Cholesterol
HDL Cholesterol
Non-HDL Cholesterol
Triglycerides
Cases
4.919 (0.020); n=4128
3.036 (0.017); n=4056
1.109 (0.005); n=4128
3.812 (0.019); n=4119
1.629 (0.020); n=4145
Controls
4.776 (0.027); n=1805
2.897 (0.023); n=1775
1.157 (0.008); n=1809
3.626 (0.025); n=1803
1.591 (0.030); n=1812
P value
1.3×10−12
9.2×10−11
3.4×10−2
6.4×10−16
Yes
4.922 (0.030); n=1920
3.078 (0.025); n=1884
1.120 (0.007); n=1920
3.807 (0.027); n=1915
1.597 (0.031); n=1927
No
4.853 (0.019); n=4012
2.954 (0.016); n=3946
1.126 (0.005); n=4015
3.731 (0.018); n=4006
1.632 (0.020); n=4029
1.5×10−4
2.2×10−7
n.s.
5.0×10−6
CAD*
5.1×10−7
MI†
P value
n.s.
Lipid levels shown are mean (SEM) in mmol/L. CAD indicates coronary artery disease; HDL, high-density lipoprotein; LDL, low-density lipoprotein; MI, myocardial infarction; and n.s., nonsignificant. *Significant CAD indicated by >50% stenosis in ≥1 coronary artery in angiography. †Prevalent or incident MI.
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46 Circ Cardiovasc Genet February 2014 Having detected that subjects of non-O type had higher ORs of significant CAD and MI as well as higher total TC, LDLc, and NHDLc levels, together with the observation that TC, LDLc, and NHDL levels were higher in subjects with significant CAD (than in those without) and in those with MI (than those without), we performed mediation analyses with TC, LDLc, and NHDLc as mediators for the associations of ABO group with CAD and MI. Mediation analysis assesses an intermediate variable as a mediator in the pathway between a risk factor and an outcome to estimate the extent to which the effect of the risk factor occurs through the mediator.17–20 This method has been used in various research fields including studies of effects of genetic variants.21–24 A mediation analysis in a model with CAD as the dependent variable, non-O versus O type as the independent variable, and LDLc level as the mediator variable, with age, sex,
and hospitals as covariates, indicated that there was a 4.9% difference in CAD susceptibility between non-O type and O type (P=4.0×10−5) and that 10% of this difference was mediated by increased LDLc level (0.5% of the difference in CAD susceptibility was mediated by LDLc level; P=7.8×10−4 for mediation effect; Table 5). In the above model, substituting LDLc with TC or NHDLc as the mediator variable showed that TC and NHDLc had a similar mediation effect size to that of LDLc (Table 5). As expected, the levels of TC, LDLc, and NHDLc were highly correlated (correction coefficient, 0.873 for TC and LDLc; 0.890 for LDLc and NHDLc; and 0.959 for TC and NHDLc; all P50% stenosis in ≥1 coronary artery in angiography. ‡Prevalent or incident MI.
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Chen et al ABO Blood Group and CHD 47 risk between non-O type and O type (P=8.0×10−3) and that 11% of this difference was mediated by increased LDLc level (0.4% of the difference in MI risk was mediated by LDLc level; P=2.0×10−3 for mediation effect; Table 5). Substituting LDLc with TC or NHDLc as the mediator variable in this model showed that TC and NHDLc had a somewhat smaller mediation effect size (6% and 7%, respectively) than that of LDLc (Table 5). In mediation analyses in which ABO group was coded as AB=2, A or B=1, and O=0, rather than non-O type=1 and O type=0 as in the models described above, the results were similar to those described above (Table 5). When the mediation analyses were performed in men and women separately, the mediation effect sizes of LDLc, TC, and NHDLc for CAD were similar; however, with regard to MI, mediation effects of LDLc, TC, and NHDLc were significant in men but not in women (Tables VI and VII in the Data Supplement). Some limitations to our study warrant mentioning. First, the usefulness of the results of the mediation analyses will be dependent on the correctness of the presumed model. Second, there were no available data for ABO subtypes (such as A1 and A2) or single-nucleotide polymorphisms at the ABO locus for analysis in this study. However, the A2 allele is very rare in the Chinese, and therefore it is likely that type A in this study was mostly the A1 type.25 Third, because this study was conducted in individuals of Chinese ancestry, the findings may not be generalizable to other races/ethnicities.
Discussion In summary, our study confirms the presence of associations of ABO blood group with cholesterol levels and CHD susceptibility, with non-O types associating with higher TC, LDLc, and NHDLc levels and greater risks of significant CAD and MI in an investigation of a large group of individuals undergoing coronary angiography. In a presumed model with cholesterol levels as mediators for the associations of ABO group with CHD, our mediation analyses indicated that ≈10% of the effect of non-O type on CAD and MI susceptibility was attributable to the mediation of LDLc level and that this 10% was significantly nonzero. These findings provide a useful insight for understanding the genetic mechanisms underlying CHD susceptibility.
Sources of Funding Work in the laboratory of author Dr Ye is supported by the British Heart Foundation and forms part of the research themes contributing to the translational research portfolio of Barts Cardiovascular Biomedical Research Unit, which is supported and funded by the National Institute of Health Research. The UK Medical Research Council has provided financial support for the MRC Centre of Epidemiology for Child Health, UCL Institute of Child Health, where Dr Ke works.
Disclosures None.
References 1. Wu O, Bayoumi N, Vickers MA, Clark P. ABO(H) blood groups and vascular disease: a systematic review and meta-analysis. J Thromb Haemost. 2008;6:62–69. 2. Reilly MP, Li M, He J, Ferguson JF, Stylianou IM, Mehta NN, et al; Myocardial Infarction Genetics Consortium; Wellcome Trust Case Control Consortium. Identification of ADAMTS7 as a novel locus for coronary atherosclerosis and association of ABO with myocardial infarction in the presence of coronary atherosclerosis: two genome-wide association studies. Lancet. 2011;377:383–392. 3. He M, Wolpin B, Rexrode K, Manson JE, Rimm E, Hu FB, et al. ABO blood group and risk of coronary heart disease in two prospective cohort studies. Arterioscler Thromb Vasc Biol. 2012;32:2314–2320. 4. Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med. 1999;340:115–126. 5. Langman MJ, Elwood PC, Foote J, Ryrie DR. ABO and Lewis blood-groups and serum-cholesterol. Lancet. 1969;2:607–609. 6. Oliver MF, Geizerova H, Cumming RA, Heady JA. Serum-cholesterol and ABO and rhesus blood-groups. Lancet. 1969;2:605–606. 7. Teslovich TM, Musunuru K, Smith AV, Edmondson AC, Stylianou IM, Koseki M, et al. Biological, clinical and population relevance of 95 loci for blood lipids. Nature. 2010;466:707–713. 8. Schunkert H, König IR, Kathiresan S, Reilly MP, Assimes TL, Holm H, et al; Cardiogenics; CARDIoGRAM Consortium. Large-scale association analysis identifies 13 new susceptibility loci for coronary artery disease. Nat Genet. 2011;43:333–338. 9. Wu Y, Waite LL, Jackson AU, Sheu WH, Buyske S, Absher D, et al. Trans-ethnic fine-mapping of lipid loci identifies population-specific signals and allelic heterogeneity that increases the trait variance explained. PLoS Genet. 2013;9:e1003379. 10. Deloukas P, Kanoni S, Willenborg C, Farrall M, Assimes TL, Thompson JR, et al. Large-scale association analysis identifies new risk loci for coronary artery disease. Nat Genet. 2013;45:25–33. 11. Willer CJ, Schmidt EM, Sengupta S, Peloso GM, Gustafsson S, Kanoni S, et al. Discovery and refinement of loci associated with lipid levels. Nat Genet. 2013;45:1274–1283. 12. Silbernagel G, Chapman MJ, Genser B, Kleber ME, Fauler G, Scharnagl H, et al. High intestinal cholesterol absorption is associated with cardiovascular disease and risk alleles in ABCG8 and ABO: evidence from the LURIC and YFS cohorts and from a meta-analysis. J Am Coll Cardiol. 2013;62:291–299. 13. Teupser D, Baber R, Ceglarek U, Scholz M, Illig T, Gieger C, et al. Genetic regulation of serum phytosterol levels and risk of coronary artery disease. Circ Cardiovasc Genet. 2010;3:331–339. 14. Strandberg TE, Tilvis RS, Pitkala KH, Miettinen TA. Cholesterol and glucose metabolism and recurrent cardiovascular events among the elderly: a prospective study. J Am Coll Cardiol. 2006;48:708–714. 15. Imai K, Kees L, Tingley D, Yamamoto T. Causal mediation analysis using R. In: Vinod HD, ed. Advances in Social Science Research Using R. New York: Springer; 2010:129–154. 16. Liu J, Sempos CT, Donahue RP, Dorn J, Trevisan M, Grundy SM. Non-high-density lipoprotein and very-low-density lipoprotein cholesterol and their risk predictive values in coronary heart disease. Am J Cardiol. 2006;98:1363–1368. 17. Baron RM, Kenny DA. The moderator-mediator variable distinction in social psychological research: conceptual, strategic, and statistical considerations. J Pers Soc Psychol. 1986;51:1173–1182. 18. Huang B, Sivaganesan S, Succop P, Goodman E. Statistical assess ment of mediational effects for logistic mediational models. Stat Med. 2004;23:2713–2728. 19. Li Y, Schneider JA, Bennett DA. Estimation of the mediation effect with a binary mediator. Stat Med. 2007;26:3398–3414. 20. Albert JM. Mediation analysis via potential outcomes models. Stat Med. 2008;27:1282–1304. 21. Stoltenberg SF, Twitchell GR, Hanna GL, Cook EH, Fitzgerald HE, Zucker RA, et al. Serotonin transporter promoter polymorphism, peripheral indexes of serotonin function, and personality measures in families with alcoholism. Am J Med Genet. 2002;114:230–234.
Downloaded from http://circgenetics.ahajournals.org/ at Queen's University--Kingston (CANADA) on August 18, 2014
48 Circ Cardiovasc Genet February 2014 22. VanderWeele TJ, Asomaning K, Tchetgen Tchetgen EJ, Han Y, Spitz MR, Shete S, et al. Genetic variants on 15q25.1, smoking, and lung cancer: an assessment of mediation and interaction. Am J Epidemiol. 2012;175:1013–1020. 23. Machiela MJ, Lindström S, Allen NE, Haiman CA, Albanes D, Barricarte A, et al. Association of type 2 diabetes susceptibility variants with advanced prostate cancer risk in the Breast and Prostate Cancer Cohort Consortium. Am J Epidemiol. 2012;176:1121–1129.
24. Siedlinski M, Tingley D, Lipman PJ, Cho MH, Litonjua AA, Sparrow D, et al; COPDGene and ECLIPSE Investigators. Dissecting direct and indirect genetic effects on chronic obstructive pulmonary disease (COPD) susceptibility. Hum Genet. 2013;132:431–441. 25. Zhu F, Tao S, Xu X, Ying Y, Hong X, Zhu H, et al. Distribution of ABO blood group allele and identification of three novel alleles in the Chinese Han population. Vox Sang. 2010;98:554–559.
CLINICAL PERSPECTIVE It has long been recognized that the ABO blood group has an influence on risk of coronary heart disease (CHD), such that individuals of a non–O-type (A, B, or AB type) are more likely to have CHD than O-type individuals. However, the mechanistic basis underlying this relationship is still unclear. Interestingly, it has also been shown that non–O-type individuals tend to have higher cholesterol absorption rates and higher levels of total cholesterol and low-density lipoprotein cholesterol in the blood. Therefore, it is thought that the effect of ABO blood group on CHD susceptibility is in part mediated by its influence on circulating cholesterol levels, a well-established risk factor that plays an important role in the development of CHD. However, it has remained unknown as to how much of the effect of non-O type on CHD susceptibility is attributable to the mediation of cholesterol levels. In this study, we sought to estimate this mediation effect size. In a large group of individuals (n=6476) undergoing diagnostic or interventional coronary angiography, we first verified the associations among ABO group, cholesterol levels, and CHD phenotypes and then performed mediation analyses in a model with cholesterol levels as a mediator for the effect of ABO group on CHD phenotypes. The mediation analyses indicated that ≈10% of the effect of non-O type on CHD susceptibility and risk of myocardial infarction was mediated by increased low-density lipoprotein cholesterol level.
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SUPPLEMENTAL MATERIAL
Table S1. Characteristics of study subjects by gender Mean (SD) or % Males (n=4296)
Females (n=2180)
Age (years)
62.95 (11.67)
66.41 (10.53)
ABO group (% of AB, A, B, and O)
7.2, 28.8, 25.9, 40.1 7.3, 27.6, 25.6, 39.5
Total cholesterol (mmol/L)
4.76 (1.24)
5.01 (1.24)
LDL-cholesterol (mmol/L)
2.95 (1.04)
3.07 (1.01)
HDL-cholesterol (mmol/L)
1.09 (0.31)
1.19 (0.33)
NHDL- cholesterol (mmol/L)
3.68 (1.16)
3.91 (1.15)
Triglycerides (mmol/L)
1.72 (1.17)
1.57 (1.50)
Systolic blood pressure (mmHg)
133.24 (23.39)
139.62 (24.27)
Diastolic blood pressure (mmHg)
79.99 (14.04)
80.49 (13.57)
Diabetes mellitus (%)
18.1
22.7
Cases with >50% coronary stenosis (%) 75.1
59.2
MI (%)
19.2
38.7
1
Table S2. Association of ABO blood group with CAD and MI, by gender Gender Males
Females
AB A B O
*CAD ‡OR (95% CI) cases controls 236 (79.5%) 61 (20.5%) 1.49 (1.10, 2.02) 858 (77.6%) 248 (22.4%) 1.28 (1.07, 1.54) 810 (75.8%) 258 (24.2%) 1.16 (0.97, 1.39) 1208 (73.2%) 443 (26.8%)
†MI ‡OR (95% CI) Yes No 119 (40.1%) 178 (59.9%) 1.25 (0.96, 1.62) 460 (41.6%) 646 (58.4%) 1.34 (1.14, 1.58) 412 (38.6%) 656 (61.4%) 1.18 (1.01, 1.39) 576 (34.9%) 1075 (65.1%)
non-O O
1904 (77.1%) 567 (22.9%) 1.25 (1.08, 1.45) 1208 (73.2%) 443 (26.8%) p=3.010-3
991 (40.1%) 1480 (59.9%) 1.26 (1.02, 1.44) 576 (34.9%) 1075 (65.1%) p=1.010-3
AB A B O
87 (56.5%) 369 (63.0%) 332 (61.1%) 477 (57.0%)
67 (43.5%) 1.00 (0.69, 1.45) 217 (37.0%) 1.31 (1.04, 1.66) 211 (38.9%) 1.21 (0.96, 1.54) 360 (43.0%)
25 (16.2%) 118 (20.1%) 112 (20.6%) 152 (18.2%)
non-O O
788 (61.4%) 477 (57.0%)
495 (38.6%) 1.23 (1.02, 1.48) 360 (43.0%) p=3.510-2
255 (19.9%) 1028 (80.1%) 1.08 (0.86, 1.36) 152 (18.2%) 685 (81.8%) n.s.
ABO type
129 (83.8%) 468 (79.9%) 431 (79.4%) 685 (81.8%)
0.85 (0.53, 1.37) 1.09 (0.82, 1.43) 1.15 (0.87, 1.52)
*significant CAD indicated by >50% stenosis in at least one coronary artery in angiography. †prevalent or incident MI. ‡odds ratio (95% confidence interval) for non-O type versus O type.
2
Table S3. Patients with one, two or three coronary arteries with >50% stenosis, by ABO types Gender
ABO type
Both
AB A B O
Males
Females
Number of coronary artery with >50% stenosis One Two Three 110 (6.8%) 104 (7.9%) 109 (7.5%) 439 (27.2%) 372 (28.4%) 416 (28.6%) 434 (26.9%) 339 (25.9%) 369 (25.4%) 631 (39.1%) 495 (37.8%) 559 (38.5%)
Non-O O
983 (60.9%) 631 (39.1%)
815 (62.2%) 495 (37.8%)
894 (61.5%) 559 (38.5%)
AB A B O
81 (7.5%) 286 (26.4%) 304 (28.1%) 412 (38.0%)
69 (7.2%) 272 (28.4%) 244 (25.5%) 372 (38.9%)
86 (8.0%) 300 (28.0%) 262 (24.4%) 424 (39.6%)
Non-O O
671 (62.0%) 412 (38.0%)
585 (61.1%) 372 (38.9%)
648 (60.4%) 424 (39.6%)
AB A B O
29 (5.5%) 153 (28.8%) 130 (24.5%) 219 (41.2%)
35 (9.9%) 100 (28.3%) 95 (26.9%) 123 (34.8%)
23 (6.0%) 116 (30.4%) 107 (28.1%) 135 (35.4%)
Non-O O
312 (58.8%) 219 (41.2%)
230 (65.2%) 123 (34.8%)
246 (64.6%) 135 (35.4%)
3
Table S4. Lipid levels according to ABO blood types, by gender Gender Males
ABO type AB A B O p-value
Total cholesterol 4.903 (0.075), n=276 4.802 (0.037), n=1015 4.792 (0.039), n=990 4.685 (0.033), n=1521 3.010-3
non-O O p-value
4.810 (0.025), n=2281 2.987 (0.022), n=2244 1.091 (0.007), n=2277 3.722 (0.024), n=2275 1.581 (0.025), n=2286 4.685 (0.033), n=1521 2.876 (0.027), n=1494 1.083 (0.008), n=1522 3.675 (0.031), n=1517 1.542 (0.031), n=1531 n.s. n.s. 4.010-4 2.210-4 1.110-4
Females AB A B O p-value non-O O p-value
5.094 (0.105), n=142 5.177 (0.055), n=515 5.181 (0.056), n=505 4.962 (0.043), n=763 2.010-3
LDL-cholesterol 3.016 (0.065), n=270 2.991 (0.032), n=999 2.976 (0.036), n=975 2.876 (0.027), n=1494 3.010-3
3.079 (0.081), n=138 3.164 (0.048), n=505 3.149 (0.046), n=497 2.965 (0.035), n=751 2.010-3
HDL-cholesterol Non-HDL-cholesterol 1.117 (0.023), n=275 3.787 (0.071), n=275 1.076 (0.009), n=1013 3.729 (0.035), n=1012 1.101 (0.010), n=989 3.697 (0.036), n=988 1.083 (0.008), n=1522 3.675 (0.031), n=1517 n.s. 1.010-3
1.192 (0.026), n=142 1.206 (0.014), n=517 1.213 (0.017), n=507 1.169 (0.012), n=764 n.s.
3.902 (0.095), n=142 3.974 (0.052), n=515 3.979 (0.051), n=505 3.801 (0.040), n=762 1.010-2
Triglycerides 1.694 (0.074), n=276 1.569 (0.035), n=1018 1.563 (0.039), n=992 1.542 (0.031), n=1531 n.s.
1.698 (0.114), n=142 1.655 (0.053), n=516 1.782 (0.078), n=508 1.725 (0.056), n=766 n.s.
5.168 (0.037), n=1162 3.147 (0.031), n=1140 1.207 (0.010), n=1166 3.967 (0.034), n=1162 1.716 (0.044), n=1166 4.962 (0.043), n=763 2.965 (0.035), n=751 1.169 (0.012), n=764 3.801 (0.040), n=762 1.725 (0.056), n=766 -4 -4 -3 n.s. 2.110 1.310 8.010 1.010-3
Lipid levels shown are mean (SEM) in mmol/L. n.s.: non-significant.
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Table S5. Lipid levels by CAD status and MI status, respectively, in males and in females Gender Males
Females
*CAD
Cases Controls p-value
Total cholesterol 4.818 (0.023), n=2971 4.601 (0.036), n=982 2.210-9
LDL-cholesterol 2.998 (0.020), n=2925 2.794 (0.031), n=961 8.410-10
HDL-cholesterol 1.083 (0.006), n=2970 1.105 (0.011), n=982 n.s.
Non-HDL-cholesterol 3.738 (0.022), n=2963 3.500 (0.034), n=980 1.610-11
Triglycerides 1.561 (0.021), n=2986 1.584 (0.039), n=984 4.510-2
†MI
Yes No p-value
4.875 (0.033), n=1541 4.693 (0.025), n=2411 1.610-5
3.071 (0.027), n=1513 2.869 (0.021), n=2372 3.810-9
1.104 (0.008), n=1541 1.079 (0.006), n=2410 n.s.
3.776 (0.030), n=1537 3.616 (0.023), n=2405 1.710-6
1.533 (0.029), n=1547 1.588 (0.024), n=2422 9.010-3
*CAD
Cases Controls p-value
5.176 (0.038), n=1157 4.985 (0.040), n=823 2.010-3
3.135 (0.032), n=1131 3.018 (0.033), n=814 3.010-2
1.178 (0.012), n=1158 1.218 (0.010), n=827 n.s.
4.004 (0.036), n=1156 3.775 (0.036), n=823 3.110-4
1.807 (0.047), n=1159 1.598 (0.047), n=828 9.810-6
†MI
Yes No p-value
5.111 (0.070), n=379 5.093 (0.030), n=1601 n.s.
3.108 (0.056), n=371 3.081 (0.025), n=1574 n.s.
1.185 (0.016), n=379 1.197 (0.009), n=1606 n.s.
3.936 (0.063), n=378 3.902 (0.028), n=1601 n.s.
1.811 (0.097), n=380 1.698 (0.035), n=1607 n.s.
Lipid levels shown are mean (SEM) in mmol/L. *significant CAD indicated by >50% stenosis in at least one coronary artery in angiography. †prevalent or incident MI. n.s.: non-significant.
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Table S6. TC, LDLc or NHDLc level as a mediator for association of ABO with CAD and MI, respectively, in males (n=3802) Effect size Mediator Model Parameter *CAD †MI -3 Total cholesterol (TC) Model 1 Total effect of ABO group 0.048, p=1.110 0.048, p=2.610-3 -3 (non-O versus O type) Effect not mediated by TC 0.043, p=2.310 0.046, p=3.810-3 Effect mediated by TC 0.005, p=8.710-3 0.003, p=5.310-2 Proportion of ABO effect mediated by TC 0.09 0.06
LDL-cholesterol (LDLc)
Non-HDL-cholesterol (NHDLc)
Model 2 Total effect of ABO group (AB=2, A or B=1, O=0) Effect not mediated by TC Effect mediated by TC Proportion of ABO effect mediated by TC
0.042, p=2.010-4 0.038, p=4.010-4 0.004, p=7.410-3 0.09
0.034, p=5.410-3 0.032, p=8.610-3 0.002, p=3.610-2 0.07
Model 1 Total effect of ABO group (non-O versus O type) Not mediated by LDLc Mediated by LDLc Proportion of ABO effect mediated by LDLc
0.046, p=1.010-3 0.042, p=3.210-3 0.005, p=7.810-3 0.10
0.045, p=5.310-3 0.041, p=1.210-2 0.005, p=1.510-2 0.10
Model 2 Total effect of ABO group (AB=2, A or B=1, O=0) Effect not mediated by LDLc Effect mediated by LDLc Proportion of ABO effect mediated by LDLc
0.039, p=2.010-4 0.036, p=1.210-3 0.004, p=1.210-2 0.09
0.029, p=1.710-2 0.026, p=3.810-2 0.004, p=1.010-2 0.12
Model 1 Total effect of ABO group (non-O versus O type) Effect not mediated by TC Effect mediated by TC Proportion of ABO effect mediated by TC
0.049, p=5.210-4 0.043, p=2.010-3 0.006, p=5.210-3 0.12
0.048, p=2.010-3 0.045, p=4.710-3 0.003, p=2.810-2 0.07
Model 2 Total effect of ABO group (AB=2, A or B=1, O=0) Effect not mediated by TC Effect mediated by TC Proportion of ABO effect mediated by TC
0.043, p=2.010-3 0.038, p=2.010-3 0.005, p=1.210-2 0.11
0.033, p=8.710-3 0.031, p=1.510-2 0.003, p=2.310-2 0.08
*the difference in disease risk. †significant CAD indicated by >50% stenosis in at least one coronary artery in angiography. ‡prevalent or incident MI.
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Table S7. TC, LDLc or NHDLc level as a mediator for association of ABO with CAD and MI, respectively, in females (n=1925) *Effect size Mediator Model Parameter †CAD ‡MI -2 Total cholesterol (TC) Model 1 Total effect of ABO group 0.013, p=0.45 0.052, p=1.210 (non-O versus O type) Effect not mediated by TC 0.047, p=2.510-2 0.014, p=0.44 Effect mediated by TC 0.006, p=4.510-2 -0.0008, p=0.55 Proportion of ABO effect mediated by TC 0.11 -0.06
LDL-cholesterol (LDLc)
Non-HDL-cholesterol (NHDLc)
Model 2 Total effect of ABO group (AB=2, A or B=1, O=0) Effect not mediated by TC Effect mediated by TC Proportion of ABO effect mediated by TC
0.029, p=0.11 0.025, p=0.16 0.004, p=0.06 0.13
0.004, p=0.76 0.004, p=0.73 -0.0005, p=0.57 -0.13
Model 1 Total effect of ABO group (non-O versus O type) Not mediated by LDLc Mediated by LDLc Proportion of ABO effect mediated by LDLc
0.054, p=8.010-3 0.050, p=1.7×10-2 0.004, p=7.110-2 0.08
0.013, p=0.48 0.013, p=0.48 -0.0001, p=0.92 -0.01
Model 2 Total effect of ABO group (AB=2, A or B=1, O=0) Effect not mediated by LDLc Effect mediated by LDLc Proportion of ABO effect mediated by LDLc
0.030, p=0.08 0.027, p=0.10 0.003, p=0.11 0.10
0.003, p=0.83 0.003, p=0.81 -0.0001, p=0.95 -0.01
Model 1 Total effect of ABO group (non-O versus O type) Effect not mediated by TC Effect mediated by TC Proportion of ABO effect mediated by TC
0.053, p=2.010-2 0.047, p=2.6×10-2 0.006, p=3.410-2 0.12
0.01, p=0.47 0.01, p=0.47 0.000004, p=0.97 0.00001
Model 2 Total effect of ABO group (AB=2, A or B=1, O=0) Effect not mediated by TC Effect mediated by TC Proportion of ABO effect mediated by TC
0.030, p=0.08 0.025, p=0.14 0.004, p=0.06 0.15
0.004, p=0.79 0.004, p=0.80 0.00003, p=0.98 0.008
*the difference in disease risk. †significant CAD indicated by >50% stenosis in at least one coronary artery in angiography. ‡prevalent or incident MI.
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