Arteriosclerosis, Thrombosis, and Vascular Biology

CLINICAL AND POPULATION STUDIES

Independent Causal Effect of Remnant

Cholesterol on Atherosclerotic Cardiovascular
Outcomes: A Mendelian Randomization Study
Eliano P. Navarese , Donna Vine , Spencer Proctor, Klaudyna Grzelakowska , Sergio Berti, Jacek Kubica, Paolo Raggi

BACKGROUND: Observational studies suggested that residual risk of cardiovascular events after LDL (low-density lipoprotein)
cholesterol lowering may be linked to remnant cholesterol (RC). We conducted a large-scale Mendelian randomization study
to investigate the causal role of RC to predict coronary artery disease (CAD), myocardial infarction (MI), and stroke risk.

METHODS: We extracted single-nucleotide polymorphisms for RC and LDL from large-scale genome-wide association
databases. We estimated the genetic association with outcomes from the CARDIoGRAMplusC4D consortium (Coronary
Artery Disease Genome-Wide Replication and Meta-Analysis Plus the Coronary Artery Disease Genetics), the Metastroke
consortium, as well as the GLGC (Global Lipids Genetics Consortium). Genetic variants were used as instruments, thereby
minimizing residual confounding and reverse causation biases of observational studies.

RESULTS: By leveraging data from a combined sample of 958 434 participants, we found evidence for a significant causal
effect of RC on the risk of CAD (odds ratio [OR], 1.51 per SD unit increase in RC [95% CI, 1.42–1.60]; P=5.3×10-5), MI (OR,
1.57 [95% CI, 1.21–2.05]; P=9.5×10-4), and stroke (OR, 1.23 [95% CI, 1.12–1.35]; P=3.72×10-6). There was no evidence
of pleiotropy. The effect of RC on CAD and MI remained consistent after accounting for the effects of RC-associated genetic
variants on LDL cholesterol: OR, 1.49 (95% CI, 1.37–1.61) for CAD and OR, 1.80 (95% CI, 1.70–19.1) for MI without a
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meaningful indirect effect exerted on these outcomes via the LDL cholesterol mediator.

CONCLUSIONS: This large-scale Mendelian randomization study showed a robust genetic causal association between RC and
cardiovascular outcomes. The effect on CAD and MI is independent of LDL cholesterol. Early screening for RC along with
long-term inhibition of RC should be the focus of future therapeutic interventions.
GRAPHIC ABSTRACT: A graphic abstract is available for this article.

Key Words: cholesterol ◼ coronary artery disease ◼ myocardial infarction ◼ risk ◼ stroke

D
espite improved patient care and optimization of
therapeutic interventions, atherosclerotic cardio-
vascular disease (ASCVD) remains one of most
burdensome noncommunicable diseases. Limitations to
early prediction models for ASCVD have been the result
of selective outcome bias, small observational samples,
and lack of validation with robust, global Mendelian ran-
domization (MR) analyses.
One of the most important advancements has been
the understanding of how remnant cholesterol (RC)
represents the most significant and independent contri-
bution to ASCVD risk after lowering of LDL (low-density
lipoprotein) cholesterol (LDL-C).1,2 RC is the cholesterol
carried in chylomicrons, chylomicron remnants, VLDL
(very low-density lipoprotein), and IDL (intermediate-
density lipoprotein). The concept of residual risk seeks to
reconcile the large proportion of clinical events despite
aggressive regime(s) of lipid-lowering pharmacothera-
pies. Early pathophysiological data demonstrated that
RC can be taken up by the arterial wall and accumulate

 
Correspondence to: Paolo Raggi, MD, PhD, Division of Cardiology, Department of Medicine, University of Alberta, 8440 112 St, Ste 2C2.09, Edmonton, AB T5N3J9.
Email raggi@ualberta.ca
Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/ATVBAHA.123.319297.
For Sources of Funding and Disclosures, see page e379.
© 2023 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at www.ahajournals.org/journal/atvb

Arterioscler Thromb Vasc Biol. 2023;43:e373–e380. DOI: 10.1161/ATVBAHA.123.319297 September 2023   e373
 Navarese et al
CLINICAL AND POPULATION
Nonstandard Abbreviations and Acronyms
STUDIES - AL
ASCVD atherosclerotic cardiovas-
cular disease
CAD coronary artery disease
CARDIoGRAMplusC4D Coronary Artery Disease
Genome-Wide Replica-
tion and Meta-Analysis
Plus the Coronary Artery
Disease Genetics
GLGC  Global Lipids Genetics
Consortium
GWAS  genome-wide association
study
IDL intermediate-density
lipoprotein
LDL low-density lipoprotein
LDL-C LDL cholesterol
L-VLDL large VLDL
MI myocardial infarction
MR Mendelian randomization
M-VLDL medium VLDL
OR odds ratio
RC remnant cholesterol
SNP  single-nucleotide
polymorphism
S-VLDL small VLDL
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VLDL  very low-density
lipoprotein
in the intima and smooth muscle layers leading to foam
cell formation and atherogenesis.3 Subsequent case-
control and intervention clinical studies underpinned the
rationale for pursuing RC as a potential therapeutic tar-
get in both adults and younger populations.4–6 Access
to global scale data sets could improve precision of
patient risk stratification and early outcomes prediction
for ASCVD.
MR analysis (based on the Mendel law of inheritance)
has been used more recently to plausibly investigate
causal inferences to ASCVD with regional data sets,
including RC.7 Critically, MR approaches can be used
to strengthen the causal inference utilizing genetic vari-
ants as instrumental variables for RC exposure, which
are randomly allocated at conception.8 Genetic studies
such as the Copenhagen Heart Study have successfully
guided new lipid-lowering practices and policies in these
regions.9 Other regions have begun to embark on similar
analyses to test local observations and inherent genetic
influences.10,11
In an effort to further transform this field, we chose to
combine multiple data sets and increase the global scal-
ability of the MR model(s), specifically for RC and ASCVD
outcomes. In particular, we leveraged the potential of
e374   September 2023 Arterioscler Thromb Vasc Biol. 2023;43:e373–e380. DOI: 10.1161/ATVBAHA.123.319297
Mendelian Randomization Study of RC and ASCVD
Highlights
• This large-scale Mendelian randomization study
showed a robust causal association between rem-
nant cholesterol and cardiovascular outcomes.
• The effect on coronary artery disease and myocar-
dial infarction was independent of LDL (low-density
lipoprotein) cholesterol.
• The findings of this study and other observational
analyses are consistent in showing the causal effect
of remnant cholesterol.
• These results should prompt a revision of current
guidelines to incorporate remnant cholesterol as an
independent risk factor for atherosclerotic cardio-
vascular disease.
naturally occurring RC-related genetic variants to predict
ASCVD risk by examining the causal role of RC using
genome-wide association study (GWAS) data set vari-
ants as instruments, thereby minimizing residual con-
founding, measurement error, and reverse causation
biases of observational studies.12 Here, we compiled a
combined sample of 958 434 participants; comprehen-
sively used genetic evaluation screening of 12 000 000
single-nucleotide polymorphisms (SNPs) from GWAS;
incorporated RC outcomes independent from that of
LDL-C; applied both direct and indirect effect strength(s)
of odds ratio (OR); and computed the equivalent of life-
long intervention for RC lowering.
METHODS
Study Design
The data that support the findings of this study are avail-
able from the corresponding author on reasonable request.
A 2-sample MR study was used to assess the causal asso-
ciations between RC, and the risk of coronary artery disease
(CAD), myocardial infarction (MI), and stroke. This innovative
approach implies that the genetic instrument-risk factor asso-
ciation and genetic instrument-outcome association are gen-
erated from different (nonoverlapping) samples to minimize
biases. The study design concept is depicted in the Graphic
Abstract. RC was regarded as the exposure, and the risk of
CAD, MI, and stroke served as the outcomes. RC was mea-
sured using nonfasting methods. Total RC was regarded as the
combination of several lipid subfractions such as VLDL, IDL,
and chylomicron remnants, as conventionally defined.13 The
causal effect of RC on the outcomes of interest was inves-
tigated as the total effect and separated into a direct and an
indirect effect using LDL-C as a potential mediator. The valid
instrumental variables for RC should satisfy 3 key assump-
tions: (1) the genetic variants must be strongly associated with
RC (relevance assumption), (2) the genetic variants must be
independent of potential environmental confounding factors
(independence assumption), and (3) the genetic variants must
influence the risk of CAD, MI, and stroke only via the change
of RC (exclusion restriction). In reporting on the findings of the
 Navarese et al
study and its assumptions, we formally adhered to international
guidelines for MR.14
Outcome Sources
Genetic instruments to test the association with RC and their
individual lipoprotein components were obtained in 19 273
subjects of European ancestry from a large GWAS15; addition-
ally, data on 115 078 individuals from the OpenGWAS public
data infrastructure, a comprehensive database of harmonized
genome-wide association summary statistics, were used
(https://gwas.mrcieu.ac.uk/datasets/met-d-Remnant_C/).
To estimate the genetic association with outcomes, publicly
available summary-level data were obtained for CAD and MI
from the CARDIoGRAMplusC4D consortium (Coronary Artery
Disease Genome-Wide Replication and Meta-Analysis Plus the
Coronary Artery Disease Genetics; www.cardiogramplusc4d.
org) including 184 305 and 171 875 patients, respectively.16
Data on ischemic stroke were available in 446 696 individuals
from a large genome-wide-association meta-analysis includ-
ing those from the Metastroke consortium.17 Serum lipid lev-
els were quantified by high-throughput nuclear magnetic
resonance spectroscopy metabolomics across included data
sources.
Genetic instruments for the association with clinical out-
comes were obtained in a total of 184 305 patients for CAD/
MI and 466 696 for stroke. The combined sample size reached
a total of 958 434 subjects. Over 12 000 000 SNPs were
screened.
Twenty-six variants were eventually used as instruments for
CAD, 30 for myocardial infarction (MI), and 24 for stroke. In
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parallel, the effect of genetic instruments on LDL-C exposure
was estimated. Instruments to proxy for LDL were retrieved
from the GLGC (Global Lipids Genetics Consortium) including
a population of 173 082 subjects (http://lipidgenetics.org/)18
and used to compare the effect of RC on outcomes and media-
tion analyses.
Genetic Instrument Selection
To construct genetic instruments, we identified genetic prox-
ies as SNPs associated with RC exposure and individual lipo-
proteins with greatest RC content including L-VLDLs (large
VLDLs), M-VLDL (medium VLDLs), S-VLDLs (small VLDLs),
and IDLs (intermediate-density lipoproteins).15 We utilized
SNPs that were associated with RC at the standard genome-
wide significance threshold (P<5×10−8) that were independent
(r2<0.001 and distance >10 000 kb) to minimize weak instru-
ment bias. SNPs in linkage disequilibrium were clumped.
Palindromic variants in which genetic ambiguity was pres-
ent based on the comparative evaluation of the minor effect
allele frequency in exposure and outcome traits were excluded
from the analysis. The SNPs used as instrumental variables are
shown in Tables S1 through S3. This study used publicly avail-
able GWAS data, and the original studies were all approved by
the relevant ethical committees.
Statistical Analyses
To ensure that the effect of an SNP on the exposure and
the outcome corresponded to the same allele, both exposure
and outcome data sets were harmonized. We formally tested
Arterioscler Thromb Vasc Biol. 2023;43:e373–e380. DOI: 10.1161/ATVBAHA.123.319297
Mendelian Randomization Study of RC and ASCVD
for heterogeneity of causal effects estimated by each of the
CLINICAL AND POPULATION
genetic variants by the Cochran Q test, which can offer a mea-
sure of potential violation of instrumental variable assumption
STUDIES - AL
and presence of confounding.14 We also tested for horizontal
pleiotropy by MR Egger regression, as formally recommended.12
Outliers were detected and removed using MR pleiotropy resid-
ual sum (and outlier) MR-radial algorithm.19
We calculated the inverse-variance point estimates of the
effect adjusted per unit higher RC by dividing the natural loga-
rithm of the unadjusted (OR; and its SE) by the effect estimate
for each exposure allele, and pooled estimates by dividing the
RC effect size associated with that exposure allele and pooled
estimates measured in reference units (1.0 mmol/L), using the
ratio of effect estimates method.20,21 To provide further sensi-
tivity analysis to account for potential residual horizontal plei-
otropy, we performed MR pleiotropy residual sum and outlier,
which is robust to pleiotropic variants by identifying and remov-
ing them.22 F value statistic was calculated which reflects the
strength of the association of SNP with RC. Values >10 indi-
cate valid genetic instruments.
A mediation analysis was conducted using multivariable MR
and established methods23,24 to derive the total effect (direct
and indirect via LDL-C) of RC on outcome and direct effect
controlling for the LDL-C mediator. Additionally, the role of
apoB as a mediator was also explored. A further sensitivity
analysis was conducted to approximate the effect of calculated
RC on clinical outcomes, by performing a multivariable MR with
genetic instruments for total cholesterol, LDL-C, and HDL-C
(high-density lipoprotein-cholesterol). To obtain the indirect
effect, a 2-step MR using the product of coefficient methods
was performed. Two MR estimates were calculated: (1) the
causal effect of the exposure (RC) on the mediator (LDL-C)
and (2) the causal effect of the mediator (LDL-C) on the out-
come. These 2 estimates were then multiplied together to esti-
mate the indirect effect on outcomes of interest.
P<0.05 was considered significant. Effects on outcomes
are expressed as SD unit increase in RC levels. OR with 95%
CI was calculated and presented for each clinical outcome.
Analyses were conducted using R Project Version 4.0.3 for
statistical computing. The following packages were used for
the analyses: 2-sample MR, MR, MR pleiotropy residual sum
and outlier, RadialMR, and MVMR. The code used for mediation
analysis has been reported elsewhere.24
RESULTS
Effect of Genetic Instruments, SNPs, on RC
Levels
Depending on the variant coverage of the outcome data
set available for analysis, up to 30 SNPs were used for
MR. All SNPs showed a strong association with RC expo-
sure (P<5×10−8). SNPs included in the genetic instru-
ments are listed in Tables S1 through S3. A description
of study cohorts is provided in the Table S4.
Each selected SNP was associated with a highly con-
sistent increased OR for risk of CAD per SD unit increase
in RC (Figure 1). The Cochran Q test did not indicate
heterogeneity, nor did the MR Egger intercept indicate
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 Navarese et al Mendelian Randomization Study of RC and ASCVD
CLINICAL AND POPULATION
STUDIES - AL
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Figure 1. Individual contribution to the risk of coronary artery disease of each selected single-nucleotide polymorphism (SNP).
The effect is per SD increment in remnant cholesterol (RC). F value reflects. The strength of the association of SNP with RC. Values >10
indicate valid genetic instruments. OR indicates odds ratio.

directional pleiotropy, which strengthens the causal infer-
ence of the MR estimate.
The genetic score showed that RC was associated
with significantly higher risk for CAD with an OR per
SD unit increase in RC of 1.51 (95% CI, 1.42–1.60),
P=5.30×10-5 (Figure 2). An SD unit increase in RC was
also associated with significantly higher OR of MI: 1.57
(95% CI, 1.21–2.05), P=9.50×10-4, and stroke: 1.23
(95% CI, 1.12–1.35), P=3.72×10-6 (Figure 2). pleiotropy
residual sum and outlier testing did not detect horizon-
tal pleiotropy for CAD (P=0.74) demonstrating that the
selected variants did not significantly affect disease out-
side of their influence on the exposure in MR.
For MI, 3 potential outliers were identified and
deleted. There was no significant distortion in the
outlier-corrected model in comparison to the causal
estimates before correction (distortion test P=0.47),
showing consistency with the overall model: OR, 1.23
(95% CI, 1.12–1.35). Overall, a genetic association was
found between genetic exposure and risk of CAD (Fig-
ure S1) with no evidence of publication bias (Figure S2).
The analysis of individual RC lipid subfractions showed
that MI risk significantly increased with S-VLDLs: OR,
1.39 (95% CI, 1.07–1.79) and IDL: OR, 1.39 (95% CI,
1.07–1.79), whereas a higher stroke risk was associ-
ated with s-VLDL only: OR, 1.39 (95% CI, 1.07–1.79;

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 Navarese et al Mendelian Randomization Study of RC and ASCVD

CLINICAL AND POPULATION

STUDIES - AL
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Figure 2. Mendelian randomization results of the effect of remnant cholesterol (RC) and its most represented subfractions as
well LDL (low-density lipoprotein) cholesterol (LDL-C) on the risk of coronary artery disease, myocardial infarction, and stroke.
IDL indicates intermediate-density lipoprotein; L, large VLDL; M-VLDL, medium VLDL; OR, odds ratio; S-VLDL, small VLDL; Tot RC, total
cholesterol; and VLDL, very low-density lipoprotein.

Figure 2). There was a directional but no significant

increase in CAD risk with individual lipoprotein subfrac-
tions (Figure 2).
Mediation Analysis
Mediation analysis showed that total effect was driven
nearly entirely by a direct effect of RC: OR was 1.49
(95% CI, 1.37–1.61) for CAD and OR was 1.80 (95%
CI, 1.70–19.1) for MI without any indirect effect for
CVD outcomes via the mediator LDL-C (Figure 3). The
multivariate total and direct effect on stroke were atten-
uated, indicating that part of the increased risk of stroke
is mediated by LDL-C. When apoB was explored as a
mediator, RC remained associated with an increased
risk of CAD and MI but not stroke (Table S5). On sen-
sitivity analysis conducted using multivariate MR, the
effect of calculated RC on clinical outcomes yielded
results highly concordant with the main analyses (Table
S6).
DISCUSSION
In this study, we used data from a combined sample
of 958 434 participants and applied MR analyses of
known SNPs related to ASCVDs as determined by
GWAS. We found strong evidence that RC is causally
associated with increased risk of CAD and MI. The
association of RC with stroke was attenuated in a medi-
ation analysis and may need further investigation. The
analysis of individual lipoprotein subfractions showed
a significant increase in MI with S-VLDL and IDL,
whereas stroke risk was increased only with S-VLDL.
The observed magnitude of risk increase was lower
than that achieved with the combined genetic score,
which allowed us to reach a greater statistical power to
predict clinical risk.
Previous observations from registry databases have
suggested causal associations between RC and isch-
emic heart disease and mortality by using selected
genes as instruments.9,25 However, these reports were

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 Navarese et al
CLINICAL AND POPULATION
STUDIES - AL
Figure 3. Mediation analysis disentangling the multivariate total, direct effect of remnant cholesterol (RC), and the fraction
of indirect effect mediated by LDL (low-density lipoprotein) cholesterol (LDL-C) on the risk of coronary artery disease (CAD),
myocardial infarction (MI), and stroke.
The effect of RC on CAD and MI is largely independent of LDL-C, whereas a direct effect on stroke becomes attenuated when separated from
the total effect. OR indicates odds ratio.
not GWAS studies; thus they were not designed to sys-
tematically investigate candidate SNPs that would allow
the selection of the most robust genetic variants associ-
ated with both RC and outcome within an MR frame-
work. The genes identified in prior analyses were not
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specific to RC and were common to other apoB-carrying
lipoproteins.
It is plausible that the sensitivity of previous studies
might have been influenced by inherent pleiotropy26,27
that can diminish a full appreciation of the independent
association of RC with ASCVDs. The current MR study
expands previous genetic evidence, in that it identi-
fies only the most robust candidate instruments for the
association between RC and ASCVD outcomes among
a large array of SNPs (>12 000 000) to calculate. The
strongest associations between SNPs and trait and out-
comes were retained to limit pleiotropy. Furthermore, the
mediation analysis enabled us to genetically disentangle
the effect of RC from that of LDL-C on ASCVD out-
comes. The influence of RC on CAD and MI was largely
independent of LDL-C levels (direct effect), with only
a negligible effect through the LDL-C mediation (indi-
rect effect). This was not true of stroke, and this aspect
may deserve further investigation. An ischemic stroke is
caused by systemic embolization and cerebrovascular
atherosclerosis, whereas a CAD event is caused almost
exclusively by atherosclerosis. We question whether
the definition of ischemic stroke may have affected the
results of our analyses.
Our findings can be seen as a genetic confirmation
of the recent observations that elevated RC is associ-
ated with worse cardiovascular outcomes.10,11 Currently,
e378   September 2023 Arterioscler Thromb Vasc Biol. 2023;43:e373–e380. DOI: 10.1161/ATVBAHA.123.319297
Mendelian Randomization Study of RC and ASCVD
RC is not included in most guidelines for assessing the
10-year risk of atherosclerotic cardiovascular disease.
The findings of this MR and other observational reports
should prompt a revision of current recommendations to
incorporate RC as an independent risk factor for athero-
sclerotic cardiovascular disease.
Our findings together with other evidence in the field
suggest that RC should be considered an independent
marker of risk in addition to LDL-C and should become
the target of long-term treatment when measured over
expected levels. A lifelong reduction in RC could trans-
late into impactful clinical benefits. Within this framework,
early detection of altered RC metabolism in childhood-
adolescence-youth may prevent development of CVD
and reduce risk of ASCVDs.
Limitations
We used summary data, but future analyses based on
available individual data might be useful to further inform
the field. However, as previously demonstrated, for a
set of genetic markers with small effect size and not in
linkage disequilibrium with each other, regression on a
genetic risk score can be reconstructed from regres-
sions on the individual SNPs without further access
to individual-level data.28 The addressed ancestry was
mixed, thus differences might arise in selected groups.
However, the mixed ancestry allowed the analyses to be
conducted in a broader population. Future analyses using
other GWAS among non-European ancestries might pro-
vide additional useful insights. Importantly, MR findings
were directionally consistent using individual SNPs and
 Navarese et al
a genetic risk score model, suggesting that the overall
effect is robust and justified.
Conclusions
This large MR study showed a robust, genetic causal
association between RC and atherosclerotic cardio-
vascular disease outcomes. The effect of RC on CAD
remained consistent after accounting for the effects of
RC-associated genetic variants on LDL-C. Our findings
suggest that a lifelong inhibition of RC may be desirable.
As a result, we propose that early screening of RC should
be implemented and that RC lowering should become
the target of future drug developments.
ARTICLE INFORMATION
Received March 14, 2023; accepted June 27, 2023.
Affiliations
Interventional Cardiology and Cardiovascular Medicine Research, Department of Cardi-
ology and Internal Medicine, Nicolaus Copernicus University, Bydgoszcz, Poland (E.P.N.,
K.G., J.K.). Division of Cardiology and Department of Medicine (E.P.N., P.R.) and Metabol-
ic and Cardiovascular Diseases Laboratory (D.V., S.P.), University of Alberta, Edmonton,
Canada. SIRIO MEDICINE Research Network, Poland (E.P.N., J.K.). Now with Clinical
and Interventional Cardiology, Sassari University Hospital, Italy (E.P.N.). Cardiology Unit,
Ospedale del Cuore, Fondazione Toscana “G. Monasterio,” Massa, Italy (S.B.).
Sources of Funding
None.
Disclosures
Downloaded from http://ahajournals.org by on August 27, 2023
E.P. Navarese reports research grants from Abbott, Amgen, and lecture fees/
honoraria from Amgen, AstraZeneca, Bayer, Pfizer, and Sanofi-Regeneron, out-
side the submitted work. P. Raggi reports lecture fees/honoraria from Amgen,
Novo Nordisk, and Novartis outside the submitted work. The other authors report
no conflicts.
Supplemental Material
Figure S1–S2
Tables S1–S6
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e380   September 2023 Arterioscler Thromb Vasc Biol. 2023;43:e373–e380. DOI: 10.1161/ATVBAHA.123.319297