Ference BA Et Al. N Eng J Med 2016

The n e w e ng l a n d j o u r na l of m e dic i n e
Original Article
Variation in PCSK9 and HMGCR and Risk

of Cardiovascular Disease and Diabetes
BrianA. Ference, M.D., JenniferG. Robinson, M.D., M.P.H.,
RobertD. Brook, M.D., AlbericoL. Catapano, Ph.D., M.John Chapman, Ph.D.,
DavidR. Neff, D.O., Szilard Voros, M.D., RobertP. Giugliano, M.D.,
George DaveySmith, M.D., D.Sc., Sergio Fazio, M.D., Ph.D.,
and MarcS. Sabatine, M.D., M.P.H.
A BS T R AC T
BACKGROUND
From the Division of Cardiovascular Pharmacologic inhibitors of proprotein convertase subtilisinkexin type 9 (PCSK9)
Medicine, Wayne State University School are being evaluated in clinical trials for the treatment of cardiovascular disease.
of Medicine, Detroit (B.A.F.), the Division
of Cardiovascular Medicine, University of The effect of lowering low-density lipoprotein (LDL) cholesterol levels by inhibiting
Michigan Medical School, Ann Arbor PCSK9 on the risk of cardiovascular events or diabetes is unknown.
(R.D.B.), and Michigan State University,
East Lansing (D.R.N.) all in Michigan; METHODS
the Departments of Epidemiology and
Medicine, College of Public Health, Uni
We used genetic scores consisting of independently inherited variants in the genes
versity of Iowa, Iowa City (J.G.R.); the De encoding PCSK9 and 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGCR;
partment of Pharmacological and Bio the target of statins) as instruments to randomly assign 112,772 participants from
molecular Sciences, University of Milan
and MultiMedica Istituto di Ricovero e
14 studies, with 14,120 cardiovascular events and 10,635 cases of diabetes, to
Cura a Carattere Scientifico, Milan (A.L.C.); groups according to the number of LDL cholesterollowering alleles that they had
INSERM, PitiSalptrire University Hos inherited. We compared the effects of lower LDL cholesterol levels that were medi-
pital, Paris (M.J.C.); the Global Genomics
Group, Richmond, VA (S.V.); the Throm
ated by variants in PCSK9, HMGCR, or both on the risk of cardiovascular events and
bolysis in Myocardial Infarction Study the risk of diabetes.
Group, Division of Cardiovascular Medi
cine, Brigham and Womens Hospital, RESULTS
Harvard Medical School, Boston (R.P.G., Variants in PCSK9 and HMGCR were associated with nearly identical protective effects
M.S.S.); the Medical Research Council
Integrative Epidemiology Unit, Univer on the risk of cardiovascular events per decrease of 10 mg per deciliter (0.26 mmol
sity of Bristol, Bristol, United Kingdom per liter) in the LDL cholesterol level: odds ratio for cardiovascular events, 0.81
(G.D.S.); and the Center for Preventive (95% confidence interval [CI], 0.74 to 0.89) for PCSK9 and 0.81 (95% CI, 0.72 to 0.90)
Cardiology, Knight Cardiovascular Institute,
Oregon Health and Science University, for HMGCR. Variants in these two genes were also associated with very similar
Portland (S.F.). Address reprint requests effects on the risk of diabetes: odds ratio for each 10 mg per deciliter decrease in
to Dr. Ference at the Division of Cardio LDL cholesterol, 1.11 (95% CI, 1.04 to 1.19) for PCSK9 and 1.13 (95% CI, 1.06 to
vascular Medicine, Wayne State University
School of Medicine, UHC, 4H-34, Detroit, 1.20) for HMGCR. The increased risk of diabetes was limited to persons with im-
MI 48202, or at bference@med.wayne.edu. paired fasting glucose levels for both scores and was lower in magnitude than the
N Engl J Med 2016;375:2144-53. protective effect against cardiovascular events. When present together, PCSK9 and
DOI: 10.1056/NEJMoa1604304 HMGCR variants had additive effects on the risk of both cardiovascular events and
Copyright 2016 Massachusetts Medical Society.
diabetes.
CONCLUSIONS
In this study, variants in PCSK9 had approximately the same effect as variants in
HMGCR on the risk of cardiovascular events and diabetes per unit decrease in the
LDL cholesterol level. The effects of these variants were independent and additive.
(Funded by the Medical Research Council and the National Heart, Lung, and Blood
Institute.)
2144 n engl j med 375;22nejm.org December 1, 2016
The New England Journal of Medicine

Downloaded from nejm.org at AMGEN INC. on November 30, 2016. For personal use only. No other uses without permission.
Copyright 2016 Massachusetts Medical Society. All rights reserved.
Variation in PCSK9 and HMGCR and Cardiovascular Risk
M
onoclonal antibodies and other compared the effect of these scores on the risk
therapies that inhibit proprotein con- of cardiovascular disease and the risk of diabe-
vertase subtilisinkexin type 9 (PCSK9) tes to make inferences about the potential clini-
have been shown to reduce low-density lipopro- cal benefit and safety of treatment with a PCSK9
tein (LDL) cholesterol levels by approximately 50 inhibitor as compared with treatment with a
to 60% in several randomized trials.1-8 Whether statin.
lowering LDL cholesterol levels by inhibiting
PCSK9 will reduce the risk of cardiovascular Me thods
events and, like statins, also increase the risk of
new-onset diabetes is unknown.9 Study Population
Exploratory and post hoc analyses of random- The study included 112,772 participants (with
ized trials have suggested that lowering LDL 14,120 cardiovascular events and 10,635 cases of
cholesterol levels by approximately 70 mg per diabetes) from 14 prospective cohort or case
deciliter (1.81 mmol per liter) with a PCSK9 in- control studies who had provided written in-
hibitor may reduce the risk of major cardiovas- formed consent for genetic studies and for whom
cular events by up to 50%.10,11 However, these individual participant-level data were available as
trials included a total of fewer than 120 cardio- part of the Database of Genotypes and Pheno-
vascular events, and data on the risk of new- types program of the National Center for Bio-
onset diabetes are very limited. Four much larger technology Information.18 A description of the
and longer-term cardiovascular outcome trials included studies and the genotyping platforms
are currently ongoing and will provide a much that were used in each study is provided in Table
more robust estimate of the clinical effect of S1 in the Supplementary Appendix.
PCSK9 inhibitors.12-15
In anticipation of the results of those trials, we Genetic Instruments
have used a mendelian randomization approach We constructed genetic scores for PCSK9 and
to test the effect of LDL cholesterollowering HMGCR by combining all variants within 100 kb
variants in PCSK9 on the risk of cardiovascular on either side of each gene that were associated
events and diabetes. The results of a mendelian with LDL cholesterol levels at a genomewide
randomization study can be interpreted as follows: level of significance (P<5.0108) as reported by
if a genetic variant (e.g., in PCSK9) is associated the Global Lipids Genetics Consortium and that
with an exposure of interest (e.g., LDL choles- were in low linkage disequilibrium (r2<0.2) with
terol levels) that is observationally associated all other variants included in the score.19,20 For
with the outcome under study (e.g., coronary heart each variant, we defined the exposure allele as
disease), then the observed association between the allele associated with lower LDL cholesterol
the exposure and the outcome is likely to be levels.19 For each study participant, we calculated
causal if the variant is also associated with the a weighted PCSK9 genetic score and a weighted
outcome. If not, the observed association be- HMGCR score by adding the number of LDL cho-
tween the exposure and the outcome is likely to lesterollowering alleles that the person had inher-
be noncausal. (For more information, see the ited at each variant that was included in either
Methods section in the Supplementary Appen- score, weighted by the effect of each variant on
dix, available with the full text of this article at LDL cholesterol levels measured in milligrams
NEJM.org.) per deciliter.19
Because PCSK9 inhibitors are designed to
recapitulate the phenotype of loss-of-function Study Design
mutations,16,17 we used the presence of LDL cho- We dichotomized each genetic score and used
lesterollowering variants in PCSK9 to estimate this instrument to divide participants into two
the biologic effect of inhibiting PCSK9 on both groups of approximately equal size on the basis
the risk of cardiovascular events and the risk of of whether their genetic score was above the
diabetes. We constructed genetic scores that median or below the median (Fig. S1 in the
mimic the effect of PCSK9 inhibitors and the Supplementary Appendix). Because each variant
effect of statins (which target 3-hydroxy-3-methyl- that was included in either genetic score is inher-
glutarylcoenzyme A reductase [HMGCR]) and ited approximately randomly at the time of con-
n engl j med 375;22nejm.org December 1, 2016 2145

ception21 and is inherited approximately indepen- All analyses were performed separately in each
dently of other variants included in the score of the included studies and then combined across
owing to low linkage disequilibrium, the number studies in a fixed-effects inverse-varianceweighted
of LDL cholesterollowering alleles that a person meta-analysis to produce summary estimates of
inherits in either score should also be random. effect. To minimize the potential for bias with
Therefore, assignment to each group with the use respect to population stratification, separate
of this instrument should be random. To evalu- analyses were performed for each included an-
ate the doseresponse relationship, we divided cestral group before being combined.
participants into four groups on the basis of the In a test of replication, we compared the effect
quartile value of their genetic score. To compare of lower LDL cholesterol levels on the risk of coro-
the separate and combined effect of variants in nary heart disease mediated by the PCSK9 and
PCSK9 and HMGCR, we conducted a 22 factorial HMGCR genetic scores in up to 62,240 case patients
analysis (Fig. S1 in the Supplementary Appendix). and 127,299 controls without such disease who
were enrolled in the Coronary Artery Disease Ge-
Study Outcomes nomewide Replication and Meta-Analysis plus the
The primary cardiovascular outcome for the study Coronary Artery Disease (CARDIoGRAMplusC4D)
was a composite of the first occurrence of myo- consortium studies and in up to 86,196 partici-
cardial infarction or death from coronary heart pants of European descent (with 22,669 cases of
disease. Key secondary cardiovascular outcomes diabetes) who were enrolled in the Diabetes Ge-
were major coronary events (defined as the first netics Replication and Meta-Analysis (DIAGRAM)
occurrence of myocardial infarction, coronary consortium studies (Table S2 in the Supplemen-
revascularization, or death from coronary heart tary Appendix).22-24 Pleiotropy was assessed with
disease) and major vascular events (defined as the use of mendelian randomizationEgger re-
the first occurrence of a major coronary event or gression.25 All analyses were performed with the
stroke). The primary safety outcome was diabetes, use of Stata 12 software or Golden Helix SNP &
defined as either a glycated hemoglobin level Variation Suite software (version 8.1.4).26 A de-
greater than 6.5% or treatment with a glucose- tailed description of the methods is provided in
lowering medication. Key secondary safety out- the Supplementary Appendix.
comes were fasting plasma glucose level, weight,
and body-mass index. To increase the statistical R e sult s
power of the analyses, we combined prevalent
and incident outcome events under the assump- Participant Characteristics
tion that all events occur incident to a genetic The weighted mean age of the study participants
exposure. was 59.9 years. The participants had weighted
mean cholesterol values as follows: LDL choles-
Statistical Analysis terol, 129.9 mg per deciliter (3.36 mmol per liter);
We assessed whether the assignment to each high-density lipoprotein (HDL) cholesterol, 52.3 mg
group was indeed random by comparing the per deciliter (1.35 mmol per liter); and non-HDL
baseline characteristics of the participants in each cholesterol, 155.3 mg per deciliter (4.02 mmol
group. We measured the difference in LDL choles- per liter) (Table S3 in the Supplementary Appen-
terol level between groups using linear regres- dix). Seven variants were included in the PCSK9
sion and compared the risk of cardiovascular genetic score and six variants in the HMGCR ge-
events or diabetes using logistic-regression analy- netic score (Tables S4 through S7 in the Supple-
ses that were adjusted for age and sex. To com- mentary Appendix). There were no significant
pare the effect of different variants or genetic differences in any nonlipid baseline characteris-
scores on the risk of cardiovascular events and tics between the groups being compared, thus
diabetes, we adjusted each effect size estimate showing that assignment to each group was in-
for a standard decrement of 10 mg per deciliter deed random (Table1).
(0.26 mmol per liter) in the LDL cholesterol level
using the usual ratio of effect estimates method Cardiovascular Events
(for details, see the Methods section in the Sup- As expected, participants in the group with higher
plementary Appendix). PCSK9 genetic scores had a lower mean LDL

Table 1. Baseline Characteristics of the Participants, According to PCSK9 Genetic Score.*
Below Median Score Above Median Score

Characteristic (N=57,064) (N=55,708) P Value
Lipids (mg/dl)
LDL cholesterol 132.635.2 128.435.4 5.61016
HDL cholesterol 52.415.6 52.915.8 5.4105
Triglycerides
Median 121.4 116.1 6.81010
Interquartile range 82164 79158
Non-HDL cholesterol 157.637.5 153.138.2 1.81016
Nonlipid characteristics
Age (yr) 61.37.2 61.47.2 0.24
Female sex (%) 58.2 58.1 0.68
Blood pressure (mm Hg)
Systolic 127.717.5 127.817.2 0.43
Diastolic 74.99.9 75.010.3 0.36
Weight (kg) 76.916.7 76.916.2 0.75
Body-mass index 27.55.3 27.75.0 0.17
Ever smoked (%) 54.1 54.3 0.28
* Plusminus values are means SD. The weighted PCSK9 genetic score was calculated for each participant by adding
the number of low-density lipoprotein (LDL) cholesterollowering alleles that the person had inherited at each variant
that was included in the score, weighted by the effect of each variant on LDL cholesterol levels measured in milligrams
per deciliter. Values in the table represent weighted mean values of the baseline characteristics for the entire study
sample (for age and sex) or from the prospective cohort studies (for all other variables) in either group, after combin
ing study-specific estimates in an inverse-varianceweighted meta-analysis. To convert the values for LDL, high-density
lipoprotein (HDL), and non-HDL cholesterol to millimoles per liter, multiply by 0.02586. To convert the values for tri
glycerides to millimoles per liter, multiply by 0.01129.
The body-mass index is the weight in kilograms divided by the square of the height in meters.
cholesterol level than those in the group with stepwise decrease in LDL cholesterol levels and
lower PCSK9 scores (difference, 4.2 mg per a corresponding stepwise decrease in the risk of
deciliter [0.11 mmol per liter]; P=5.61016), as myocardial infarction or death from coronary
well as a lower mean level of non-HDL cholesterol heart disease (Fig.1A). Indeed, when the effects
(difference, 4.5 mg per deciliter [0.12 mmol of each PCSK9 score and the individual PCSK9
per liter]; P=1.81016), a lower median level of variants included in these scores were plotted,
triglycerides (difference, 5.3 mg per deciliter there was a dose-dependent log-linear associa-
[0.06 mmol per liter]; P=6.81010), and a higher tion between PCSK9-mediated lower LDL choles-
mean level of HDL cholesterol (difference, 0.5 mg terol levels and the risk of myocardial infarction
per deciliter [0.01 mmol per liter]; P=5.4105) or death from coronary heart disease (Fig. S3 in
(Table1). Participants in the group with higher the Supplementary Appendix). The effect of the
PCSK9 genetic scores had an 8.4% lower risk of PCSK9 score on the risk of myocardial infarction
myocardial infarction or death from coronary or death from coronary heart disease was similar
heart disease (odds ratio, 0.92; 95% confidence in all subgroups studied (Fig. S4 in the Supple-
interval [CI], 0.88 to 0.95), as well as similarly mentary Appendix).
lower risks of major coronary events, major vascu- In similar analyses using the HMGCR genetic
lar events, myocardial infarction, and death from score, participants in the group with higher
coronary heart disease (Fig. S2 in the Supplemen- HMGCR scores had a mean LDL cholesterol
tary Appendix). In doseresponse analyses, in- level that was lower by 3.2 mg per deciliter
creasing PCSK9 scores were associated with a (0.08 mmol per liter) than participants with lower

A PCSK9 Score
Difference in LDL Cholesterol vs. Odds Ratio for Myocardial Infarction
Score below Median or Reference or Death from CHD (95% CI)
mg/dl
PCSK9 score above median 4.2 0.92 (0.880.95)
Quartile of PCSK9 scores
4 5.8 0.89 (0.840.94)
3 3.9 0.93 (0.880.98)
2 1.8 0.97 (0.911.03)
1 Reference Reference
0.20 0.15 0.10 0.05 0 0.05
Natural Logarithm of Odds Ratio
B HMGCR Score
Score below Median or Reference or Death from CHD (95% CI)
mg/dl
HMGCR score above median 3.2 0.93 (0.900.97)
Quartile of HMGCR scores
4 4.6 0.90 (0.850.95)
3 3.1 0.93 (0.880.98)
2 1.2 0.98 (0.921.04)
0.20 0.15 0.10 0.05 0 0.05
C Effect of PCSK9 and HMGCR Scores on Risk of Myocardial Infarction or Death from CHD per Unit Change
in LDL Cholesterol
Standardized Difference Odds Ratio for Myocardial Infarction or Death from
in LDL Cholesterol CHD (95% CI) per Decrease in LDL Cholesterol of 10 mg/dl
mg/dl
PCSK9 genetic score 10.0 0.81 (0.740.89)
HMGCR genetic score 10.0 0.81 (0.720.90)
0.40 0.30 0.20 0.10 0 0.10
Figure 1. Effect of PCSK9 and HMGCR Genetic Scores on the Risk of Myocardial Infarction or Death from Coronary
Heart Disease.
For each study participant, we calculated a weighted PCSK9 genetic score and a weighted HMGCR score by adding
the number of low-density lipoprotein (LDL) cholesterollowering alleles that the person had inherited at each vari
ant that was included in either score, weighted by the effect of each variant on LDL cholesterol levels measured in
milligrams per deciliter. A total of 10,401 primary cardiovascular events (myocardial infarction or death from coro
nary heart disease [CHD]) were included in the analysis. Of these events, 5508 (1528 prevalent myocardial infarc
tions and 3980 incident myocardial infarctions or deaths from CHD) occurred in the prospective cohort studies and
4893 in the casecontrol studies. Across the included studies, the median weighted PCSK9 score was 12.7 (range,
0 to 24.5), and the median weighted HMGCR score was 16.8 (range, 4.1 to 26.7). Boxes represent point estimates
of effect. Lines represent 95% confidence intervals (CIs).
HMGCR scores (P=2.91015) and a 6.6% lower After adjustment for a standard decrement of
risk of myocardial infarction or death from 10 mg per deciliter in the LDL cholesterol level,
coronary heart disease (odds ratio, 0.93; 95% CI, PCSK9 variants were associated with an 18.9%
0.90 to 0.97) (Fig.1B). As with the PCSK9 score, decrease in the risk of myocardial infarction or
the HMGCR score had a very consistent effect on death from coronary heart disease (odds ratio,
each of the secondary outcomes and a similar 0.81; 95% CI, 0.74 to 0.89) and HMGCR variants
effect in all subgroups studied. were associated with a nearly identical 19.1%

A Myocardial Infarction or Death from CHD

Group Both Scores below Median or Death from CHD (95% CI)
mg/dl
Both scores above median 7.1 0.88 (0.830.93)
0.20 0.15 0.10 0.05 0 0.05
B Diabetes
Difference in LDL Cholesterol vs. Odds Ratio for Diabetes
Group Both Scores below Median (95% CI)
mg/dl
Both scores above median 7.1 1.11 (1.041.19)
0.05 0 0.05 0.10 0.15 0.20 0.25
Figure 2. 22 Factorial Analysis of the Separate and Combined Effects of PCSK9 and HMGCR Genetic Scores
on the Risk of Cardiovascular Events and Diabetes.
Boxes represent point estimates of effect. Lines represent 95% CIs.
decrease in risk (odds ratio, 0.81; 95% CI, 0.72

those in the group with lower PCSK9 scores
to 0.90) (Fig.1C). The effects of the PCSK9 and
(odds ratio, 1.06; 95% CI, 1.02 to 1.11). After
HMGCR scores were very similar for all of the adjustment for a standard decrement of 10 mg
cardiovascular outcomes studied (Fig. S5 in theper deciliter in the LDL cholesterol level, PCSK9
Supplementary Appendix). In the 22 factorial variants were associated with an 11.2% increase
analysis, the PCSK9 and HMGCR genetic scores in the risk of diabetes (odds ratio, 1.11; 95% CI,
had additive effects on LDL cholesterol and the1.04 to 1.19). This effect was very similar to the
corresponding risk of cardiovascular events 12.7% increase in the risk of diabetes per 10 mg
(Fig.2A). per deciliter decrease in the LDL cholesterol level
that was associated with HMGCR variants (odds
In external validation analyses involving up to
ratio, 1.13; 95% CI, 1.06 to 1.20) (Fig.3). Both the
62,240 case patients with coronary heart disease
and 127,299 controls without such disease, the PCSK9 and HMGCR genetic scores appeared to
PCSK9 genetic score (odds ratio, 0.84; 95% CI, have dose-dependent effects on the risk of dia-
0.80 to 0.88) and HMGCR genetic score (odds betes. When present together in the 22 facto-
ratio, 0.84; 95% CI, 0.81 to 0.89) had nearly rial analysis, the PCSK9 and HMGCR variants had
additive effects on the risk of diabetes (Fig.2B).
identical associations with the risk of coronary
heart disease per decrease of 10 mg per deciliter Among persons without prevalent diabetes at
baseline, neither the PCSK9 genetic score nor the
in the LDL cholesterol level (Figs. S6, S7, and S8
in the Supplementary Appendix). Mendelian ran- HMGCR genetic score was significantly associat-
ed with baseline fasting plasma glucose levels
domizationEgger analyses showed that the effect
of both PCSK9 and HMGCR variants on the risk of(difference in plasma glucose level for each 10 mg
cardiovascular disease was due entirely to their
per deciliter decrease in LDL cholesterol level for
LDL cholesterollowering effect, with no evidence
PCSK9 score, 0.26 mg per deciliter [0.01 mmol
for any significant pleiotropic effects (Figs. S9
per liter]; P=0.33; difference for HMGCR score,
and S10 in the Supplementary Appendix). 0.42 mg per deciliter [0.02 mmol per liter];
P=0.10). Among persons with impaired fasting
Risk of Diabetes glucose levels at baseline (100 mg per deciliter
Participants in the group with higher PCSK9 [5.6 mmol per liter]), both the PCSK9 and the
scores had a 6.1% higher risk of diabetes than HMGCR genetic scores were associated with a

A PCSK9 Score
Score below Median or Reference (95% CI)
mg/dl
Quartile of PCSK9 scores
4 5.8 1.07 (1.001.14)
3 3.9 1.06 (1.001.12)
2 1.8 1.02 (0.971.08)
0.05 0 0.05 0.10 0.15
B HMGCR Score
Score below Median or Reference (95% CI)
mg/dl
Quartile of HMGCR scores
4 4.6 1.07 (1.001.14)
3 3.0 1.08 (1.011.15)
2 1.4 1.03 (0.981.09)
0.05 0 0.05 0.10 0.15
C Effect of PCSK9 and HMGCR Scores on Risk of Diabetes per Unit Change in LDL Cholesterol
Standardized Difference Odds Ratio for Diabetes (95% CI)
in LDL Cholesterol per Decrease in LDL Cholesterol of 10 mg/dl
mg/dl
PCSK9 genetic score 10.0 1.11 (1.041.19)
HMGCR genetic score 10.0 1.13 (1.061.20)
0.10 0 0.10 0.20 0.30
Figure 3. DoseResponse Relationship between PCSK9 and HMGCR Scores and Risk of Diabetes.
A total of 10,635 cases of diabetes were included in the analysis. Of these cases, 4340 were prevalent at baseline
and 6295 incident cases occurred during follow-up in the prospective cohort studies. The casecontrol studies pro
vided data on cardiovascular disease but did not provide information on diabetes status and thus were not included
in the analysis of diabetes risk. Boxes represent point estimates of effect. Lines represent 95% CIs.
higher risk of incident diabetes per decrease ofpotential common mechanism by which PCSK9
10 mg per deciliter in the LDL cholesterol leveland HMGCR inhibition may increase the risk of
(odds ratio for PCSK9 score, 1.22; 95% CI, 1.03 to
diabetes, a genetic score consisting of variants
1.45; odds ratio for HMGCR score, 1.19; 95% CI, in the gene encoding the LDL receptor (LDLR) had
1.00 to 1.41). By contrast, among persons with a very similar effect on the risk of diabetes per
normal fasting glucose levels at baseline, neither
unit decrease in the LDL cholesterol level as com-
the PCSK9 nor the HMGCR genetic score was as- pared with the PCSK9 and HMGCR genetic scores
sociated with an increased risk of incident dia-(Fig. S11 in the Supplementary Appendix).
betes (odds ratio for PCSK9 score, 0.99; 95% CI, In a test of replication involving up to 86,196
0.84 to 1.17; odds ratio for HMGCR score, 1.04; participants of European descent (with 22,669
95% CI, 0.89 to 1.22) (Fig.4). cases of diabetes) who were enrolled in the
DIAGRAM consortium studies, the PCSK9 and
Replication and Additional Analyses HMGCR genetic scores had very similar effects
In additional analyses designed to evaluate the on the risk of diabetes per decrease of 10 mg per
role of LDL receptormediated pathways as a deciliter in the LDL cholesterol level (odds ratio

Odds Ratio for Diabetes (95% CI) per Decrease

Glucose Level No. of Incident Cases in LDL Cholesterol of 10 mg/dl
Overall 6295
PCSK9 genetic score 1.11 (1.001.26)
HMGCR genetic score 1.12 (1.001.25)
Impaired fasting glucose: 100 mg/dl 2319
(N=7383)
Normal fasting glucose: <100 mg/dl 1608
(N=23,694)
0.10 0.0 0.10 0.20 0.30 0.40
Figure 4. Effect of PCSK9 and HMGCR Scores on the Risk of Incident Diabetes.
A total of 6295 incident cases of diabetes occurred during follow-up in the prospective cohort studies. After the exclu
sion of participants with prevalent diabetes, baseline fasting plasma glucose levels were available for 31,077 partici
pants. The main analysis included all the participants after the exclusion of 4340 participants with prevalent diabetes;
the subgroup analysis that was stratified according to fasting plasma glucose level included the 31,077 participants
without prevalent diabetes for whom baseline fasting plasma glucose levels were available. Boxes represent point
estimates of effect. Lines represent 95% CIs.
for PCSK9 score, 1.08; 95% CI, 1.02 to 1.15; odds Discussion
ratio for HMGCR score, 1.08; 95% CI, 1.02 to 1.14)
(Figs. S12, S13, and S14 in the Supplementary We found that genetic variants that mimic the
Appendix). In additional analyses involving up to effect of PCSK9 inhibitorshad remarkably simi-
133,100 persons enrolled in the Meta-Analyses of lar effects on the risk of cardiovascular events
Glucose and Insulin-Related Traits Consortium and the risk ofdiabetes as compared with vari-
studies,27 the PCSK9 and HMGCR genetic scores ants that mimic the effect of statins when mea-
had very similar effects on plasma glucose levels sured per unit change in the LDL cholesterol
2 hours after an oral glucose challenge (effect level. Furthermore, we found that when variants
of PCSK9 score per 10 mg per deciliter decrease that mimic the effect of PCSK9 inhibitors and
in LDL cholesterol, 1.55 mg per deciliter; 95% statins were present together, they had indepen-
CI, 0.46 to 2.63 [0.09 mmol per liter; 95% CI, dent and additive effects on the risk of both
0.03 to 0.15]; effect of HMGCR score per 10 mg per cardiovascular events and diabetes.
deciliter decrease in LDL cholesterol, 1.44 mg Our finding that PCSK9 and HMGCR variants
per deciliter; 95% CI, 0.11 to 2.77 [0.08 mmol were associated with approximately the same
per liter; 95% CI, 0.01 to 0.15]). These effects effect on the risk of cardiovascular disease per
were quantitatively similar to the effect of the unit decrease in the LDL cholesterol level sug-
PCSK9 46L loss-of-function allele alone on both gests that treatment with a PCSK9 inhibitor
the risk of diabetes (odds ratio, 1.09; 95% CI, should reduce the risk of cardiovascular events
0.98 to 1.23) and on plasma glucose levels 2 hours by approximately the same amount as treatment
after an oral glucose challenge (effect per 46L with a statin. Therefore, treatment with a PCSK9
allele, 1.51 mg per deciliter; 95% CI, 0.00 to 3.02 inhibitor, used either alone or in combination
[0.08 mmol per liter; 95% CI, 0.00 to 0.17]). with a statin, should reduce the risk of cardio-
Unlike the HMGCR genetic score, the PCSK9 vascular events by approximately 20% per de-
genetic score was not associated with weight, crease of 1.0 mmol per liter (39 mg per deciliter)
body-mass index, waist circumference, or waist- in the LDL cholesterol level.29
to-hip ratio in up to 339,224 participants enrolled Our finding that variants in PCSK9 and
in the Genetic Investigation of Anthropometric HMGCR were associated with very similar effects
Traits consortium studies (Table S8 in the Sup- on the risk of diabetes per unit decrease in the
plementary Appendix).28 LDL cholesterol level implies that, like statins,

PCSK9 inhibitors may also increase the risk of oral glucose challenge) without materially in-
new-onset diabetes. However, the increased risk creasing fasting glucose levels, which may then
of diabetes that was associated with both PCSK9 lead to an increased likelihood of incident diabe-
and HMGCR variants appeared to be confined to tes among persons who have impaired fasting
persons with impaired fasting glucose levels. glucose levels. This conclusion is consistent with
Therefore, as with statins, any potential increased data from the Justification for the Use of Statins
risk of new-onset diabetes during treatment with in Prevention: an Intervention Trial Evaluating
a PCSK9 inhibitor is likely to be confined to Rosuvastatin (JUPITER), in which treatment with
persons with impaired fasting glucose levels. rosuvastatin was associated with an increased
Although variants that mimic the effect of risk of diabetes but not an increase in fasting
PCSK9 inhibitors and statins were associated plasma glucose levels, and virtually all of the
with an increased risk of diabetes, the corre- increased risk of diabetes occurred among per-
sponding proportional reduction in cardiovascu- sons with impaired fasting glucose levels.33
lar risk was much greater than the increased risk Our study has limitations. Lifelong exposure
of diabetes. Therefore, as with statins, the reduc- to decreased levels of LDL cholesterol that are
tion in cardiovascular risk with PCSK9 inhibitors mediated by genetic variants is associated with
should far exceed any potential increased risk of much greater reductions in the risk of cardiovas-
diabetes. Furthermore, we found that both PCSK9 cular disease per unit decrease in the LDL cho-
and HMGCR variants were associated with a de- lesterol level than is short-term pharmacologic
creased risk of cardiovascular events among per- treatment.34 Therefore, the effect of PCSK9 vari-
sons with diabetes and those without diabetes. ants on the risk of cardiovascular events (and
This finding is consistent with the results of probably diabetes) that was estimated is likely to
meta-analyses showing that statins are associated be quantitatively much larger than the effect of
with the same proportional reduction in the risk treatment with a PCSK9 inhibitor observed in the
of cardiovascular events in persons with diabe- ongoing outcome trials as measured according
tes as in those without diabetes.30 Therefore, like to the unit decrease in the LDL cholesterol level.
statins, PCSK9 inhibitors should reduce the risk However, having first established that variants
of cardiovascular events equally well among per- that mimic the effect of PCSK9 inhibitors and
sons with diabetes and those without diabetes. statins have biologically equivalent effects on the
The mechanism by which PCSK9 and HMGCR risk of cardiovascular events and diabetes per unit
variants increase the risk of diabetes is unclear. decrease in the LDL cholesterol level, we believe
However, it is unlikely to be mediated by weight it is reasonable to anticipate that PCSK9 inhibi-
gain because unlike HMGCR variants, PCSK9 vari- tors and statins are likely to have therapeutically
ants are not associated with obesity or its sub- equivalent effects on the risk of cardiovascular
phenotypes, such as weight, body-mass index, or events and diabetes per unit decrease in the LDL
waist circumference. Instead, the mechanism may cholesterol level.35,36 In addition, it is important
involve an LDL receptormediated pathway. We to note that monoclonal antibodies bind extra-
found that each set of gene-specific variants in cellular PCSK9 and therefore may not have the
PCSK9, HMGCR, and LDLR had a very similar effect same biologic effect as PCSK9 variants that lower
as the other sets on the risk of diabetes per unit LDL cholesterol levels.
decrease in the LDL cholesterol level. This find- In conclusion, we found that variants in PCSK9
ing is consistent with the fact that both PCSK9 and HMGCR were associated with approximately
and HMGCR inhibitors ultimately reduce plasma the same effects on the risk of cardiovascular
LDL cholesterol levels by increasing the density events and very similar effects on the risk of
of LDL receptors.31 It is also consistent with the diabetes per unit decrease in the LDL cholesterol
observation that persons with familial hypercho- level. We also found that these effects were in-
lesterolemia appear to have a lower prevalence of dependent and additive.
diabetes than unaffected relatives.32
Supported by a grant (MC_UU_12013/1, to Dr. Davey Smith)
The genetic evidence suggests that PCSK9 and from the Medical Research Council and a grant (R01HL132985,
HMGCR inhibition, possibly acting through an to Dr. Fazio) from the National Heart, Lung, and Blood Institute.
LDL receptormediated pathway, may cause mild- Funders for the various studies that are discussed in this article
are listed in the Supplementary Appendix.
ly impaired glucose tolerance (as suggested by Disclosure forms provided by the authors are available with
higher plasma glucose levels 2 hours after an the full text of this article at NEJM.org.

References
1. Roth EM, McKenney JM, Hanotin C, et al. Effect of alirocumab, a monoclonal 25. Bowden J, Davey Smith G, Burgess S.
Asset G, Stein EA. Atorvastatin with or antibody to PCSK9, on long-term cardio- Mendelian randomization with invalid in-
without an antibody to PCSK9 in primary vascular outcomes following acute coro- struments: effect estimation and bias de-
hypercholesterolemia. N Engl J Med 2012; nary syndromes: rationale and design of tection through Egger regression. Int J
367:1891-900. the ODYSSEY outcomes trial. Am Heart J Epidemiol 2015;44:512-25.
2. Giugliano RP, Desai NR, Kohli P, et al. 2014;168:682-9. 26. SNP & Variation Suite, (version 8.1.4).
Efficacy, safety, and tolerability of a mono- 13. Sabatine MS, Giugliano RP, Keech A, Bozeman, MT:Golden Helix (http://www
clonal antibody to proprotein convertase et al. Rationale and design of the Further .goldenhelix.com/SNP_Variation/i ndex
subtilisin/kexin type 9 in combination cardiovascular OUtcomes Research with .html).
with a statin in patients with hypercholes- PCSK9 Inhibition in subjects with Elevat- 27. Scott RA, Lagou V, Welch RP, et al.
terolaemia (LAPLACE-TIMI 57): a random ed Risk trial. Am Heart J 2016;173:94-101. Large-scale association analyses identify
ised, placebo-controlled, dose-ranging, 14. ClinicalTrials.gov. The evaluation of new loci influencing glycemic traits and
phase 2 study. Lancet 2012;380:2007-17. bococizumab (PF-04950615;RN316) in provide insight into the underlying bio-
3. Koren MJ, Lundqvist P, Bolognese M, reducing the occurrence of major cardio- logical pathways. Nat Genet 2012;44:991-
et al. Anti-PCSK9 monotherapy for hyper- vascular events in high risk subjects 1005.
cholesterolemia: the MENDEL-2 random- (SPIRE-1) (https://clinicaltrials.gov/ct2/ 28. Locke AE, Kahali B, Berndt SI, et al.
ized, controlled phase III clinical trial of show/NCT01975376). Genetic studies of body mass index yield
evolocumab. J Am Coll Cardiol 2014;63: 15. ClinicalTrials.gov. The evaluation of new insights for obesity biology. Nature
2531-40. bococizumab (PF-04950615; RN316) in 2015;518:197-206.
4. Robinson JG, Nedergaard BS, Rogers reducing the occurrence of major cardio- 29. The Cholesterol Treatment Trialists
WJ, et al. Effect of evolocumab or ezetimibe vascular events in high risk subjects (CTT) Collaboration. Efficacy and safety
added to moderate- or high-intensity statin (SPIRE-2) (https://w ww.clinicaltrials.gov/ of more intensive lowering of LDL choles-
therapy on LDL-C lowering in patients ct2/results?term=NCT01975389&Search= terol: a meta-analysis of data from
with hypercholesterolemia: the LAPLACE-2 Search). 170,000 participants in 26 randomised
randomized clinical trial. JAMA 2014;311: 16. Cohen J, Pertsemlidis A, Kotowski IK, trials. Lancet 2010;376:1670-81.
1870-82. Graham R, Garcia CK, Hobbs HH. Low 30. The Cholesterol Treatment Trialists
5. Koren MJ, Giugliano RP, Raal FJ, et al. LDL cholesterol in individuals of African (CTT) Collaboration. Efficacy of choles-
Efficacy and safety of longer-term admin- descent resulting from frequent nonsense terol-lowering therapy in 18,686 people
istration of evolocumab (AMG 145) in pa- mutations in PCSK9. Nat Genet 2005;37: with diabetes in 14 randomised trials of
tients with hypercholesterolemia: 52-week 161-5. statins: a meta-analysis. Lancet 2008;371:
results from the Open-Label Study of Long- 17. Cohen JC, Boerwinkle E, Mosley TH Jr, 117-25.
Term Evaluation Against LDL-C (OSLER) Hobbs HH. Sequence variations in PCSK9, 31. Tavori H, Fan D, Blakemore JL, et al.
randomized trial. Circulation 2014;129: low LDL, and protection against coronary Serum proprotein convertase subtilisin/
234-43. heart disease. N Engl J Med 2006;354: kexin type 9 and cell surface low-density
6. Blom DJ, Hala T, Bolognese M, et al. 1264-72. lipoprotein receptor: evidence for a recip-
A 52-week placebo-controlled trial of evo- 18. Mailman MD, Feolo M, Jin Y, et al. rocal regulation. Circulation 2013; 127:
locumab in hyperlipidemia. N Engl J Med The NCBI dbGaP database of genotypes 2403-13.
2014;370:1809-19. and phenotypes. Nat Genet 2007;39:1181-6. 32. Besseling J, Kastelein JJ, Defesche JC,
7. Kereiakes DJ, Robinson JG, Cannon CP, 19. The Global Lipids Genetics Consor- Hutten BA, Hovingh GK. Association be-
et al. Efficacy and safety of the proprotein tium. Discovery and refinement of loci tween familial hypercholesterolemia and
convertase subtilisin/kexin type 9 inhibitor associated with lipid levels. Nat Genet prevalence of type 2 diabetes mellitus.
alirocumab among high cardiovascular 2013;45:1274-83. JAMA 2015;313:1029-36.
risk patients on maximally tolerated statin 20. Johnson AD, Handsaker RE, Pulit SL, 33. Ridker PM, Pradhan A, MacFadyen JG,
therapy: the ODYSSEY COMBO I study. Nizzari MM, ODonnell CJ, de Bakker PI. Libby P, Glynn RJ. Cardiovascular bene-
Am Heart J 2015;169(6):906-915.e13. SNAP: a Web-based tool for identification fits and diabetes risks of statin therapy in
8. Cannon CP, Cariou B, Blom D, et al. and annotation of proxy SNPs using Hap- primary prevention: an analysis from the
Efficacy and safety of alirocumab in high Map. Bioinformatics 2008;24:2938-9. JUPITER trial. Lancet 2012;380:565-71.
cardiovascular risk patients with inade- 21. Lawlor DA, Harbord RM, Sterne JA, 34. Ference BA, Yoo W, Alesh I, et al. Ef-
quately controlled hypercholesterolaemia Timpson N, Davey Smith G. Mendelian fect of long-term exposure to lower low-
on maximally tolerated doses of statins: randomization: using genes as instru- density lipoprotein cholesterol beginning
the ODYSSEY COMBO II randomized con- ments for making causal inferences in early in life on the risk of coronary heart
trolled trial. Eur Heart J 2015;36:1186-94. epidemiology. Stat Med 2008;27:1133-63. disease: a Mendelian randomization analy-
9. Swerdlow DI, Preiss D, Kuchenbaecker 22. The CARDIoGRAMplusC4D Consor- sis. J Am Coll Cardiol 2012;60:2631-9.
KB, et al. HMG-coenzyme A reductase in- tium. Large-scale association analysis 35. Cannon CP, Blazing MA, Giugliano
hibition, type 2 diabetes, and bodyweight: identifies new risk loci for coronary ar- RP, et al. Ezetimibe added to statin therapy
evidence from genetic analysis and ran- tery disease. Nat Genet 2013;45:25-33. after acute coronary syndromes. N Engl J
domised trials. Lancet 2015;385:351-61. 23. Nikpay M, Goel A, Won HH, et al. A Med 2015;372:2387-97.
10. Sabatine MS, Giugliano RP, Wiviott SD, comprehensive 1,000 Genomes-based 36. Ference BA, Majeed F, Penumetcha R,
et al. Efficacy and safety of evolocumab in genome-wide association meta-analysis of Flack JM, Brook RD. Effect of naturally
reducing lipids and cardiovascular events. coronary artery disease. Nat Genet 2015; random allocation to lower low-density
N Engl J Med 2015;372:1500-9. 47:1121-30. lipoprotein cholesterol on the risk of
11. Robinson JG, Farnier M, Krempf M, 24. Morris AP, Voight BF, Teslovich TM, coronary heart disease mediated by poly-
et al. Efficacy and safety of alirocumab in et al. Large-scale association analysis pro- morphisms in NPC1L1, HMGCR, or both:
reducing lipids and cardiovascular events. vides insights into the genetic architec- a 22 factorial Mendelian randomization
N Engl J Med 2015;372:1489-99. ture and pathophysiology of type 2 diabe- study. J Am Coll Cardiol 2015;65:1552-61.
12. Schwartz GG, Bessac L, Berdan LG, tes. Nat Genet 2012;44:981-90. Copyright 2016 Massachusetts Medical Society.

Supplementary Appendix
This appendix has been provided by the authors to give readers additional information about their work.
Supplement to: Ference BA, Robinson JG, Brook RD, et al. Variation in PCSK9 and HMGCR and risk of cardiovas-
cular disease and diabetes. N Engl J Med 2016;375:2144-53. DOI: 10.1056/NEJMoa1604304
Supplementary Appendix
Table of Contents
Supplemental Methods 3
Data Sources and Acknowledgements 13
Figure S1: Design of the Naturally Randomized Trials 20
Figure S2: Effect of PCSK9 genetic score on the risk of cardiovascular events 23
Figure S3: Log-linear association between lower LDL-C mediated by PCSK9 24
polymorphisms and the risk of coronary death or MI
Figure S4: Effect of PCSK9 genetic score on coronary death and MI in selected subgroups 25
Figure S5: Comparison of the effect of PCSK9 and HMGCR genetic scores on the risk of 26
various cardiovascular outcomes adjusted per 10 mg/dL lower LDL-C
Figure S6: Effect of PCSK9 genetic score on risk of cardiovascular disease in up to 62,240 27
case and 127,299 control subjects enrolled in the CARDIoGRAMplusC4D consortium
studies adjusted per 10 mg/dL lower LDL-C
Figure S7: Effect of HMGCR genetic score on risk of cardiovascular disease in up to 28
62,240 case and 127,299 control subjects enrolled in the CARDIoGRAMplusC4D
consortium studies adjusted per 10 mg/dL lower LDL-C
Figure S8: Comparison of effect of PCSK9 and HMGCR genetic scores on risk of 29
cardiovascular events in up to 62,240 case and 127,299 control subjects enrolled in the
CARDIoGRAMplusC4D consortium studies adjusted per 10 mg/dL lower LDL-C
Figure S9: PCSK9 genetic score MR-Egger Evaluation of Pleiotropic Effects 30
Figure S10: HMGCR genetic score MR-Egger Evaluation of Pleiotropic Effects 31
Figure S11: Comparison of LDLR, PCSK9, HMGCR and NPC1L1 genetic scores on risk of 32
diabetes
Figure S12: Effect of PCSK9 genetic score on risk of diabetes in up to 86,197 subjects 33
enrolled in the DIAGRAM consortium studies
Figure S13: Effect of HMGCR genetic score on risk of diabetes in up to 181,111 subjects 34
enrolled in DIAGRAM consortium and other studies
Figure S14: Comparison of effect of PCSK9 and HMGCR genetic scores on risk of diabetes 35
in up to 181,111 subjects enrolled in DIAGRAM consortium and other studies
Figure S15: Comparison of effect of unweighted PCSK9 and HMGCR scores on the risk 36
1
cardiovascular events and diabetes
Table S1: Included studies and genotyping platforms 37
Table S2: Consortia studies used for external validation 38
Table S3: Baseline characteristics of study sample participants 39
Table S4: PCSK9 polymorphisms included in genetic score and their association with LDL- 40
C in the Global Lipids Genetics Consortium
Table S5: Linkage disequilibrium matrix for polymorphisms included in the PCSK9 genetic 40
score
Table S6: HMGCR polymorphisms included in genetic score and their association with 41
LDL-C in the Global Lipids Genetics Consortium
Table S7: Linkage disequilibrium matrix for polymorphisms included in the HMGCR 41
genetic score
Table S8: Association between PCSK9 and HMGCR genetic scores and cardiometabolic 42
outcomes in consortia data per 10 mg/dl lower LDL-C
Supplemental References 43
Supplement to:
PCSK9 Polymorphisms, HMGCR Polymorphisms, Protection from Cardiovascular Disease, and Risk of
Diabetes
Brian A. Ference, MD, MPhil, MSc, Jennifer G. Robinson, MD, MPH, Robert D. Brook, MD, Alberico L.
Catapano, MSc, M. John Chapman, PhD, David R. Neff, DO, Szilard Voros, MD, Robert P. Giugliano, MD,
SM, George Davey Smith, MD, DSc, Sergio Fazio, MD, PhD, Marc S. Sabatine, MD, MPH
2
Supplemental Methods:
I. Principle of Mendelian randomization

Observational epidemiologic studies measure the association between an exposure and an outcome.
However, these studies are vulnerable to confounding, reverse causation and other forms of bias. As a
result, epidemiologic studies do not provide compelling evidence that an observed association is causal.
Mendelian randomization studies are designed to introduce a randomization scheme into an

observational study specifically to assess whether an observed association between an exposure and an
outcome is likely to be causal. The concept of Mendelian randomization is based on the random
allocation of genetic variants from parents to offspring as described in Mendels law of independent
assortment. These studies use genetic polymorphisms that are associated with an exposure of interest
as an instrument to randomly allocate study participants to higher or lower levels of the exposure under
study.1 Because allocation to genetic variation in levels of the exposure is random, this study design
should be less susceptible to confounding. In addition, because allocation of the polymorphism occurs
at conception this study design should not be vulnerable to reverse causation.
The results of a Mendelian randomization study can be interpreted as follows. If a polymorphism is

associated with an exposure of interest that is observationally associated with the outcome under study;
then the observed association between the exposure and outcome is likely to be causal if the
polymorphism is also associated with the outcome. If not, then the observed association between the
exposure and outcome is likely to be an artefact of confounding, reverse causation or other study bias.
Perhaps the most intuitive way to explain the potential clinical relevance of a Mendelian randomization
study is by way of analogy with a randomized trial. Indeed, Mendelian randomization studies have been
called natures randomized trials.2,3
There are many polymorphisms that are associated with plasma LDL-C cholesterol levels, including
polymorphisms in the PCSK9 gene.4 For example, the C allele of the rs11206510 polymorphism in the
PCSK9 gene is associated with lower LDL-C. This polymorphism, like all other polymorphisms, is
inherited approximately randomly at the time of conception in the process sometimes referred to as
Mendelian randomization. Therefore, inheriting an LDL-C lowering C allele at this polymorphism in the
3
PCSK9 gene is analogous to being randomly allocated to a PCSK9 inhibitor while inheriting the other
allele is analogous to being randomly allocated to usual care. If this polymorphism is associated only
with LDL-C, and not with any other biomarkers or other pleiotropic effects, then the only difference
between persons with and without one or more C alleles at this PCSK9 polymorphism will be their
plasma LDL-C level. If allocation is indeed random, then measuring the association between this PCSK9
polymorphism and the risk of cardiovascular disease should provide a naturally randomized and
unconfounded estimate of the causal effect of lower LDL-C mediated by polymorphisms in the PCSK9
gene on the risk of cardiovascular disease in a manner analogous to a long-term randomized trial.
As in a randomized trial, one can evaluate the success of the Mendelian randomization scheme by
comparing a table of baseline characteristics among persons with and without the LDL-C lowering allele.
If the baseline characteristics are well balanced, then allocation must have been random and one can
reasonably assume that all known and unknown confounders between the exposure and outcomes of
interest are equally distributed between the groups being compared.
Ironically, Mendelian randomization studies have very little to do with genetics. They are not designed
to identify persons at higher or lower risk of disease based on genotype (or genetic scores). Instead,
Mendelian randomization studies are designed to evaluate the causal association between an exposure
and the risk of disease. The genetic polymorphisms in a Mendelian randomization study are merely
convenient instruments that are used to randomly allocate persons to higher or lower levels of the non-
genetic exposure under study.
It is important to note that Mendelian randomization studies have several limitations. Despite
presumed random allocation of genetic polymorphisms according to Mendels law of independent
assortment, this study design is still vulnerable confounding by population structure, pleiotropy and
linkage disequilibrium. Confounding by population structure can be addressed by performing studies
within ethnically homogeneous study populations. Confounding by pleiotropy can be addressed by
selecting polymorphisms that are only associated with the exposure of interest, but not with other
exposures that are known to be causally associated with the outcome under study. Both confounding
by pleiotropy and confounding by linkage disequilibrium can be assessed by measuring the effect of the
genetic instrument on known potential confounding exposures.
4
Another important limitation of Mendelian randomization studies is the problem of weak instruments.
If a polymorphism does not have a quantitatively large enough effect on the exposure of interest, it will
not be a useful instrument to reliably assess the causal effect of that exposure on an outcome. The
problem of weak instruments can be addressed by combining multiple polymorphisms that are
associated with the exposure of interest into a genetic score to create an instrument that has a
quantitatively much larger effect on the exposure of interest.1
II. Constructing the genetic scores

To create a genetic instrument with the largest possible effect on LDL-C, we constructed genetic scores.
We combined multiple independently inherited polymorphisms in the PCSK9 gene to create a PCSK9
genetic score, and combined multiple independently inherited polymorphisms in the HMGCR gene to
create an HMGCR genetic score. These genetic scores are instruments that reflect the combined effect
of the polymorphisms included in either score, respectively, on circulating LDL-C levels. As a result,
each score has a much larger effect on plasma LDL-C than any individual polymorphism included in the
score.
We selected polymorphisms for inclusion in each genetic score according to the following protocol. First
we identified all polymorphisms within 100Kb of the target gene (PCSK9 or HMGCR). We ranked each
polymorphism by its p value for the association with LDL-C as reported by the Global Lipid Genetics
Consortium (GLGC).4 We then iteratively selected for inclusion (in order of decreasing magnitude of
association with LDL-C ) all polymorphisms that satisfied both of two criteria: 1) a p-value for association
with LDL-C of < 5x10-8; and 2) low linkage disequilibrium with all other polymorphisms included in the
score (defined as r2 < 0.2 for all comparisons with all other SNPs included in the score).5 We iteratively
confirmed that each polymorphism added to a score had an independent effect on LDL-C and
contributed additional information to the score using forward step-wise regression.
We defined the exposure allele for each polymorphism as the allele associated with lower LDL-C as
reported in the GLGC. To calculate the PCSK9 and HMGCR genetic score for each participant, we
multiplied the number of exposure alleles that a person inherited at each polymorphism included in
either score by the effect of that polymorphism on LDL-C measured in mg/dL as estimated in the GLGC.
We then summed these values to create a weighted genetic score for each participant. For sensitivity
analyses, we also constructed unweighted PCSK9 and HMGCR genetic scores for each participant by
5
summing the number of exposure alleles that a person inherited at each polymorphism included in
either score, respectively.
If a polymorphism included in the PCSK9 or HMGCR genetic scores was not included on the genotyping
platform(s) used in a particular study, we selected the closest proxy polymorphism that was genotyped
on at least one of the available genotyping platforms available for that study.
III. Allocation into exposure groups

Because all polymorphisms included in either genetic score are inherited approximately randomly at the
time of conception in a process sometimes referred to as Mendelian randomization,1 and because each
polymorphism is inherited approximately independently of the other polymorphisms included in the
score by virtue of low linkage disequilibrium, the number of exposure alleles that a person inherits in
either score should also be random.
We dichotomized each genetic score as having a value above or below the median score for participants
in the population under study. Because the number of exposure alleles that a person inherits in either
score should be random, dichotomizing the genetic score as above and below the median should
therefore randomly allocate the population under study into two approximately equal sized groups.
We chose to dichotomize the genetic scores in the primary analysis and use the dichotomized score as
an instrument to randomly allocate study participants into two approximately equal sized groups for
two reasons. First, using either genetic score to randomly allocate the population into two
approximately equal sized groups permitted us to directly estimate the separate and combined effect of
polymorphisms that mimic the effect of PCSK9 inhibitors and statins using a 2x2 factorial study design.
Second, randomly allocating study participants into two approximately equal sized groups would give
our study the same structure as a randomized trial. We designed our Mendelian randomization study to
have the same structure as a randomized trial to facilitate clarity of presentation and ease of
interpretation for a clinical audience.
To evaluate dose-response, we allocated participants into four groups based on the quartile value of
their PCSK9 and HMGCR genetic scores, respectively. Because the number of exposure alleles that a
6
person inherits in either score should be random, allocation to each quartile score should also be
random.
To conduct the 2x2 factorial analyses, study participants were first randomly allocated into two groups
based on whether their PCSK9 genetic score was above or below the median value. Subjects in either of
these two groups were then randomly allocated into two further groups based on whether their HMGCR
genetic score was above or below the median value. Because all polymorphisms included in either score
are inherited approximately randomly and approximately independently of each other due to low
linkage disequilibrium; and because PCSK9 and HMGCR polymorphisms are located on different
chromosomes and therefore inherited independently of the other, this process should randomly
allocate the study population into 4 approximately equal-sized groups: the reference group (analogous
to a placebo group), a group with lower LDL-C mediated polymorphisms in the HMGCR gene (analogous
to treatment with a statin), a group with lower LDL-C mediated polymorphisms in the PCSK9 gene
(analogous to treatment with PCSK9 inhibitor), and a group with lower LDL-C mediated by the combined
effect of polymorphisms in the both the HMGCR and PCSK9 genes (analogous to treatment with
combination statin and a PCSK9 inhibitor).6
The success of the naturally random allocation scheme was assessed by comparing baseline
characteristics among persons in each of the groups being compared. Continuous variables were
compared using a t-test, dichotomous (and ordinal) variables were compared using a chi-square test,
and non-normally distributed variables were compared using non-parametric rank tests or empirical
resampling.
IV. Harmonized definition of cardiovascular outcome events

As part of a larger project, we first harmonized the definition of all cardiovascular-related outcome
variables in each of the 14 studies listed in Table S1. We then re-coded individual level data for each
study participant as necessary to satisfy the harmonized variable definitions to the extent possible, as
described below.
In general, and where possible, we only included the outcomes of coronary heart disease death (as
adjudicated by the individual studies); definite myocardial infarction (excluding silent MI, possible
MI, probable MI, ECG-detected prior MI and resuscitated cardiac arrest); coronary
7
revascularization (defined as angioplasty, percutaneous coronary intervention or coronary artery
bypass grafting); stroke (using ischemic stroke only in studies that sub-divided stroke by type
specifically excluding haemorrhagic stroke, embolic stroke or unknown stroke type). We created
new reconciled study outcome variables in each data set using the definitions described above.
The primary cardiovascular outcome for our study was a composite of the first occurrence of coronary
death or MI. We used both prevalent and incident cases of MI in the primary composite to meet the
definition of first occurrence (understanding that all coronary deaths during follow-up were
necessarily incident events) in the cohort studies. Therefore, the primary cardiovascular outcome is a
composite of prevalent MI or the first occurrence of incident MI or coronary death.
The key secondary composite cardiovascular outcomes included a) major coronary events (MCE) defined
as the first occurrence of coronary death, MI or coronary revascularization; b) major vascular events
(MVE) defined as the first occurrence of coronary death, MI, coronary revascularization or stroke; and c)
the first occurrence of coronary death, MI, or stroke.
Therefore, MCE is a composite of prevalent MI or prevalent coronary revascularization; or the first

occurrence of incident MI, incident coronary revascularization or coronary death. (Coronary
revascularization was defined as coronary angioplasty, percutaneous coronary intervention, or
coronary artery bypass graft surgery).
Similarly, MVE is therefore a composite of prevalent MI, prevalent coronary revascularization or

prevalent stroke; or the first occurrence of incident MI, incident coronary revascularization, incident
stroke or coronary death.
Finally, the outcome of coronary death, MI, or stroke is therefore a composite of the first occurrence of
either prevalent MI or prevalent stroke; or the first occurrence of incident MI, incident stroke or
coronary death.
Tertiary cardiovascular outcomes included the individual components of the composite cardiovascular
outcomes: coronary death; MI; stroke; and coronary revascularization.
8
Coronary death included only incident events by definition. Myocardial infarction included prevalent MI
or the first occurrence of an incident MI (recurrent events were not included). Stroke included
prevalent stoke or the first occurrence of an incident stroke (recurrent events were not included).
Coronary revascularization included either prevalent or the first occurrence of an incident coronary
revascularization (recurrent events were not included).
We did not recode the case definition for the case-control studies. In the six (6) Myocardial Infarction
Genetics (MIGEN) Consortium case-control studies, all cases were MI.7 Therefore, these cases were
included in the primary composite outcome.
In the Wellcome Trust Case-Control Consortium (WTCCC) study, case subjects had a history of either
myocardial infarction or coronary revascularization before the age of 66 years, and a family history of
coronary artery disease.8 Of the 1,926 WTCCC case subjects, 1,377 had MI (71.5%) and the remaining
549 had coronary revascularization (202 PCI; 347 CABG) as the case ascertainment event. Because the
vast majority of events in the WTCCC were MI, and to avoid excluding the 1,377 cases of MI, all 1,926
WTCCC CAD cases were included in the composite primary cardiovascular event outcome.
V. Analytic methods
The association between each dichotomized weighted genetic LDL-C score and plasma LDL-C level was
evaluated using linear regression, and the association with the cardiovascular outcomes and diabetes
was evaluated using logistic regression (for combined prevalent and incident outcomes) or proportional
hazards models (for incident events). All analyses were adjusted for age and gender.
All analyses were conducted separately in each of the 14 studies listed in Table S1, and then combined
using a fixed-effects inverse variance-weighted meta-analysis to produce summary estimates of effect.
Within each study population, all analyses were conducted separately among each included ethnic
group to minimize the potential for confounding by population stratification bias before being combined
to produce the overall summary estimate of effect.
In the main analyses, we combined prevalent and incident cardiovascular outcome events and cases of
diabetes. The rational for using both prevalent and incident events in the main analyses is four-fold.
First, we assumed that all events occurred incident to the genetic exposure. Second, as mentioned
9
above, the primary and secondary composite outcome definitions included the first occurrence of a
component event. Therefore, persons with a prevalent event at the time of enrolment into one of the
prospective cohort studies would have satisfied the outcome definition. Third, combining prevalent and
incident events in genetic analyses is a common strategy to maximize the number of events and
therefore to maximize power. Fourth, the primary analysis combined data from both prospective cohort
studies and case-control studies, and therefore excluding prevalent cases from the cohort studies would
be incongruent.
We did not to adjust for the use of lipid-lowering therapy. We chose not to adjust for the use of lipid-
lowering therapy for three reasons. First, recognize that the use of lipid-lowering therapy has the
potential to bias our effect estimates toward the null when measuring the effect of either genetic score
on both LDL-C and the risk of cardiovascular events and diabetes. We were willing to accept this
potential bias toward the null to adopt the most conservative analysis strategy possible. Second, we
wanted to perform the same analyses using the same methods in all 14 of the included studies to avoid
introducing any potential bias. Because we did not have data on lipid-lowering therapy for participants
in the case-control studies, we did not want to analyze the case-control and cohort studies differently by
adjusting for lipid lowering therapy only in the cohort studies. Third, we were willing to accept that our
point estimates of effect may be biased toward the null because estimating the precise magnitude of
these point estimates of effect was not the primary goal of the study. Instead, the primary goal of the
study was to compare the relative magnitude of the point estimates of effect for the PCSK9 and HMGCR
genetic scores on the risk of both cardiovascular events and diabetes in order to make inferences about
the potential relative magnitude of the benefit of treatment with a PCSK9 inhibitor as compared to a
statin.
VI. External Validation Analyses

To provide external validation, we compared the effect of lower LDL-C on the risk of cardiovascular
events mediated by the HMGCR and PCSK9 genetic scores in up to 62,240 case and 127,299 control
subjects enrolled in the CARDIoGRAMplusC4D consortia studies,9,10 and in up to 86,196 Caucasian
subjects (22,669 cases of diabetes) enrolled in the DIAGRAM consortia studies (Supplemental Table
S2).11
10
As has been previously demonstrated, for a set of genetic markers with small effect size and in linkage
equilibrium with each other, regression on a genetic risk score can be reconstructed from regressions on
the individual polymorphisms without further access to individual-level data.12 This is accomplished by
weighting the association between each exposure allele and the risk of the outcome of interest by the
effect size of the exposure allele on the modifiable exposure of interest, and then combining these
weighted effect estimates to produce an overall weighted summary estimate of effect.
To calculate PCSK9 and HMGCR genetic scores on the risk of CHD using the available summary data, we
looked-up the association between each polymorphism included in either genetic score, respectively,
and the risk of coronary heart disease (CHD) as reported by the CARDIoGRAMplusC4D consortium
(www.CARDIOGRAMPLUSC4D.org).9,10 We adjusted the reported CHD effect size (and the corresponding
standard error) by the effect of that polymorphism on LDL-C (measured in mg/dl) as reported by the
GLGC using the usual ratio of effect estimates method.4 We then combined the adjusted effect
estimates in a fixed-effects inverse variance-weighted meta-analysis to produce PCSK9 and HMGCR
genetic scores that represent a summary estimate of the effect of each unit lower LDL-C on the risk of
coronary events mediated by the combined effect of the polymorphisms included in either genetic score,
respectively. In sensitivity analyses, we also calculated the effect of lower LDL-c mediated by the
combined effect of the polymorphisms included in either genetic score, respectively, using linear
regression analysis forced to pass through the origin (by leaving out the constant term in the regression
equation). These two methods provide numerically and computationally equivalent results. We also
performed the same linear regression analyses that retained the constant term in the regression
equation. In these MR-Egger analyses, the constant term represents an estimate of the total pleiotropic
effect of the genetic score on the risk of CHD not mediated by LDL-C.13
To evaluate the effect of the PCSK9 and HMGCR genetic scores on the risk of diabetes using the available
summary data, we looked-up the association between each polymorphism included in either genetic
score, respectively, and the risk of diabetes as reported by the DIAbetes Genetics Replication And Meta-
analysis (DIAGRAM) consortium (www.diagram-consortium.org).11 We then repeated the procedure
described above using diabetes as the outcome definition.
11
VII. Calculating Standardized Effect Estimates
To directly compare the effect of each genetic score or individual polymorphism on various outcomes
measured per unit change in LDL-C, we adjusted the effect estimate for a standard decrement of 10
mg/dL in LDL-C using the usual ratio of effect estimates method. The same standardized effect estimate
methods were applied using the individual participant data in the primary analyses, and using the
summary-level data in external validation analyses.
Specifically, for dichotomous outcome variables, we multiplied the natural logarithm of the odds ratio
(and the corresponding standard error) for the association with coronary events or diabetes,
respectively, by the effect the effect of that genetic score (or polymorphism) on LDL-C measured in units
of 10 mg/dL. We then exponentiated this value to produce the adjusted odds ratio (and 95% confidence
interval) for the association between that genetic score (or polymorphism) and the risk of cardiovascular
events or diabetes per 10 mg/dL lower LDL-C.
For continuous outcome variables (e.g. fasting glucose), we multiplied the effect estimate in measured
units (and the corresponding standard error) by the effect the effect of the genetic score (or
polymorphism) on LDL-C measured in units of 10 mg/dL, to produce an adjusted estimate of the effect
of the genetic score (or polymorphism) and the continuous outcome variable per 10 mg/dL lower LDL-C.
In sensitivity analyses, the standardized effect estimates were also calculated using linear regression
analysis forced to pass through the origin (by leaving out the constant term in the regression equation)
and coding the effect of each genetic score (or polymorphism) on LDL-C in units of 10 mg/dl (rather than
mg/dL). These two methods provide numerically and computationally equivalent results.
12
Data Sources and Acknowledgements
ARIC Atherosclerosis Risk in Communities Study (ARIC)14
DbGaP dataset reference: The datasets used for the analyses described in this manuscript were obtained
from dbGaP at http://www.ncbi.nlm.nih.gov/sites/entrez?db=gap through dbGaP Study Accession:
phs000280.v2.p1
The research reported in this article was supported by contract numbers HHSN268201100006C,
HHSN268201100007C, HHSN268201100008C, HHSN268201100009C, HHSN268201100010C,
HHSN268201100011C, and HHSN268201100012C; all from the National Heart, Lung, and Blood Institute;
National Institutes of Health; Bethesda, MD, USA. A full list of principal ARIC investigators and
institutions can be found at https://www2.cscc.unc.edu/aric/. This manuscript was not prepared in
collaboration with ARIC investigators and does not necessarily reflect the opinions or views of ARIC, or
the NHLBI.
Candidate Gene Association Resource (CARe) - Support for the genotyping through the CARe Study was
provided by NHLBI Contract N01-HC-65226.
GENEVA (Gene-Environment Association Studies) - Support for the genotyping through the GENEVA
Study was provided by the NIH GEI U01HG004438, U01HG04424, and HHSN268200782096C
Cardiovascular Health Study (CHS) 15
phs000287.v4.p1
The research reported in this article was supported by contract numbers N01-HC-
85079, N01-HC-85080, N01-HC-85081, N01-HC-85082, N01-HC-85083, N01-HC-
85084, N01-HC-85085, N01-HC-85086, N01-HC-35129, N01 HC-15103, N01 HC-
13
55222, N01-HC-75150, N01-HC-45133, N01-HC-85239 and HHSN268201200036C; grant numbers U01
HL080295 from the National Heart, Lung, and Blood Institute and R01 AG-023629 from the National
Institute on Aging, with additional contribution from the National Institute of Neurological Disorders and
Stroke. A full list of principal CHS investigators and institutions can be found at http://www.chs-
nhlbi.org/pi.htm. This manuscript was not prepared in collaboration with CHS investigators and does not
necessarily reflect the opinions or views of CHS, or the NHLBI.
CHS Candidate gene Association Resource (CARe): Support for the genotyping through the CARe Study
was provided by NHLBI Contract N01-HC-65226.
Support for the Cardiovascular Health Study Whole Genome Study was provided by NHLBI grant
HL087652. Additional support for infrastructure was provided by HL105756 and additional genotyping
among the African-American cohort was supported in part by HL085251.
DNA handling and genotyping at Cedars-Sinai Medical Center was supported in part by National Center
for Research Resources grant UL1RR033176, now at the National Center for Advancing Translational
Technologies CTSI grant UL1TR000124; in addition to the National Institute of Diabetes and Digestive
and Kidney Diseases grant DK063491 to the Southern California Diabetes Endocrinology Research Center.
The Framingham Heart Study (FHS) 16,17
phs000007.v23.p8
The Framingham Heart Study is conducted and supported by the National Heart, Lung, and Blood
Institute (NHLBI) in collaboration with Boston University (Contract No. N01-HC-25195). This manuscript
was not prepared in collaboration with investigators of the Framingham Heart Study and does not
necessarily reflect the opinions or views of the Framingham Heart Study, Boston University, or NHLBI.
14
FHS SNP Health Association Resource (SHARe): Funding for SHARe Affymetrix genotyping was provided
by NHLBI Contract N02-HL-64278. SHARe Illumina genotyping was provided under an agreement
between Illumina and Boston University.
Candidate gene Association Resource (CARe): Funding for CARe genotyping was provided by NHLBI
Contract N01-HC-65226.
Multi-Ethnic Study of Atherosclerosis (MESA) 18
phs000209.v12.p3
MESA and the MESA SHARe project are conducted and supported by the National Heart, Lung, and
Blood Institute (NHLBI) in collaboration with MESA investigators. Support for MESA is provided by
contracts N01-HC-95159, N01-HC-95160, N01-HC-95161, N01-HC-95162, N01-HC-95163, N01-HC-95164,
N01-HC-95165, N01-HC-95166, N01-HC-95167, N01-HC-95168, N01-HC-95169 and CTSA UL1-RR-024156.
MESA SNP Health Association Resource (SHARe): Funding for SHARe genotyping was provided by NHLBI
Contract N02-HL-64278. Genotyping was performed at Affymetrix (Santa Clara, California, USA) and the
Broad Institute of Harvard and MIT (Boston, Massachusetts, USA) using the Affymetric Genome-Wide
Human SNP Array 6.0.
Candidate gene Association Resource (CARe): The MESA CARe data used for the analyses described in
this manuscript were obtained through dbGaP (accession numbers). Funding for CARe genotyping was
15
Coronary Artery Risk Development in Young Adults (CARDIA) 19
phs000285.v3.p2
The research reported in this article was supported by contract numbers HHSN268201100006C,
HHSN268201100007C, HHSN268201100008C, HHSN268201100009C, HHSN268201100010C,
HHSN268201100011C, and HHSN268201100012C; all from the National Heart, Lung, and Blood Institute;
National Institutes of Health; Bethesda, MD, USA. A full list of principal ARIC investigators and
institutions can be found at http://www.cardia.dopm.uab.edu/. This manuscript was not prepared in
collaboration with CARDIA investigators and does not necessarily reflect the opinions or views of
CARDIA, or the NHLBI.
Candidate Gene Association Resource (CARe) - Support for the genotyping through the CARe Study was
GENEVA (Gene-Environment Association Studies) - Support for the genotyping through the GENEVA
Study was provided by the NIH GEI U01HG004438, U01HG04424, and HHSN268200782096C
Women's Health Initiative (WHI) 20
phs000200.v9.p3
The WHI program is funded by the National Heart, Lung, and Blood Institute, National Institutes of
Health, U.S. Department of Health and Human Services through contracts N01WH22110, 24152, 32100-
2, 32105-6, 32108-9, 32111-13, 32115, 32118-32119, 32122, 42107-26, 42129-32, and 44221. This
manuscript was not prepared in collaboration with investigators of the WHI, has not been reviewed
and/or approved by the Womens Health Initiative (WHI), and does not necessarily reflect the opinions
of the WHI investigators or the NHLBI.
16
PAGE: WHI PAGE is funded through the NHGRI Population Architecture Using Genomics and
Epidemiology (PAGE) network (Grant Number U01 HG004790). Assistance with phenotype
harmonization, SNP selection, data cleaning, meta-analyses, data management and dissemination, and
general study coordination, was provided by the PAGE Coordinating Center (U01HG004801-01).
GARNET: Funding support for WHI GARNET was provided through the NHGRI Genomics and Randomized
Trials Network (GARNET) (Grant Number U01 HG005152). Assistance with phenotype harmonization and
genotype cleaning, as well as with general study coordination, was provided by the GARNET
Coordinating Center (U01 HG005157). Assistance with data cleaning was provided by the National
Center for Biotechnology Information. Funding support for genotyping, which was performed at the
Broad Institute of MIT and Harvard, was provided by the NIH Genes, Environment and Health Initiative
[GEI] (U01 HG004424).
SHARe: Funding for WHI SNP Health Association Resource (SHARe) genotyping was provided by NHLBI
Contract N02-HL-64278.
Myocardial Infarction Genetics Consortium (MIGen) 7
phs000294.v1.p1
Funding Source: R01 HL087676. National Institutes of Health, Bethesda, MD, USA
Wellcome Trust Case Control Consortium (WTCCC) 8
The principal funder of this project was the Wellcome Trust. Case collections were funded by: Arthritis
Research Campaign, BDA Research, British Heart Foundation, British Hypertension Society, Diabetes UK,
Glaxo-Smith Kline Research and Development, Juvenile Diabetes Research Foundation, National
17
Association for Colitis and Crohn's disease, SHERT (The Scottish Hospitals Endowment Research Trust), St
Bartholomew's and The Royal London Charitable Foundation, UK Medical Research Council, UK NHS
R&D and the Wellcome Trust.
Global Lipid Genetic Consortium (GLGC) 4
Data on coronary artery disease / myocardial infarction have been contributed by Global Lipids Genetics
Consortium investigators and have been downloaded from:
www.sph.umich.edu/csg/abecasis/public/lipids2013/
CARDIoGRAMplusC4D Consortium 9,10
External validation study data on coronary artery disease / myocardial infarction have been contributed
by CARDIoGRAMplusC4D investigators and have been downloaded from:
www.CARDIOGRAMPLUSC4D.ORG
DIAbetes Genetics Replication And Meta-analysis (DIAGRAM) Consortium11
External validation study data on diabetes have been contributed by DIAGRAM investigators and have
been downloaded from: diagram-consortium.org/downloads.html
Meta-Analyses of Glucose and Insulin-related traits Consortium (MAGIC) 21-23
External validation data on glycaemic traits have been contributed by MAGIC investigators and have
been downloaded from: www.magicinvestigators.org
The Genetic Investigation of ANthropometric Traits (GIANT) consortium 24-26
External validation data on anthropomorphic traits have been contributed by GIANT investigators and
have been downloaded from:
www.broadinstitute.org/collaboration/giant/index.php/GIANT_consortium_data_files
18
Additional Data Sources:
Data on SNP annotation, proxy search and estimates of pairwise linkage disequilibrium metrics was
obtained from SNAP: SNP Annotation and Proxy Search Version 2.2 5
https://www.broadinstitute.org/mpg/snap/index.php
SNAP development is funded, in part, by the NHLBI CARe (Candidate Gene Resource) grant (N01-HC-
65226) and by NHLBI's Framingham Heart Study (N01-HC-25195).
Data on genotyping platform specific SNP identification was obtained from NCBI dbSNP Human Build
141: http://www.ncbi.nlm.nih.gov/SNP/
19
Figure S1: Design of the Naturally Randomized Trials
A. PCSK9 genetic score
B. HMGCR genetic score
20
C. 2x2 factorial analysis
Legend: A. We dichotomized the PCSK9 genetic score, and used this instrument to randomly allocate
subjects into two approximately equal sized groups based on whether their PCSK9 genetic score was
above or below the median so that our study would have the same structure as a randomized trial. B.
Similarly, we dichotomized the HMGCR genetic score, and used this instrument to randomly allocate
subjects into two approximately equal sized groups based on whether their HMGCR genetic score was
above or below the median so that this part of the study would also have the same structure as a
randomized trial. C. To conduct the 2x2 factorial analysis comparing the separate and combined
effects of PCSK9 and HMGCR genetic scores, we first randomly allocated all subjects into two groups
based on whether their HMGCR genetic score was above or below the median value and then randomly
allocated subjects in these two groups into two further groups based on whether their PCSK9 genetic
21
score was above or below the median value. This process had the effect of randomly allocating all
subjects into one of 4 groups: the reference group (analogous to a placebo group), a group with lower
LDL-C mediated polymorphisms in the PCSK9 gene (analogous to treatment with a PCSK9 inhibitor), a
group with lower LDL-C mediated polymorphisms in the HMGCR gene (analogous to treatment with
statin), and a group with lower LDL-C mediated by the combined effect of polymorphisms in the both
genes (analogous to treatment with combination PCSK9 inhibitor and statin).
22
Figure S2: Effect of PCSK9 genetic score on the risk of cardiovascular events
Legend: Boxes represent point estimates of effect. Lines represent 95% confidence intervals (CI). Please
see text for details.
23
Figure S3: Log-linear association between lower LDL-C mediated by PCSK9 polymorphisms and the risk
of coronary death or MI
Legend: Plot of LDL-C effect size by proportional risk reduction. Boxes represent proportional risk
reduction (1 OR) for each exposure allele or genetic score plotted against the absolute magnitude of
lower LDL-C associated with that allele or score, respectively. Vertical lines represent one SE above and
below point estimate of proportional risk reduction. SNPs and scores are plotted in order of increasing
absolute magnitude of exposure to lower LDL-C. The line (which is forced to pass through the origin)
represents the increase in proportional risk reduction of coronary death or MI per unit exposure to
lower LDL-C (plotted on the log scale).
24
Figure S4: Effect of PCSK9 genetic score on coronary death and MI in selected subgroups
25
Figure S5: Comparison of the effect of PCSK9 and HMGCR genetic scores on the risk of various
cardiovascular outcomes adjusted per 10 mg/dL lower LDL-C
26
Figure S6: Effect of PCSK9 genetic score on risk of cardiovascular disease in up to 62,240 case and
127,299 control subjects enrolled in the CARDIoGRAMplusC4D consortium studies adjusted per 10
mg/dL lower LDL-C
ORCHD (per 10 mg/dl lower LDL-C): 0.843 (0.805- 0.882), p = 1.6x10-14
27
Figure S7: Effect of HMGCR genetic score on risk of cardiovascular disease in up to 62,240 case and
127,299 control subjects enrolled in the CARDIoGRAMplusC4D consortium studies adjusted per 10
mg/dL lower LDL-C
ORCHD (per 10 mg/dl lower LDL-C): 0.844 (0.806-0.885), p = 3.2x10-14
28
Figure S8: Comparison of effect of PCSK9 and HMGCR genetic scores on risk of cardiovascular events in
up to 62,240 case and 127,299 control subjects enrolled in the CARDIoGRAMplusC4D consortium studies
adjusted per 10 mg/dL lower LDL-C
A. Comparison of PCSK9 and HMGCR scores
B. Comparison ofmultiple genetic scores and polymorphisms involving lower LDL-C mediated through
the common final pathway of the LDL recptor
29
Figure S9: PCSK9 genetic score MR-Egger Evaluation of Pleiotropic Effects in up to 62,240 case and
127,299 control subjects enrolled in the CARDIoGRAMplusC4D consortium studies
Legend: The constant term in these regression analyses is a measure of the effect of the PCSK9 genetic
score on the risk of coronary death or MI not related to its effect on the biomarker in the regression
equation; and is therefore an estimate of the total pleiotropic effect of the PCSK9 genetic score not
related to lower LDL-C.
30
Figure S10: HMGCR genetic score MR-Egger Evaluation of Pleiotropic Effects in up to 62,240 case and
127,299 control subjects enrolled in the CARDIoGRAMplusC4D consortium studies
Legend: The constant term in these regression analyses is a measure of the effect of the HMGCR
genetic score on the risk of coronary death or MI not related to its effect on the biomarker in the
regression equation; and is therefore an estimate of the total pleiotropic effect of the HMGCR genetic
score not related to lower LDL-C.
31
Figure S11: Comparison of LDLR, PCSK9, HMGCR and NPC1L1 genetic scores on risk of diabetes
Legend: The LDL receptor (LDLR) genetic score includes 5 polymorphisms (rs6511720, rs8110695,
rs1122608, rs688 and rs7188); and the NPC1L1 genetic score includes 5 polymorphisms (rs217386,
rs2073547, rs7791240, rs10234070 and rs2300414).6 The effect of each score on the risk of diabetes
(combined prevalent or incident cases) was estimated using the individual participant data.
Polymorphisms in the LDLR gene and genetic scores that mimic the effect of PCSK9 inhibitors, statins
(HMGCR) and ezetimibe (NPC1L1), each of which lower LDL-C via a common final pathway involving the
LDLR, appear to have very similar effects on the risk of diabetes per unit lower LDL-C.
32
Figure S12: Effect of PCSK9 genetic score on risk of diabetes in up to 86,197 subjects enrolled in the
DIAGRAM consortium studies
ORDM (per 10 mg/dl lower LDL-C): 1.085(1.023 - 1.150), p = 0.006
33
Figure S13: Effect of HMGCR genetic score on risk of diabetes in up to 181,111 subjects enrolled in
DIAGRAM consortium and other studies
Sample
SNP Size (n) OR (95% CI)
rs12916 151,329 1.09 (1.00, 1.18)
rs17238484 181,111 1.16 (1.03, 1.31)
rs5909 54640 0.90 (0.71, 1.14)
rs2303152 73794 0.86 (0.63, 1.16)
rs10066707 80650 1.06 (0.91, 1.24)
Overall (I-squared = 31.7%, p = 0.210) 1.08 (1.02, 1.14)
.7 .8 .9 1 1.1 1.2 1.3 1.4
ORDM (per 10 mg/dl lower LDL-C): 1.078(1.016 - 1.144), p = 0.013
NB: data for rs12916 and rs17238484 include data from the DIAGRAM consortium supplemented by non-overlapping data from
additional studies as described in the study by Swerdlow, et al (supplementary Table 10).27
34
Figure S14: Comparison of effect of PCSK9 and HMGCR genetic scores on risk of diabetes in up to
181,111 subjects enrolled in DIAGRAM consortium and other studies
35
Figure S15: Comparison of effect of unweighted PCSK9 and HMGCR scores on the risk cardiovascular
events and diabetes
A. Risk of coronary death or MI
B. Risk of Diabetes
Legend: For each study participant, unweighted PCSK9 and HMGCR genetic scores, respectively, were
calculated by summing the number of LDL-C lowering alleles that person inherited at each
polymorphism included in either score (without weighting by each polymorphisms effect on LDL-C)
36
Table S1: Included studies and genotyping platforms
Total Total No. Total No.

No. Primary CV Cases of
Study Subjects Events Diabetes Follow-up Included Genetic Sub-studies Genotyping Platforms
(Incident (Incident (Years)
Cases) Cases)
Atherosclerosis Risk in Communities 15,676 1,590 (1,063) 2,794 (1,311) 16 phs000090 GENEVA_ARIC Affymetrix AFFY_6.0
Study (ARIC) phs000557 ARIC_CARe Illumina CVDSNP55v1_A
Cardiovascular Health Study (CHS) 5,592 1,462 (964) 1318 (709) 14 phs000377 CARe Cardiovascular Health Study Illumina CVDSNP55v1_A
phs000226 STAMPEED: Cardiovascular Health Study Illumina HumanOmni1-Quad_v1-0_B
The Framingham Heart Study (FHS): phs000342 Framingham SHARe Affymetrix HuGeneFocused 50K_Affy
phs000282 Framingham CARe Affymetrix 500K Set
Original Cohort 5,209 416 (416) 464 (460) 54 (Mapping250K_Nsp and
Offspring Cohort 5,124 407 (393) 486 (439) 32 Mapping250K_Sty Arrays)
Illumina CVDSNP55v1_A
Multi-Ethnic Study of Atherosclerosis 8,296 206 (206) 1439 (736) 10 phs000420 MESA SHARe Affymetrix AFFY_6.0
(MESA) phs000283 MESA CARe Illumina CVDSNP55v1_A
Coronary Artery Risk Development in 3,622 21 (21) 99 (99) 15 phs000309 GENEVA_CARDIA Affymetrix AFFY_6.0
Young Adults (CARDIA) phs000613 CARDIA_CARe Illumina CVDSNP55v1_A
Women's Health Initiative (WHI) 53,318 1,406 (917) 4035 (2541) 16 phs000386 WHI SHARe Affymetrix AFFY_6.0
phs000315 WHI GARNET Illumina HumanOmni1-Quad_v1-0_B
phs000227 PAGE WHI Illumina Cardio-
Metabo_Chip_11395247_A
Myocardial Infarction Genetics N/A phs000294 STAMPEED: Myocardial Infarction Affymetrix AFFY_6.0
Consortium (MIGen) Genetics Consortium (MIGen)
ATVB 3,361 1,693 Case-control
FINRISK 339 167 Case-control
HARPS 1,064 505 Case-control
MALMO 185 86 Case-control
MGH-PCOD 464 204 Case-control
REGICOR 629 312 Case-control
Wellcome Trust Case Control 5,002 1926 N/A Case-control 1958 British Birth Cohort controls (1504) Affymetrix 500K (Mapping250K_Nsp
Consortium (WTCCC) UK National Blood Service controls (1500) and Mapping250K_Sty Arrays)
Coronary Artery Disease (CAD) cases (1998)
37
Table S2: Consortia studies used for external validation
Trait Consortium No. Studies No. No. Ethnicities Link to the Data
Inlcuded Participants Cases Included
Lipids Global Lipids Genetics 60 188,577 European http://csg.sph.umich.edu/abecasis/public/lipids2013/

Consortium (GLGC)
Coronary Heart CARDIoGRAMplusC4D 48 194,427 63,746 European http://www.cardiogramplusc4d.org/data-downloads/
Disease Consortium (2013)
Metabochip meta-
analysis
CARDIoGRAMplusC4D 48 184,305 60,801 Mixed http://www.cardiogramplusc4d.org/data-downloads/
Consortium (2015)
1000 Genomes-based
GWAS
Type II Diabetes DIAbetes Genetics 38 94,742 22,669 European http://diagram-consortium.org/downloads.html
Replication And Meta-
analysis (DIAGRAM)
Consortium
Metabolic Traits Meta-Analyses of 56 46,368 European http://www.magicinvestigators.org/downloads
Glucose and Insulin-
related traits
Consortium (MAGIC)
Anthropomorphic Genetic Investigation 125 339,224 European http://www.broadinstitute.org/collaboration/
Traits of ANthropometric giant/index.php/GIANT_consortium_data_files
Traits (GIANT)
consortium
38
Table S3: Baseline characteristics of study sample participants
Baseline Characteristic Mean (SD or IQR)
Sample Size 112,772

No. Included Studies 14
Age (years) 59.9 (6.5)
Women (%) 58.2%
LDL-C (mg/dl) 129.9 (32.0)
HDL-C (mg/dl) 52.3 (15.4)
triglycerides (mg/dl)* 117.0 (85-162)
total cholesterol (mg/dl) 207.8 (36.8)
non-HDL-C (mg/dl) 155.3 (37.6)
Systolic Blood Pressure (mmHg) 127.0 (18.7)
Diastolic Blood pressure (mmHg) 75.2 (10.2)
Weight (lbs) 169.2 (33.1)
Body mass index (kg/m2) 27.7 (5.2)
Prevalent Diabetes (%) 5.7
Prevalent Cardiovascular disease (%) 1.9
Ever smoker (%) 54.3
Legend: Prevalent Diabetes and Prevalent Cardiovascular Disease refer to the percentage of participants with a history of diabetes (Prevalent
Diabetes), or a history of myocardial infarction, stroke or coronary revascularization (Prevalent Cardiovascular Disease) at the time of enrollment
into the included prospective cohort studies. Triglycerides are given as median (inter-quartile range); all other continuous variables are given as
mean ( standard deviation). Dichotomous variables are given as percentages. Values in the table represent weighted mean values of the
baseline characteristics for the entire study sample, after combining study specific estimates in an inverse variance-weighted meta-analysis.
39
Table S4: PCSK9 polymorphisms included in genetic score and their association with LDL-C in the Global Lipids Genetics Consortium
Frequency
Sample size of LDL-C LDL-C effect
SNP position (N) lowering size (mg/dl) SE P
allele
rs11206510 chr1_55268627 172812 0.1544 2.6592 0.005 2.38E-53
rs2479409 chr1_55277238 172970 0.6675 2.0544 0.0041 2.52E-50
rs2149041 chr1_55274725 172903 0.8391 2.0352 0.0049 1.44E-35
rs2479394 chr1_55258652 172953 0.715 1.2352 0.0041 1.58E-19
rs10888897 chr1_55285649 165232 0.3945 1.6224 0.0042 8.43E-31
rs7552841 chr1_55291340 140234 0.6346 1.1776 0.0044 5.40E-15
rs562556 chr1_55296825 99192 0.1939 2.048 0.0066 6.16E-21
Legend: LDL-C effect size, measured in mg/dl, was used to weight each polymorphism in the PCSK9 genetic score
Table S5: Linkage disequilibrium matrix for polymorphisms included in the PCSK9 genetic score
rs11206510 rs2479409 rs2149041 rs2479394 rs10888897 rs7552841 rs562556

rs11206510 0.074 0.033 0.074 0.108 0.046 0.017
rs2479409 0.074 0.135 0.006 0.097 0.004 0.008
rs2149041 0.033 0.135 0.042 0.137 0.042 0.016
rs2479394 0.074 0.006 0.042 0.000 0.039 0.000
rs10888897 0.108 0.097 0.137 0.000 0.013 0.104
rs7552841 0.046 0.004 0.042 0.039 0.013 0.056
rs562556 0.017 0.008 0.016 0.000 0.104 0.056
Legend: Values represent r2 values, a measure of linkage disequilibrium. r2 values range from 0 to 1; with 0 representing complete equilibrium
(no evidence for linkage disequilibrium) and 1 representing complete disequilibrium. Polymorphisms were included in the score if they had an r2
value < 0.2 with all other polymorphisms included in the score (thus representing approximately independent inheritance)
40
Table S6: HMGCR polymorphisms included in genetic score and their association with LDL-C in the Global Lipids Genetics Consortium
Frequency
Sample size of LDL-C LDL-C effect
SNP position (N) lowering size (mg/dl) SE P
allele
rs12916 chr5:74656539 168357 0.5686 2.3456 0.1216 7.79E-78
rs17238484 chr5:74648496 80959 0.7467 2.0064 0.1984 1.35E-21
rs5909 chr5:74656175 89875 0.8984 1.9744 0.2816 4.93E-13
rs2303152 chr5:74641707 160116 0.8799 1.3536 0.2048 1.04E-09
rs10066707 chr5:74560579 89888 0.5831 1.5904 0.1728 2.97E-19
rs2006760 chr5:74562029 89885 0.8140 1.7056 0.2432 1.67E-13
Legend: LDL-C effect size, measured in mg/dl, was used to weight each polymorphism in the HMGCR genetic score
Table S7: Linkage disequilibrium matrix for polymorphisms included in the HMGCR genetic score
rs12916 rs17238484 rs5909 rs2303152 rs10066707 rs2006760

rs12916 0.368 0.239 0.082 0.289 0.176
rs17238484 0.368 0.036 0.222 0.244 0.024
rs5909 0.239 0.036 0.008 0.168 0.272
rs2303152 0.082 0.222 0.008 0.020 0.010
rs10066707 0.289 0.244 0.168 0.020 0.297
rs2006760 0.176 0.024 0.272 0.010 0.297
Legend: rs17238484 was included in the HMGCR score despite an r2 = 0.368 with rs12916 because these two polymorphisms were previously
shown to be associated with an increased risk of diabetes.27 To accommodate the inclusion of rs17238484 into the HMHCR score, the r2
threshold for other SNPs was relaxed to < 0.30 (as opposed to < 0.20 for the PCSK9 score). Use of an alternative HMGCR score not including
rs17238484 and with an r2 threshold < 0.20 for all included polymorphisms returned essentially identical results for all analyses. In forward step-
wise regression models using participant-level data, all polymorphisms included in the HMGCR score (including rs17238484) were confirmed to
have independent effects on LDL-C.
41
Table S8: Association between PCSK9 and HMGCR genetic scores and cardiometabolic outcomes in consortia data per 10 mg/dl lower LDL-C
Sample PCSK9 HMGCR

Consortium Phenotype Size (n) Effect Estimate p Effect Estimate p Units
(95% CI) (95% CI)
GIANT BMI 339,224 -0.002 (-0.018-0.013) 0.774 0.073 (0.056-0.090) 6.27x10-17 INVT
GIANT BMI (males) 152,835 0.002 (-0.018-0.022) 0.830 0.077 (0.054-0.100) 5.38x10-11 INVT
GIANT BMI (females) 171,936 -0.006 (-0.026-0.014) 0.587 0.064 (0.042-0.087) 1.80x10-8 INVT
GIANT Waist Circumference 243,899 0.005 (-0.011-0.021) 0.560 0.070 (0.051-0.089) 1.71x10-13 INVT
GIANT Waist-to-hip 226,541 0.015 (-0.001-0.031) 0.067 0.040 (0.021-0.059) 3.18x10-5 INVT
GIANT Weight 133,010 0.013 (-0.011-0.036) 0.302 0.067 (0.044-0.091) 2.14x10-8 INVT
GIANT Weight (men) 58,297 0.006 (-0.028-0.040) 0.728 0.056 (0.022-0.090) 0.001 INVT
GIANT Weight (women) 67,573 0.019 (-0.015-0.052) 0.269 0.077 (0.045-0.110) 3.01x10-6 INVT
MAGIC Fasting Glucose 133,010 -0.126 (-0.324-0.090) 0.282 0.306 (0.036-0.576) 0.025 mg/dL
MAGIC 2h Glucose 42,854 1.548 (0.468-2.628) 0.005 1.440 (0.108-2.754) 0.033 mg/dL
MAGIC Hemoglobin A1c 46,368 -0.005 (-0.023-0.013) 0.609 0.001 (-0.017-0.019) 0.926 %
MAGIC HOMA-B 46,186 -0.007 (-0.026-0.012) 0.458 0.006 (-0.012-0.023) 0.517 NA
MAGIC HOMA-IR 46,186 -0.001 (-0.024-0.021) 0.895 0.027 (0.006-0.048) 0.013 NA
MAGIC log(Fasting insulin) 108,557 -0.002 (-0.015-0.012) 0.821 0.030 (0.014-0.046) 3.06x10-4 pmol/l
MAGIC log(Proinsulin) 10,701 -0.027 (-0.058-0.004) 0.089 0.040 (0.003-0.078) 0.033 pmol/l
Legend: IVNT is inverse-normal transformed values. Nominally significant (p 0.05) results are highlighted in red.
42
Supplemental References
1. Palmer TM, Lawlor DA, Harbord RM, Sheehan NA, Tobias JH, Timpson NJ, Davey Smith G, Sterne JA.
Using multiple genetic variants as instrumental variables for modifiable risk factors. Stat Methods Med
Res. 2012;21:223-242.
2. Hingorani A, Humphries S. Nature's randomised trials. Lancet 2005;366:1906-8.
3. Thanassoulis G, O'Donnell CJ. Mendelian Randomization: Nature's Randomized Trial in the Post
Genome Era. JAMA 2009;301:2386-8.
4. Global Lipids Genetics Consortium, Willer CJ, Schmidt EM, Sengupta S, et al. Discovery and
refinement of loci associated with lipid levels. Nat Genet. 2013;45:1274-83.
5. Johnson, A. D., Handsaker, R. E., Pulit, S., Nizzari, M. M., O'Donnell, C. J., de Bakker, P. I. W.
SNAP: A web-based tool for identification and annotation of proxy SNPs using HapMap
Bioinformatics 2008 24(24):2938-2939.
6. Ference BA, Majeed F, Penumetcha R, Flack JM, Brook RD. Effect of naturally random allocation to
lower low-density lipoprotein cholesterol on the risk of coronary heart disease mediated by
polymorphisms in NPC1L1, HMGCR, or both: a 2 x 2 factorial Mendelian randomization study. J Am Coll
Cardiol 2015;65:155261.
7. Myocardial Infarction Genetics Consortium. Genome-wide association of early-onset myocardial

infarction with single nucleotide polymorphisms and copy number variants. Nat Genet. 2009 Mar;
41(3):334-41.
8. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls.
Wellcome Trust Case Control Consortium. Nature 447:2007 661-678.
9. CARDIoGRAMplusC4D Consortium, 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.
43
10. CARDIoGRAMplusC4D Consortium. A comprehensive 1000 Genomes-based genome-wide
association meta-analysis of coronary artery disease. Nat Genet 2015;47:1121-30.
11. DIAbetes Genetics Replication And Meta-analysis (DIAGRAM) Consortium. Large-scale association
analysis provides insights into the genetic architecture and pathophysiology of type 2 diabetes. Nat
Genet 2012;44:981-90.
12. International Consortium for Blood Pressure Genome-Wide Association Studies, Ehret GB, Munroe
PB, Rice KM, et al. Genetic variants in novel pathways influence blood pressure and cardiovascular
disease risk. Nature. 2011;478:103-109.
13. Bowden J, Davey Smith G, Burgess S. Mendelian randomization with invalid instruments: effect
estimation and bias detection through Egger regression. Int J Epidemiol. 2015;44:512-25.
14. Sharrett AR. The Atherosclerosis Risk in Communities (ARIC) Study. Introduction and objectives of
the hemostasis component. Ann Epidemiol. 1992 Jul; 2(4):467-9.
15. Fried LP, Borhani NO, Enright P, Furberg CD, Gardin JM, Kronmal RA, Kuller LH, Manolio TA,
Mittelmark MB, Newman A. The Cardiovascular Health Study: design and rationale. Ann Epidemiol.
1991 Feb; 1(3):263-76.
16. Kannel WB, McGee D, Gordon T. A general cardiovascular risk profile: the Framingham Study. Am J
Cardiol. 1976 Jul; 38(1):46-51.
17. Kannel WB, Feinleib M, McNamara PM, Garrison RJ, Castelli WP. An investigation of coronary heart
disease in families. The Framingham offspring study. Am J Epidemiol. 1979 Sep; 110(3):281-90.
18. Bild DE, Bluemke DA, Burke GL, Detrano R, Diez Roux AV, Folsom AR, Greenland P, Jacob DR Jr,
Kronmal R, Liu K, Nelson JC, O'Leary D, Saad MF, Shea S, Szklo M, Tracy RP. Multi-ethnic study of
atherosclerosis: objectives and design. Am J Epidemiol. 2002 Nov 1; 156(9):871-81.
44
19. Friedman GD, Cutter GR, Donahue RP, Hughes GH, Hulley SB, Jacobs DR Jr, Liu K, Savage PJ.
CARDIA: study design, recruitment, and some characteristics of the examined subjects.
J Clin Epidemiol. 1988; 41(11):1105-16.
20. Design of the Women's Health Initiative clinical trial and observational study. The Women's Health
Initiative Study Group. Control Clin Trials. 1998 Feb; 19(1):61-109.
21. Scott RA, Lagou V , Welch RP, et al. Large-scale association analyses identify new loci influencing
glycemic traits and provide insight into the underlying biological pathways. Nature Genet 2012;44:991-
1005.
22. Scott RA, Lagou V , Welch RP, et al. Large-scale association analyses identify new loci influencing
glycemic traits and provide insight into the underlying biological pathways. Nature Genet 2012;44:991-
1005.
23. Saxena R , Hivert MF , Langenberg C , et al. Genetic variation in GIPR influences the glucose and
insulin responses to an oral glucose challenge. Nature Genet 2010;42:142-148.
24. Dupuis J , Langenberg C , Prokopenko I , et al. New genetic loci implicated in fasting glucose
homeostasis and their impact on type 2 diabetes risk. Nature Genet 2010;42:105-16
25. Dupuis J , Langenberg C , Prokopenko I , et al. New genetic loci implicated in fasting glucose
homeostasis and their impact on type 2 diabetes risk. Nature Genet 2010;42:105-16
26. Shungin D, Winkler TW, Croteau-Chonka DC, et al. New genetic loci link adipose and insulin biology
to body fat distribution. Nature 2015;518:187-96.
27. Swerdlow DI, Preiss D, Kuchenbaecker KB, etal. HMG-coenzyme A reductase inhibition, type 2
diabetes, and bodyweight: evidence from genetic analysis and randomised trials. Lancet 2015 Jan
24;385:351-61.
45

Ference BA Et Al. N Eng J Med 2016

Încărcat de

Informații document

Titlu original

Drepturi de autor

Formate disponibile

Partajați acest document

Partajați sau inserați document

Opțiuni de partajare

Vi se pare util acest document?

Este necorespunzător acest conținut?

Drepturi de autor:

Formate disponibile

Ference BA Et Al. N Eng J Med 2016

Încărcat de

Drepturi de autor:

Formate disponibile

The n e w e ng l a n d j o u r na l of m e dic i n e

Variation in PCSK9 and HMGCR and Risk

2144 n engl j med 375;22nejm.org December 1, 2016

The New England Journal of Medicine

n engl j med 375;22nejm.org December 1, 2016 2145

2146 n engl j med 375;22nejm.org December 1, 2016

The New England Journal of Medicine

Table 1. Baseline Characteristics of the Participants, According to PCSK9 Genetic Score.*

Below Median Score Above Median Score

n engl j med 375;22nejm.org December 1, 2016 2147

Natural Logarithm of Odds Ratio

2148 n engl j med 375;22nejm.org December 1, 2016

The New England Journal of Medicine

A Myocardial Infarction or Death from CHD

decrease in risk (odds ratio, 0.81; 95% CI, 0.72

n engl j med 375;22nejm.org December 1, 2016 2149

Natural Logarithm of Odds Ratio

2150 n engl j med 375;22nejm.org December 1, 2016

The New England Journal of Medicine

Odds Ratio for Diabetes (95% CI) per Decrease

n engl j med 375;22nejm.org December 1, 2016 2151

2152 n engl j med 375;22nejm.org December 1, 2016

The New England Journal of Medicine

n engl j med 375;22nejm.org December 1, 2016 2153

I. Principle of Mendelian randomization

Mendelian randomization studies are designed to introduce a randomization scheme into an

The results of a Mendelian randomization study can be interpreted as follows. If a polymorphism is

II. Constructing the genetic scores

III. Allocation into exposure groups

IV. Harmonized definition of cardiovascular outcome events

Therefore, MCE is a composite of prevalent MI or prevalent coronary revascularization; or the first

Similarly, MVE is therefore a composite of prevalent MI, prevalent coronary revascularization or

VI. External Validation Analyses

ARIC Atherosclerosis Risk in Communities Study (ARIC)14

Cardiovascular Health Study (CHS) 15

The Framingham Heart Study (FHS) 16,17

Multi-Ethnic Study of Atherosclerosis (MESA) 18

Women's Health Initiative (WHI) 20

Myocardial Infarction Genetics Consortium (MIGen) 7

Wellcome Trust Case Control Consortium (WTCCC) 8

Global Lipid Genetic Consortium (GLGC) 4

CARDIoGRAMplusC4D Consortium 9,10

DIAbetes Genetics Replication And Meta-analysis (DIAGRAM) Consortium11

Meta-Analyses of Glucose and Insulin-related traits Consortium (MAGIC) 21-23

The Genetic Investigation of ANthropometric Traits (GIANT) consortium 24-26

A. PCSK9 genetic score

B. HMGCR genetic score

genes (analogous to treatment with combination PCSK9 inhibitor and statin).

see text for details.

lower LDL-C (plotted on the log scale).

see text for details.

see text for details.

ORCHD (per 10 mg/dl lower LDL-C): 0.843 (0.805- 0.882), p = 1.6x10-14

ORCHD (per 10 mg/dl lower LDL-C): 0.844 (0.806-0.885), p = 3.2x10-14

A. Comparison of PCSK9 and HMGCR scores

related to lower LDL-C.

score not related to lower LDL-C.

ORDM (per 10 mg/dl lower LDL-C): 1.085(1.023 - 1.150), p = 0.006

SNP Size (n) OR (95% CI)

rs12916 151,329 1.09 (1.00, 1.18)

rs17238484 181,111 1.16 (1.03, 1.31)

rs5909 54640 0.90 (0.71, 1.14)

rs2303152 73794 0.86 (0.63, 1.16)