Genetic invalidation of Lp-PLA2 as a therapeutic target: Large-scale study of five functional Lp-PLA2-lowering alleles

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Article

Reference

Genetic invalidation of Lp-PLA2 as a therapeutic target: Large-scale study of five functional Lp-PLA2-lowering alleles

EPIC-CVD consortium and the CHD Exome+ consortium EHRET, Georg Benedikt (Collab.)

Abstract

Aims Darapladib, a potent inhibitor of lipoprotein-associated phospholipase A2 (Lp-PLA2), has not reduced risk of cardiovascular disease outcomes in recent randomized trials. We aimed to test whether Lp-PLA2 enzyme activity is causally relevant to coronary heart disease. Methods In 72,657 patients with coronary heart disease and 110,218 controls in 23 epidemiological studies, we genotyped five functional variants: four rare loss-of-function mutations (c.109+2T >

C (rs142974898), Arg82His (rs144983904), Val279Phe (rs76863441), Gln287Ter (rs140020965)) and one common modest-impact variant (Val379Ala (rs1051931)) in PLA2G7, the gene encoding Lp-PLA2. We supplemented de-novo genotyping with information on a further 45,823 coronary heart disease patients and 88,680 controls in publicly available databases and other previous studies. We conducted a systematic review of randomized trials to compare effects of darapladib treatment on soluble Lp-PLA2 activity, conventional cardiovascular risk factors, and coronary heart disease risk with corresponding effects of Lp-PLA2-lowering alleles. Results Lp-PLA2 activity was decreased [...]

EPIC-CVD consortium and the CHD Exome+ consortium, EHRET, Georg Benedikt (Collab.).

Genetic invalidation of Lp-PLA2 as a therapeutic target: Large-scale study of five functional Lp-PLA2-lowering alleles. European Journal of Preventive Cardiology , 2017, vol. 24, no. 5, p. 492-504

DOI : 10.1177/2047487316682186 PMID : 27940953

Available at:

http://archive-ouverte.unige.ch/unige:128976

Disclaimer: layout of this document may differ from the published version.

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SUPPLEMENT: Genetic invalidation of Lp-PLA₂ as a therapeutic target: large-scale

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study of five functional Lp-PLA2 lowering alleles

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Systematic review of East Asian studies

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We searched the Medline electronic library on 04/07/2014 by combining the following terms related to

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the PLA2G7 genetic variant (see eFigure 1), without language restriction:

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(Lp-PLA2 OR LpPLA2 OR Lp-PLA(2) OR 1-alkyl-2-acetylglycerophosphocholine esterase OR PAFAH OR

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PAF-AH OR PLA2G7 OR platelet activating factor acetylhydrolase OR lipoprotein associated

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phospholipase A2)

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AND

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(gene OR genes OR polymorph* OR allel* OR phenotyp* OR SNP OR chromosom* OR variant* OR

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mutat* OR locus OR loci OR Genes [Mesh] OR Polymorphism, genetic [Mesh] OR RFLP OR V279F OR

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PLA2G7 or rs16875954)

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Titles, abstracts and full text versions of identified articles were reviewed independently by at least two

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of three investigators (JG, AT, DFF). Studies were included if they reported on, or allowed calculation

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of, the association of the loss of function variant V279F with coronary heart disease (provided

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myocardial infarction was part of the outcome definition), or the risk factors in healthy individuals.

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Additionally, we searched the NIHR GWAS catalogue (https://www.genome.gov/26525384; accessed

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04/07/2014) to identify additional GWAS studies of coronary disease in East Asians. eFigure 1

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describes the selection process, which identified 16 independent studies overall that contributed data.

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Systematic review of randomized controlled darapladib trials

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We searched the Medline electronic library on 23/02/2015 for the term “darapladib” without language

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restriction. Titles, abstracts and full text versions of identified articles were reviewed independently by

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two investigators (JG, DFF). The search strategy identified 75 publications in total. Five of these

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publications reported on the results of five randomized placebo controlled clinical trials of darapladib.

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Three of those trials reported on Lp-PLA2 activity and cardiovascular risk factors, two trials reported on

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coronary heart disease endpoints.

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Proxy variants

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In cases where information on the genetic variants was not available, we identified suitable proxy

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variants for the relevant population using 1000 Genomes, Phase I release data. For the modest impact

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variant rs1051931, which is common in Europeans, we identified suitable proxy variants in Europeans:

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rs7756935 (r2=1.00, D’=1.00) and rs3799277 (r2=0.96, D'=1.00). For the loss of function variant

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rs76863441 (previously rs16874954), common in East Asians, we identified rs1805018 as a suitable

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proxy variant in Japanese populations (r2=0.94, D’=1.0).

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Adjustment for principal components in genetic analyses

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Principal component analysis (PCA) was performed (separately for Europeans and South Asians) in the

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component studies of the CHD Exome+ Consortium to identify ethnic outliers. Following standard

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sample and variant QC, further stringent variant QC (call rate>99% and Hardy-Weinberg p-

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value<1x10^-4) was performed on common variants (minor allele frequency>0.05), followed by

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stepwise pruning until no residual linkage disequilbrium (r²>0.2) was observed. We further excluded

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variants in known coronary disease loci, leaving 19,256 variants within the Europeans and 17,968

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variants within the South Asians for the PCA. PCA was then performed on a standardised genotype

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matrix created using the Singular Value Decomposition (SVD) function implemented in the R package

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'irlba'. 219 Europeans and 10 South Asians who were ancestral outliers, defined as being at least 3

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standard deviations away from the cluster mean, were excluded. The PCA was rerun after excluding

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these samples to obtain principal components that could be used to account for population

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substructure. The first principal component was included in the statistical model for each ancestral

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group.

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ACKNOWLEDGEMENTS

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We thank Dr. Michael Sweeting and Dr. Angela Wood for helpful comments on an earlier version of this

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manuscript, and Dr. Richard Gibbs and Dr. Dawn Waterworth for contributing tabular data.

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The MORGAM consortium: Dr. Salomaa was supported by the Finnish Foundation for Cardiovascular

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Research. This work has been sustained by the MORGAM Project's recent funding: European Union FP

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7 projects ENGAGE (HEALTH-F4-2007-201413), CHANCES (HEALTH-F3-2010-242244) and

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BiomarCaRE (278913). This has supported central coordination, workshops and part of the activities of

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the The MORGAM Data Centre, at THL in Helsinki, Finland. MORGAM Participating Centres are funded

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by regional and national governments, research councils, charities, and other local sources. The PRIME

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Study was supported between 1992 and 1997 by grants from an agreement between the Institut

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National de la Santé et de la Recherche Médicale (Inserm) and the Merck, Sharp and Dohme-Chibret

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Laboratory. The KORA research platform (KORA, Cooperative Research in the Region of Augsburg) was

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initiated and financed by the Helmholtz Zentrum München - German Research Center for

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Environmental Health, which is funded by the German Federal Ministry of Education and Research and

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by the State of Bavaria. Furthermore, KORA research was supported within the Munich Center of

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Health Sciences (MC Health), Ludwig-Maximilians-Universität, as part of LMUinnovativ. MORGAM Italy:

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EPIMED Research Center, Department of Clinical and Experimental Medicine, University of Insubria,

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Varese: M. Ferrario (principal investigator), G. Veronesi, F. Gianfagna. University of Milano-Bicocca,

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Monza, Italy, Giancarlo Cesana, Paolo Brambilla and Stefano Signorini. This study was supported by

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the Health Administration of Regione Lombardia [grant numbers 9783/1986, 41795/1993,

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31737/1997,17155/2004 and 10800/2009].

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The ARIC study: The ARIC study was supported by a sponsored project (RC2HL102419) from the

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National Heart, Lung, and Blood Institute (NHLBI), contracts (HHSN268201100005C,

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HHSN268201100006C, HHSN268201100007C, HHSN268201100008C, HHSN268201100009C,

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HHSN268201100010C, HHSN268201100011C, and HHSN268201100012C) with the NHLBI, and a

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grant (U54 HG003273) from the National Human Genome Research Institute.

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Drs Kato and Takeuchi thank Dr. Eitaro Nakashima, Dr. Tomohiro Katsuya, and Dr. Masato Isono for

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their assistance in collecting the DNA samples and the accompanying clinical information and in

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genotyping the SNPs.

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Drs Kimura, Sasaoka thank Dr. Shigeru Hohda (Department of Molecular Pathogenesis, Medical

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Research Institute, Tokyo Meidcal and Dental University) for his contribution in genotyping.

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CKDGen consortium: Mathias Gorski was supported by the Else-Kröner-Fresenius-Stiftung

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(2012_A147)

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ICBP: Patricia B Munroe: This work forms part of the research programme of the NIHR Cardiovascular

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Biomedical Research Unit at Barts; George B Ehret is supported by the University of Geneva, NHLBI

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(RO1HL086694), The Swiss National Foundation (FN 33CM30-124087), and the Fondation pour

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Recherches Medicales and the GeCor Foundation.

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inhibitor darapladib on human coronary atherosclerotic plaque. Circulation 2008;118(11):1172-1182.

96 4. Mohler ER, III, Ballantyne CM, Davidson MH et al. The effect of darapladib on plasma lipoprotein-associated 97

phospholipase A2 activity and cardiovascular biomarkers in patients with stable coronary heart disease or 98

coronary heart disease risk equivalent: the results of a multicenter, randomized, double-blind, placebo- 99

controlled study. J Am Coll Cardiol 2008;51(17):1632-1641.

100 5. Daida H, Iwase T, Yagi S, Ando H, Nakajima H. Effect of darapladib on plasma lipoprotein-associated 101

phospholipase A2 activity in Japanese dyslipidemic patients, with exploratory analysis of a PLA2G7 gene 102

polymorphism of Val279Phe. Circ J 2013;77(6):1518-1525.

103 6. O'Donoghue ML, Braunwald E, White HD et al. Effect of darapladib on major coronary events after an acute 104

coronary syndrome: the SOLID-TIMI 52 randomized clinical trial. JAMA 2014;312(10):1006-1015.

105 7. White HD, Held C, Stewart R et al. Darapladib for preventing ischemic events in stable coronary heart disease.

106 N Engl J Med 2014;370(18):1702-1711.

107 8. Willer CJ, Schmidt EM, Sengupta S et al. Discovery and refinement of loci associated with lipid levels. Nat 108

Genet 2013;45(11):1274-1283.

109 9. Dehghan A, Dupuis J, Barbalic M et al. Meta-analysis of genome-wide association studies in >80 000 subjects 110

identifies multiple loci for C-reactive protein levels. Circulation 2011;123(7):731-738.

111 10. Dupuis J, Langenberg C, Prokopenko I et al. New genetic loci implicated in fasting glucose homeostasis and 112

their impact on type 2 diabetes risk. Nat Genet 2010;42(2):105-116.

113 11. Pattaro C, Kottgen A, Teumer A et al. Genome-wide association and functional follow-up reveals new loci for 114

kidney function. PLoS Genet 2012;8(3):e1002584.

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Supplementary figures and tables

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eFigure1: Search strategy and study flow diagram for systematic review and meta-analysis of V279F

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(rs76863441) and cardiovascular disease

120

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eFigure 2: Meta-analysis of associations of genetic variants in PLA2G7 or darapladib with Lp-PLA2 activity.

122

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* proxy variant rs7756935 (r²=1.00); MORGAM = MONICA, Risk, Genetics, Archiving, and Monograph; PROMIS = Pakistan Risk of Myocardial Infarction Study; PROSPER = Pravastatin in

124

elderly individuals at risk of vascular disease trial

125

+Estimate of difference in Lp-PLA2 activity per rare V279F allele using a random-effects meta-analysis with inverse-variance weighting is -46.3% (95% CI: -50.5% to -42.0%)

126

Study

participants N

difference (95% CI) Percentage mean

Satoh 1999 Unno 2006 Hou2009 Fan 2010 Jang 2006 Wang 2009 Osoegawa 2004 Ichihara 1998 Yoshida 1998 Paik 2010 Yamada 1998& 2000 Zhang 2007

111 158 403 148 670 89 82 226 270 2914 1379 2018

-47.79 (-100.00, 21.60) -59.52 (-87.99, -31.05) -46.62 (-66.26, -26.98) -43.96 (-53.46, -34.46) -49.77 (-56.84, -42.70) -51.47 (-55.44, -47.50) -46.06 (-49.03, -43.09) -49.41 (-51.36, -47.47) -49.99 (-51.77, -48.22) -31.00 (-32.71, -29.28) -49.44 (-50.69, -48.19) -44.80 (-45.68, -43.93) -45.39 (-45.96, -44.82)

0 -100 -75 -50 -25 0 Overall (I-squared = 97.0%, p<0.0001)

Val379Ala

Four LoFvariants combined Val279Phe (LoF)

Darapladib

Mean difference (%) in Lp-PLA2 activity per allele

Overall (I-squared = 82.8%, p = 0.001) Mohler

Daidasubgroup 1 Daidasubgroup 2 Serruys

480 14 37 323

64.58 (-65.98, -63.18) 66.00 (-67.67, -64.33) 69.90 (-83.65, -56.15) 67.00 (-72.50, -61.50) 59.00 (-62.00, -56.00)

Mean difference (%) in Lp-PLA2 activity per 160mg dose Study

participants N

difference (95% CI) Percentage mean Study

participants N

Study

participants N

Overall (I-squared = 0.0%, p = 0.580) Rotterdam Study*

Cardiovascular Health Study*

Framingham Heart study*

PROSPER MORGAM

1538 3217 6907 1229 944

-2.74 (-3.51, -1.98) -4.30 (-6.65, -1.95) -3.00 (-4.96, -1.04) -2.30 (-3.28, -1.32) -2.86 (-5.79, 0.07) -3.60 (-6.53, -0.67)

0

-6 -4 -2 0 2

Overall (I-squared = 0.0%, p = 0.448) PROSPER

ARIC

1240

8564

-64.35 (-76.44, -52.27) -50.07 (-88.90, -11.23)

-65.88 (-78.60, -53.17)

-75 -50 - 25 0 -100

- - -

-

- - -

-

0

-75 --50 0

-100 -25

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eFigure 3: Meta-analysis of associations of genetic variants in PLA2G7 or darapladib with coronary heart disease risk.

127

128

BRAVE = Bangladesh Risk of Acute Vascular Events Study; C4D = the Coronary Artery Disease Genetics consortium; CARDIoGRAM = the transatlantic Coronary Artery Disease Genome-wide

129

Replication and Meta-analysis consortium; CCHS = Copenhagen City Heart Study; CHS= Cardiovascular Health Study; CGPS = Copenhagen General Population Study; CIHDS = Copenhagen

130

Ischaemic Heart Disease Study; EPIC = European Prospective Investigation into Cancer and Nutrition Study; MORGAM = MONICA, Risk, Genetics, Archiving, and Monograph; PROMIS =

131

Pakistan Risk of Myocardial Infarction Study; PROSPER = Pravastatin in elderly individuals at risk of vascular disease trial; WOSCOPS = West of Scotland Coronary Prevention Study; * proxy

132

variant rs3799277 (r²=0.96 with rs1051931 in Europeans); ** proxy variant rs1805018 (r²=0.94 with rs76863441 in Japanese). CHD estimate from darapladib clinical trials uses the

133

endpoint fatal CHD, MI or urgent revascularisation (primary endpoint for SOLID-TIMI-52, and pre-specified secondary endpoint for STABILITY).

134

Overall (I-squared = 30.0%, p = 0.179) Yamada

Jang (Subset 2) Hou 2009 Li 2011

Jang (Subset 1) Males only Liu 2006 Study

Jang (Subset 1) Females only Takeuchi 2012**

Hohda 2003 N

2517 / 6076 877 / 1217 810 / 914 803 / 482

2809 / 3077 200 / 200

1130 / 1680 806 / 1335 136 / 218 Cases / controls

0.97 (0.91, 1.02) 1.01 (0.93, 1.10) 0.88 (0.73, 1.07) 0.89 (0.66, 1.20) 1.46 (1.02, 2.07)

0.89 (0.79, 0.99) 0.95 (0.67, 1.36) rare allele (95% CI)

1.09 (0.85, 1.39) 0.92 (0.79, 1.08) 1.11 (0.75, 1.63) Odds ratio per

100.00 39.55

8.29 3.40 2.41

24.62 2.42 Weight

4.98 12.30 2.02

%

Lp-PLA2 reducing allele favourable Lp-PLA2 reducing allele unfavourable 1

.6 .8 1 1.2 1.4 1.6

Odds ratio per loss of function allele

Overall (I-squared = 57.8%, p = 0.124) STABILITY

SOLID-TIMI52 1551 / 14277

1813 / 11213

0.95 (0.89, 1.02) 0.90 (0.82, 1.00)

1.00 (0.91, 1.09)

100.00 45.27

54.73

0.95 (0.89, 1.02) 0.90 (0.82, 1.00)

1.00 (0.91, 1.09)

100.00 45.27

54.73

1

.75 .9 1 1.1 1.25

de novo genotyping, consortium level data de novo genotyping, participant level data

Heterogeneity between groups: p = 0.765 Overall (I-squared = 0.0%, p = 0.524) existing summary data

Subtotal (I-squared = 0.0%, p = 0.489) CIHDS/CGPS

PROMIS EPIC CCHS

Subtotal (I-squared = 0.0%, p = 0.754)

CARDIoGRAM Discovery GWAS BioVU

PROSPER

MHI WOSCOPS

Duke EGCUT

HUNT

C4D Discovery GWAS*

GoDARTS BRAVE

PAS

BioMe

de novo genotyping, study level data de novo genotyping, participant level data

MORGAM

8079 / 10367 5863 / 7813 9810 / 10970 2020 / 6087

20315 / 58419 4583 / 16550 641 / 638

3990 / 6585 659 / 687

660 / 515 392 / 777

2351 / 2348

15420 / 15062 1568 / 2772 2971 / 2784

728 / 808 704 / 1729 2153 / 2118

1.00 (0.98, 1.02) 0.99 (0.97, 1.02) 1.03 (0.98, 1.09) 1.03 (0.98, 1.09) 1.01 (0.96, 1.06) 0.97 (0.89, 1.06)

1.00 (0.96, 1.03)

1.00 (0.97, 1.04) 0.97 (0.91, 1.02) 1.09 (0.89, 1.33)

1.01 (0.95, 1.08) 0.80 (0.66, 0.97)

0.91 (0.73, 1.12) 0.96 (0.76, 1.21)

1.02 (0.92, 1.12)

0.98 (0.93, 1.03) 1.06 (0.94, 1.19) 1.00 (0.90, 1.12)

1.04 (0.86, 1.25) 1.01 (0.98, 1.03)

1.06 (0.89, 1.26) 0.93 (0.83, 1.03)

100.00 34.52 9.15 10.50 12.32 3.65

23.57

23.54 8.94 0.72

6.42 0.77

0.64 0.54

3.05

10.99 2.15 2.39

0.85 41.90

0.98 2.42

1.00 (0.98, 1.02) 0.99 (0.97, 1.02) 1.03 (0.98, 1.09) 1.03 (0.98, 1.09) 1.01 (0.96, 1.06) 0.97 (0.89, 1.06)

1.00 (0.96, 1.03)

1.00 (0.97, 1.04) 0.97 (0.91, 1.02) 1.09 (0.89, 1.33)

1.01 (0.95, 1.08) 0.80 (0.66, 0.97)

0.91 (0.73, 1.12) 0.96 (0.76, 1.21)

1.02 (0.92, 1.12)

0.98 (0.93, 1.03) 1.06 (0.94, 1.19) 1.00 (0.90, 1.12)

1.04 (0.86, 1.25) 1.01 (0.98, 1.03)

1.06 (0.89, 1.26) 0.93 (0.83, 1.03)

100.00 34.52 9.15 10.50 12.32 3.65

23.57

23.54 8.94 0.72

6.42 0.77

0.64 0.54

3.05

10.99 2.15 2.39

0.85 41.90

0.98 2.42

1

.65 .75 1 1.25 1.5

Lp-PLA2 reduction favourable Lp-PLA2 reduction unfavourable

Odds ratio per 160mg daily darapladib

Lp-PLA2 reducing allele favourable Lp-PLA2 reducing allele unfavourable

Odds ratio per loss of function allele

Lp-PLA2 reducing allele favourable Lp-PLA2 reducing allele unfavourable

Odds ratio per Lp-PLA₂lowering allele Val379Ala

Four LoF variants combined Val279Phe (LoF)

Darapladib

Study

N

Cases / controls rare allele (95% CI)

Odds ratio per Weight

% Study

N

%

Study

N

%

Heterogeneity between groups: p = 0.995 Overall (I-squared = 0.0%, p = 0.847) Subtotal (I-squared = 0.0%, p = 0.759)

MICAD Exome consortium BRAVE MORGAM WOSCOPS

PROMIS CIHDS_CGPS CCHS

EPIC

Subtotal (I-squared = .%, p = .) 35533 / 64130 2971 / 2784 2153 / 2118 659 / 687

10137 / 11935 8079 / 10367 2020 / 6087

9810 / 10970

0.92 (0.74, 1.16) 0.92 (0.68, 1.25)

0.92 (0.66, 1.30) 1.39 (0.74, 2.60) 0.74 (0.17, 3.23) 2.20 (0.23, 21.25)

0.85 (0.46, 1.57) 0.97 (0.38, 2.47) 0.53 (0.11, 2.62)

0.75 (0.43, 1.29)

0.92 (0.66, 1.30)

100.00 55.31

44.69 12.99 2.34 1.00

13.71 5.91 1.99

17.38

44.69

0.92 (0.74, 1.16) 0.92 (0.68, 1.25)

0.92 (0.66, 1.30) 1.39 (0.74, 2.60) 0.74 (0.17, 3.23) 2.20 (0.23, 21.25)

0.85 (0.46, 1.57) 0.97 (0.38, 2.47) 0.53 (0.11, 2.62)

0.75 (0.43, 1.29)

0.92 (0.66, 1.30)

100.00 55.31

44.69 12.99 2.34 1.00

13.71 5.91 1.99

17.38

44.69

1

.1 .5 .75 1 1.5 2 3

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eFigure 4: Individual and combined estimates for loss of function variants in Europeans and South Asians

135

136

*Estimate based on one carrier of Val279Phe, hence the confidence interval has been omitted. There were no individuals with Lp-PLA2 activity levels and the splice donor loss of function

137

variant c.109+2T>C (rs142974898) measured. With an allele frequency of 0.01% (~8 times less common than the other loss of function variants), this variant was too rare for individual

138

variant testing. The variant contributed, however, to the aggregated test over all four loss of function variants presented in the main manuscript

139

Heterogeneity between groups: p = 0.216 Overall (I-squared = 0.0%, p = 0. 452) Subtotal (I-squared = 0.0%, p = 0.573) ARIC

Val279Phe PROSPER ARIC

Arg82His

ARIC Gln287Ter

Percentage difference (95% CI)

Heterogeneity between groups: p = 0.847 Overall (I-squared = 0.0%, p = 0.851) MHI

Subtotal (I-squared = 0.0%, p = 0.481) EPIC

MHI

German CAD South

BRAVE PROCARDIS BioVU

Subtotal (I-squared = 0.0%, p = 0.880) German CAD North

BHF

HUNT Ottawa CCHS

EPIC WHI European

German CAD North BRAVE South Asian MORGAM

German CAD South German CAD North

Subtotal (I-squared = 0.0%, p = 0.752) BHF

European CIHDS_CGPS

Ottawa PROMIS

PAS EGCUT

PROMIS South Asian WHI European

EPIC

0.98 (0.77, 1.25) 0.99 (0.32, 3.04)

0.91 (0.62, 1.33) 0.46 (0.14, 1.49)

0.80 (0.20, 3.19) 1.07 (0.59, 1.95)

0.70 (0.06, 8.45)

0.97 (0.40, 2.34) 0.96 (0.39, 2.39) 0.67 (0.15, 3.03)

1.37 (0.63, 2.95) 0.38 (0.09, 1.60) 0.80 (0.07, 8.89)

1.02 (0.06, 16.37) 1.25 (0.11, 13.91) 0.79 (0.11, 5.88)

1.05 (0.34, 3.22) 7.29 (1.63, 32.64)

3.11 (0.33, 29.55) 2.02 (0.82, 4.97) 0.94 (0.13, 6.61)

0.71 (0.24, 2.13) 1.27 (0.11, 14.15)

1.07 (0.56, 2.03) 0.49 (0.09, 2.60) 0.82 (0.44, 1.50)

1.09 (0.40, 3.02)

2.06 (0.13, 33.85) 0.66 (0.30, 1.46)

1.77 (0.36, 8.58) Odds ratio (95% CI)

1.94 (0.12, 31.34)

1.19 (0.46, 3.08) 5.13 (0.32, 81.99)

0.68 (0.29, 1.59)

0.98 (0.77, 1.25) 0.99 (0.32, 3.04)

0.91 (0.62, 1.33) 0.46 (0.14, 1.49)

0.80 (0.20, 3.19) 1.07 (0.59, 1.95)

0.70 (0.06, 8.45)

0.97 (0.40, 2.34) 0.96 (0.39, 2.39) 0.67 (0.15, 3.03)

1.37 (0.63, 2.95) 0.38 (0.09, 1.60) 0.80 (0.07, 8.89)

1.02 (0.06, 16.37) 1.25 (0.11, 13.91) 0.79 (0.11, 5.88)

1.05 (0.34, 3.22) 7.29 (1.63, 32.64)

3.11 (0.33, 29.55) 2.02 (0.82, 4.97) 0.94 (0.13, 6.61)

0.71 (0.24, 2.13) 1.27 (0.11, 14.15)

1.07 (0.56, 2.03) 0.49 (0.09, 2.60) 0.82 (0.44, 1.50)

1.09 (0.40, 3.02)

2.06 (0.13, 33.85) 0.66 (0.30, 1.46)

1.77 (0.36, 8.58) 1.94 (0.12, 31.34)

1.19 (0.46, 3.08) 5.13 (0.32, 81.99)

0.68 (0.29, 1.59)

Arg82His

Gln287Ter

Val279Phe

Odds ratio for CHD per loss of function allele Lp-PLA2 activity (%) per loss of function allele

1 .25 .5 .75 1 1.5 2 3 4 -59.59 (-70.24, -48.94)

-50.07 (-88.90, -11.23) -68.45 (-86.05, -50.84)

-69.46*

-54.48 (-67.85, -41.11) -55.07 (-69.32, -40.83) -50.07 (-88.90, -11.23) -68.45 (-86.05, -50.84)

-54.48 (-67.85, -41.11) -55.07 (-69.32, -40.83)

0

-100 -50 0

(10)

eTable 1: Loss of function variants in PLA2G7 and annotation

140

Protein change or context*

Rsid (if available) Observed in 1KG or ESP†

Primary source Reference for in vivo or in vitro evidence Selected for present study

69 D>G rs201315851 Neither Re-sequencing study Song Pharmacogenomics 2011 No

82 R>H rs144983904 1KG, ESP Re-sequencing study Song Pharmacogenomics 2011 Yes

134 W>STOP rs200454121 Neither Re-sequencing study Song Pharmacogenomics 2011 No

181 D>G NA Neither Re-sequencing study Song Pharmacogenomics 2011 No

273 S>A NA Neither UniProt Tjoelker LW, JBC 1995 No

273 S>F NA Neither Re-sequencing study Song Pharmacogenomics 2011 No

279 V>F rs76863441 / rs16874954 1KG Uniprot Several Yes

281 Q>R rs201256712 Neither Uniprot Yamada Y, Biochem Biophys Res Commun 1997 No

283 L>P rs200303358 Neither Re-sequencing study Song Pharmacogenomics 2011 No

286 D>A NA Neither UniProt Tjoelker LW, JBC 1995 No

286 D>N NA Neither Uniprot Tjoelker LW, JBC 1995 No

287 Q>STOP rs140020965 1KG, ESP ExAC consortium Yes

296 D>A NA Neither Uniprot Tjoelker LW, JBC 1995 No

296 D>N NA Neither Uniprot Tjoelker LW, JBC 1995 No

351 H>A NA Neither Uniprot Tjoelker LW, JBC 1995 No

Splice donor variant

rs142974898 1KG ExAC consortium Yes

* refers to the canonical transcript ENST00000274793 †(1KG = observed in 1000 Genomes project; ESP = observed in Exome sequencing project). Only loss of function variants reported in

141

either 1KG or ESP were included in this study (entries shaded in light blue).

142

(11)

eTable 2: Summary of Caucasian & South Asian studies/consortia

143

Endpoint / phenotype

Type of data Study / consortium

Endpoint definition N

cases

N controls

Reference (PMID) Coronary

disease

De-novo genotyping

BRAVE Confirmed MI meeting all of the following criteria:

i) presented within 24 hours of the onset of sustained clinical symptoms suggestive of MI lasting longer than 20 minutes, including chest pain and breathlessness; ii) had ECG changes indicative of MI (new pathologic Q waves, at least 1 mm ST elevation in any 2 or more contiguous limb leads or a new left bundle branch block, or new persistent ST-T wave changes diagnostic of a non-Q wave MI) with a subsequent confirmation by troponin-I measurements; and iii) had no previous cardiovascular diseases; defined as self- reported history of angina, MI, coronary revascularisation, transient ischaemic attack, stroke or evidence of CHD on prior ECG or in other medical records.

2971 2784 25930055

CCHS MI or major coronary event defined according to IHD (World Health Organization International Classification of Diseases-Eighth Revision, codes 410 to 414; International Classification of Diseases- Tenth Revision, codes I20 to I25) ascertained by reviewing all hospital admissions and diagnoses entered in the national Danish Patient Registry and all causes of death entered in the national Danish Causes of Death Registry.

2020 6087 19509380

CIHDS/CGPS WHO-ICD 8 codes 410 to 414 or ICD 10 codes I20 to I25. Diagnosis of acute coronary syndrome and stenosis or atherosclerosis on coronary

angiography and/or positive results on exercise electrocardiography.

8079 10,367 23265341;

17635890

EPIC Incident CHD cases defined as fatal and non-fatal MI and other major acute coronary events, according to ICD-10 codes I20-I25. All centres have recorded cause-specific mortality through mortality registries and/or active follow-up, and have ascertained and validated incident fatal and non-fatal CHD through a combination of methods (eg, morbidity registers, general practice records, MONICA registries, self-report, clinical records).

9810 10,970 17295097

MORGAM Incident definite or possible MI or coronary death, or unstable angina during follow‐up, coronary revascularization during follow‐up, documented MI at baseline, or an unclassifiable coronary death during follow‐up

2153 2118 15561751

PROMIS Confirmed MI meeting all of the following criteria:

i) presented within 24 hours of the onset of sustained clinical symptoms suggestive of MI lasting longer than 20 minutes, including chest pain and breathlessness; ii) had ECG changes indicative of MI (new pathologic Q waves, at least 1 mm ST elevation in any 2 or more contiguous limb leads or a new left bundle branch block, or new persistent ST-T wave changes diagnostic of a non-Q wave MI) with a subsequent confirmation by troponin-I measurements; and iii) had no previous cardiovascular diseases; defined as self- reported history of angina, MI, coronary revascularisation, transient ischaemic attack, stroke or evidence of CHD on prior ECG or in other medical records.

10,137 11,935 19404752

PROSPER Death from coronary heart disease or nonfatal MI. 641 638 12457784 De-novo

genotyping

WOSCOPS Death from coronary heart disease or nonfatal MI. 659 687 7566020 Summary C4D MI and other major coronary events (~90% of 11,146 10,940 21378988

(12)

data, published

cases); angiographic stenosis only (~10% of cases) Summary

data, published

CARDIoGRAM MI and other major coronary events (~90% of cases); angiographic stenosis only (~10% of cases)

20,315 58,415 21378990

Lp-PLA2 activity

Summary data, published

CHARGE consortium, composed of:

Cardiovascular Health Study, Framingham Heart Study and Rotterdam Study

Validated colourimetric or radioactive assays (diaDexus CAM Kit, diaDexus, Inc., San Francisco, CA, USA or Perkin Elmer Life Sciences, Inc., Waltham, MA, USA)

12,113 22003152

Tabular data, published

ARIC Automated Colorimetric Activity Method assay (diaDexus Inc., South San Francisco, CA) using a Beckman Coulter (Olympus) AU400e autoanalyzer

8564 25587968

MORGAM (FINRISK component)

Colorimetric Activity Method assay, Diadexus, Inc., San Francisco, CA

944 15561751

PROSPER Colorimetric Activity Method assay, Diadexus, Inc., San Francisco, CA

1229 20005516

LDL-C

GLGC Validated commercially available assays 89,888 24097068

BRAVE Validated enzymatic assay (Roche Diagnostics) 5737 25930055 De-novo

genotyping

CCHS Validated colorimetric assay 8056 19509380

CIHDS/CGPS Validated colorimetric assay 18021 23265341;

17635890 De-novo

genotyping

EPIC Validated assay (Roche Diagnostics) 17886 17295097

PROMIS Validated enzymatic assay (Roche Diagnostics) 20885 19404752 De-novo

genotyping

PROSPER Calculated using Friedewald formula 1268 12457784

WOSCOPS Calculated using Friedewald formula 1337 7566020

HDL-C

BRAVE Validated enzymatic assay (Roche Diagnostics) 5739 25930055 De-novo

genotyping

17635890 De-novo

genotyping

MORGAM Validated enzymatic assay after isolation of HDL 4269 15561751 De-novo

genotyping

PROSPER Validated assayat the Centre for Disease Control certified central lipoprotein laboratory in Glasgow.

1268 12457784 De-novo

genotyping

WOSCOPS Validated assayat the Centre for Disease Control certified central lipoprotein laboratory in Glasgow.

1337 7566020

Triglycerides

BRAVE Validated enzymatic assay (Roche Diagnostics) 5738 De-novo

genotyping

17635890 De-novo

genotyping

(13)

MORGAM Validated assays 2316 15561751

genotyping

PROSPER Validated assayat the Centre for Disease Control certified central lipoprotein laboratory in Glasgow.

1268 12457784 De-novo

genotyping

WOSCOPS Validated assayat the Centre for Disease Control certified central lipoprotein laboratory in Glasgow.

1337 7566020

BMI

GIANT Predominantly measured (few individual studies with self-report)

126,142 23754948

BRAVE Measured 5266 25930055

CCHS Measured 8082 19509380

CIHDS/CGPS Measured 14254 23265341;

17635890 De-novo

genotyping

EPIC Measured or self-report 21117 17295097

MORGAM Measured 4264 15561751

PROMIS Measured 20994 19404752

PROSPER Measured 1268 12457784

WOSCOPS Measured 1337 7566020

Diastolic blood pressure

ICBP Validated methods (eg, mercury

sphygmomanometer, automated blood pressure monitoring systems)

69,239 21909115

BRAVE Standard sphygmomanometer 5250 25930055

CCHS Standard sphygmomanometer 8070 19509380

CIHDS/CGPS Standard sphygmomanometer 13215 23265341;

17635890 De-novo

genotyping

EPIC Standard sphygmomanometer 15674 17295097

MORGAM Simple mercury sphygmomanometer, random zero sphygmomanometer, or automated device

5755 15561751 De-novo

genotyping

PROMIS Standard sphygmomanometer 20676 19404752

PROSPER Standard mercury sphygmomanometer 1264 12457784

WOSCOPS Standard mercury sphygmomanometer 1337 7566020

Systolic blood pressure

ICBP Validated methods (eg, mercury

sphygmomanometer, automated blood pressure monitoring systems)

69,245 21909115

BRAVE Standard sphygmomanometer 5250 25930055

CCHS Standard sphygmomanometer 8070 19509380

CIHDS/CGPS Standard sphygmomanometer 13218 23265341;

17635890 De-novo

genotyping

EPIC Standard sphygmomanometer 15676 17295097

MORGAM Simple mercury sphygmomanometer, random zero sphygmomanometer, or automated device

5755 15561751 De-novo

genotyping

PROMIS Standard sphygmomanometer 20686 19404752

PROSPER Standard mercury sphygmomanometer 1264 12457784

WOSCOPS Standard mercury sphygmomanometer 1337 7566020

Insulin

MAGIC Validated commercially available assays 38,238 20081858

(14)

PROMIS Validated assay 8073 19404752

PROSPER Validated assay, Mercodia; Diagenics, Milton Keynes, UK

1236 12457784

Fasting glucose

MAGIC Validated commercially available assays 46,186 20081858

PROMIS Validated assay (Roche Diagnostics) 4234 19404752

PROSPER Validated assay 1222 12457784

WOSCOPS Validated assay 1346 7566020

eGFR

CKDGen Serum creatinine calibrated to the US nationally representative National Health and Nutrition Examination Study (NHANES) standards. eGFR was estimated using the four-variable MDRD Study equation.

74,354 22479191

EPIC Serum creatinine (Roche Diagnostics). eGFR was estimated using the four-variable MDRD Study equation.

17133 17295097

PROMIS Creatinine via Jaffe method. eGFR was estimated using the four-variable MDRD Study equation.

12444 19404752 De-novo

genotyping

PROSPER Creatinine via Jaffe method. eGFR was estimated using the four-variable MDRD Study equation.

1267 12457784 De-novo

genotyping

WOSCOPS Creatinine via Jaffe method. eGFR was estimated using the four-variable MDRD Study equation.

1346 7566020

C-reactive protein

CHARGE Validated ELISA or similar methods 66,185 21300955

CCHS Turbidimetry (Dako, Glostrup, Denmark) 7349 19509380

CIHDS/CGPS Nephelometry (Dade Behring, Deerfield, Ill) 11,425 23265341;

17635890 De-novo

genotyping

MORGAM Validated assays 971 15561751

PROMIS Validated assay 1017 19404752

PROSPER Turbidimetry (Roche UK, Welwyn Garden City, UK) 1239 12457784 De-novo

genotyping

WOSCOPS Standardized in-house ELISA 1209 7566020

BRAVE = Bangladesh Risk of Acute Vascular Events Study; C4D = the Coronary Artery Disease Genetics consortium;

144

CARDIoGRAM = the transatlantic Coronary Artery Disease Genome-wide Replication and Meta-analysis consortium; CCHS =

145

Copenhagen City Heart Study; CHS= Cardiovascular Health Study; CGPS = Copenhagen General Population Study; CIHDS =

146

Copenhagen Ischaemic Heart Disease Study; ELISA = Enzyme-linked immunosorbent assay; EPIC = European Prospective

147

Investigation into Cancer and Nutrition Study; MORGAM = MONICA Risk, Genetics, Archiving and Monograph (subcohorts

148

include: ATBC, Augsburg, Brianza, FINRISK, PRIME-Belfast, PRIME-Lille, PRIME-Strasbourg, PRIME-Toulouse); PROMIS =

149

Pakistan Risk of Myocardial Infarction Study; PROSPER = Pravastatin in elderly individuals at risk of vascular disease trial;

150

WOSCOPS = West of Scotland Coronary Prevention Study; Details for assessment methods used in the MORGAM cohort are

151

available at: http://www.thl.fi/publications/morgam/qa/contents.htm

152

153

(15)

eTable 3 : Characteristics of participants from the Exome+ consortium by case-status

154

155

Numbers are mean (SD), NR=Not recorded

156 South Asian European

BRAVE PROMIS CCHS CGPS/CIHDS EPIC-CVD MORGAM PROSPER WOSCOPS

Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls

N 2971 2874 10,137 11,935 2020 6087 8079 10,367 9810 10,970 2153 2118 641 638 659 687

Male (n (%)) 2640 (88.9)

2467 (88.6)

8524 (84.1)

9054 (75.9)

1040 (51.5)

2553 (41.9)

5570 (68.9)

4706 (45.4)

6013 (61.3)

3779 (34.5)

1899 (89.2)

1753 (82.8)

403 (62.9)

305 (47.8)

659 (100)

687 (100) Age (years) 52.1

(10.5)

50.2 (10.1)

53.7 (10.2)

56.2 (9.1)

65.7 (10.9)

55.3 (15.4)

63.8 (10.9)

55.4 (12.7)

58.8 (8.6)

51.4 (9.6)

59.2 (7.7)

58.4 (8.4)

75.6 (3.5)

76.4 (3.6)

56.3 (5.4)

55.0 (5.8) Height (cm) 162.3

(7.3)

162.1 (7.3)

166.6 (7.6)

166.2 (9.1)

167.1 (9.4)

168.6 (9.6)

171.2 (8.9)

171.2 (9.4)

168.8 (9.2)

165.5 (9.2)

170.4 (7.8)

170.8 (8.4)

166.3 (9.0)

166.0 (9.4)

171.5 (7.0)

172.4 (6.9) Weight (kg) 59.5

(10.6)

59.9 (11.1)

71.9 (11.1)

71.9 (13.3)

75.0 (14.9)

71.8 (14.0)

80.7 (15.5)

76.8 (15.2)

77.3 (13.9)

71.5 (13.4)

80.7 (13.3)

78.9 (13.4)

74.7 (13.1)

73.4 (13.0)

76.5 (11.3)

76.6 (10.6) BMI (kg/m2) 22.5

(3.5)

22.8 (3.8)

25.9 (4.0)

26.1 (4.8)

26.8 (4.5)

25.2 (4.2)

27.2 (4.4)

26.1 (4.3)

27.1 (4.1)

26.1 (4.4)

27.8 (4.1)

27.0 (3.9)

27.0 (4.0)

26.6 (4.1)

26.0 (3.3)

25.7 (3.0)

WHR (-) 0.97

(0.07)

0.95 (0.07)

0.97 (0.06)

0.95 (0.06)

NR NR NR NR 0.90

(0.09)

0.84 (0.09)

0.95 (0.08)

0.91 (0.09)

NR NR NR NR

SBP (mmHg) 120.9 (23.0)

121.8 (18.3)

126.8 (20.2)

128.5 (17.1)

147.4 (22.6)

136.2 (21.9)

142.7 (21.9)

138.4 (20.4)

141.3 (20.2)

130.3 (18.9)

143.8 (20.8)

140.1 (19.8)

152.7 (23.0)

157.1 (21.4)

137.4 (17.6)

134.3 (17.7) DBP (mmHg) 79.7

(13.8)

78.3 (10.3)

80.4 (11.2)

81.2 (9.8)

86.2 (12.2)

83.5 (12.2)

80.9 (12.7)

82.6 (11.2)

85.2 (10.9)

81.0 (10.7)

85.1 (11.1)

84.2 (11.0)

81.9 (12.0)

84.7 (11.6)

84.7 (10.2)

83.1 (10.4) LDL (mmol/l) 3.18

(1.03)

2.70 (0.85)

3.22 (1.13)

2.77 (1.02)

4.08 (1.15)

3.65 (1.16)

2.89 (1.10)

3.31 (0.95)

4.77 (1.11)

4.16 (1.08)

3.93 (0.97)

3.76 (0.91)

3.72 (0.74)

3.78 (0.80)

4.99 (0.45)

4.60 (0.45) HDL (mmol/l) 0.85

(0.21)

0.87 (0.22)

0.91 (0.27)

0.93 (0.29)

1.48 (0.49)

1.61 (0.50)

1.36 (0.45)

1.65 (0.51)

1.27 (0.38)

1.49 (0.41)

1.18 (0.34)

1.30 (0.35)

1.20 (0.32)

1.32 (0.37)

1.09 (0.24)

1.15 (0.24) Triglycerides

(mmol/l)

2.14 (1.34)

2.27 (1.32)

2.27 (1.40)

2.34 (1.40)

2.12 (1.30)

1.75 (1.13)

1.79 (1.22)

1.77 (1.19)

1.89 (1.18)

1.32 (0.88)

1.98 (1.25)

1.66 (1.08)

1.61 (0.76)

1.47 (0.65)

1.92 (0.78)

1.79 (0.75) Total

cholesterol (mmol/l)

5.13 (1.12)

4.71 (0.99)

5.06 (1.35)

4.66 (1.30)

6.23 (1.28)

6.05 (1.28)

4.86 (1.28)

5.73 (1.06)

6.41 (1.17)

5.91 (1.12)

6.10 (1.11)

5.92 (1.05)

5.57 (0.84)

5.68 (0.90)

7.03 (0.59)

7.00 (0.59)

(16)

eTable 4: Characteristics of participants from the Exome+ consortium by carriage of loss of function (LoF) variants

157 European South Asians

At least 1 LoF variant No LoF variants At least 1 LoF variant No LoF variant

N 84 54,145 80 27,747

Male (n (%)) 41 (48.8) 43 (51.2) 69 (86.3) 22,616 (81.5)

Age (years) 55.8 (11.9) 57.6 (11.9) 53.2 (10.1) 54.3 (10.0)

Height (cm) 168.0 (9.1) 168.8 (9.4) 165.7 (7.6) 165.5 (8.4)

Weight (kg) 74.8 (13.5) 75.7 (14.5) 66.4 (14.5) 69.4 (13.0)

BMI (kg/m

²

) 26.6 (4.6) 26.5 (4.3) 24.1 (4.7) 25.3 (4.5)

WHR (-) 0.87 (0.10) 0.88 (0.10) 0.96 (0.06) 0.96 (0.06)

SBP (mmHg) 138.6 (23.0) 138.8 (21.1) 122.2 (18.7) 126.4 (19.3)

DBP (mmHg) 83.6 (12.5) 83.3 (11.4) 79.0 (10.0) 80.5 (10.9)

LDL (mmol/l) 3.88 (1.17) 3.81 (1.22) 2.84 (1.00) 2.97 (1.07)

HDL (mmol/l) 1.34 (0.40) 1.44 (0.46) 0.93 (0.30) 0.91 (0.27)

Triglycerides

(mmol/l) 1.79 (1.18) 1.71 (1.13) 2.09 (1.53) 2.29 (1.38)

Total cholesterol

(mmol/l) 5.78 (1.15) 5.86 (1.25) 4.79 (1.16) 4.86 (1.29)

Numbers are mean (SD)

158

(17)

eTable 5: Study-level characteristics of studies of rs76863441 (Val279Phe) and CHD or cardiovascular risk factors

159

Study/Author name

Country Control population Case definition N cases N controls

Overall minor allele frequency

Mean age

% Male Lp-PLA2 activity assay

Genotyping method

Phenotypes reported by genotype*

Lp-PLA2

activity

Lp-PLA2 mass

Blood pressure

Lipids Inflamm ation

Glucose /Diabetes Studies with information on V279F and CVD

Yamada 1998 &

2000$

Japan Healthy hospital based* MI or stroke 2517 6076 17% 56 66 Spec. PCR √ √ √ √

Hohda 2003 Japan General population MI 136 218 18% 47 76 NA PCR

Liu 2006 Taiwan Healthy hospital based* MI before age 45 200 200 17% 41 84 NA Puregene DNA Isolation Kit Li 2011 China Healthy hospital based* AP (with stenosis >

50%) /MI

804 482 6% 61 69 NA TaqMan

Hou 2009 (Beijing atherosclerosis study)

China General population Non-fatal MI, CAD defined by stenosis (>70%)

810 914 5% 53 76 CAM PCR √

Jang 2006 &

2011 (Study subset 1 & 2)

South Korea

Healthy volunteers MI, CAD defined by stenosis (>50%)

5874 5222 12% 55 75 Spec. TaqMan √ √

Takeuchi 2012 Japan MI or angina with

stenosis >75%

806 1335 20%** 66 64 NA GWAS-

Illumina Studies with information on V279F and CHD risk factors

Ichihara 1998 Japan Healthy hospital based NA NA 226 13% 54 76 Spec. PCR √

Yoshida 1998 Japan Healthy hospital based* NA NA 270 18% 61 56 Spec. PCR √

Satoh 1999 Japan General population NA NA 111 18% - - Spec. - √

Osoegawa 2004 Japan General population NA NA 82 14% - - Spec. PCR √

Unno 2006 Japan Healthy hospital based* NA NA 158 17% 71 86 TCA PCR √

Zhang 2007 Japan Healthy hospital based* NA NA 2018 17% 58 55 Azwell PCR √ √ √ √

Wang 2009 (Shimane study)

Japan Healthy hospital based* NA NA 800 19% 64 40 Cayman PCR/Taqma

n

√ √ √ √

Paik 2010 Korea Healthy hospital based* NA NA 2914 13% 57 40 Radiome

tric

SNaPShot assay kit

√ √ √ √ √ √

^{Fan 2010} ^China Hospital Infertile otherwise healthy

NA NA 148 4% 28 0 TCA PCR √ √ √

*Attending routine check up or screening test and found to lack any serious disorders; **Figures for Takeuchi relate to rs1805018 which is a proxy of rs16874954 in Japanese (r²=0.94) + stenosis refers to stenosis >50% in at least one

160

major coronary artery, except in the Beijing atherosclerosis study where it is at least 70%. $Non-overlapping subjects obtained via a data request. AP=angina pectoris; CAD=coronary artery disease; CAM=calorimetric assay method;

161

MI=myocardial infarction; PAD=Peripheral arterial disease; PCR=polymerase chain reaction ; Spec.=Spectophometric; TCA=tricalorimetric assay

162 163

(18)

eTable 6: Number of cases and controls carrying loss of function variants Study Participants

with loss of function variants

Participants without loss of function variants Cases Controls Cases Controls Gln287Ter

CCHS 1 4 2,019 6,083

CIHDS/CGPS 7 8 8,072 10,359

EPIC-CVD 9 10 9,801 10,960

MORGAM 0 1 2,153 2,117

PROSPER 0 0 641 638

WOSCOPS 0 1 659 686

MICAD consortium

32 51 36,155 64,581

TOTAL 49 75 59,500 95,424

rs142974898

CCHS 0 0 2,020 6,087

CIHDS/CGPS 0 0 8,079 10,367

EPIC-CVD 3 4 9,807 10,966

MORGAM 1 0 2,152 2,118

PROSPER 0 0 641 638

WOSCOPS 0 0 659 687

MICAD

consortium Not assayed

European total 4 4 23,358 30,863

BRAVE 0 0 2,971 2,782

PROMIS 0 0 10,135 11,928

South Asian

total 0 0 13,106 14,710

TOTAL 4 4 36,464 45,573

Arg82His

CCHS 0 2 2,020 6,085

CIHDS/CGPS 1 0 8,078 10,367

EPIC-CVD 2 8 9,808 10,962

MORGAM 2 2 2,151 2,116

PROSPER 1 0 640 638

WOSCOPS 2 0 657 687

MICAD consortium

14 23 36,173 64,609 European total 22 35 59,527 95,464

BRAVE 13 6 2,958 2,776

PROMIS 10 14 10,125 11,914

South Asian

total 23 20 13,083 14,690

TOTAL 45 55 72,610 110,154

Val279Phe

CCHS 0 1 2,020 6,086

CIHDS/CGPS 0 2 8,079 10,365

EPIC-CVD 5 6 9,805 10,964

MORGAM 0 1 2,153 2,117

PROSPER 0 0 641 638

WOSCOPS 0 0 659 687

MICAD

consortium 11 20 36,176 64,612 European total 16 30 59,533 95,469

(19)

BRAVE 10 10 2,961 2,772

PROMIS 7 10 10,128 11,918

South Asian total

17 20 13,089 14,690 TOTAL 33 50 72,622 110,159