Genome-wide association of early-onset myocardial infarction with single nucleotide polymorphisms and copy number variants Myocardial Infarction Genetics Consortium* We conducted a genome-wide association study testing single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) for association with early-onset myocardial infarction in 2,967 cases and 3,075 controls. We carried out replication in an independent sample with an effective sample size of up to 19,492. SNPs at nine loci reached genome-wide significance: three are newly identified (21q22 near MRPS6-SLC5A3-KCNE2, 6p24 in PHACTR1 and 2q33 in WDR12) and six replicated prior observations1���4 (9p21, 1p13 near CELSR2-PSRC1-SORT1, 10q11 near CXCL12, 1q41 in MIA3, 19p13 near LDLR and 1p32 near PCSK9). We tested 554 common copy number polymorphisms (41% allele frequency) and none met the pre-specified threshold for replication (P o 10 3). We identified 8,065 rare CNVs but did not detect a greater CNV burden in cases compared to controls, in genes compared to the genome as a whole, or at any individual locus. SNPs at nine loci were reproducibly associated with myocardial infarction, but tests of common and rare CNVs failed to identify additional associations with myocardial infarction risk. Myocardial infarction is a leading cause of death and disability worldwide5, with family history being an independent risk factor6. The inherited basis for myocardial infarction remains incompletely understood. Whereas the majority of myocardial infarctions occur in individuals 465 y old, 1���5% of younger individuals report a history of myocardial infarction5,7. These latter events are associated with substantially greater heritability8. Thus, early-onset myocardial infarc- tion is a promising phenotype for genetic mapping. Genome-wide association studies (GWASs) of common SNPs have been reported for myocardial infarction and coronary artery disease (CAD), with each study finding common SNPs on chromosome 9p21.3 associated with myocardial infarction or CAD1���3. In addition to 9p21.3, Samani et al. reported six other loci as harboring SNPs associated with CAD3. Some of these loci await definitive replication, but even if all were valid, they would explain a small fraction of the risk for myocardial infarction. Structural variants, another class of human DNA sequence varia- tion, may account for some of the unexplained heritability in myocardial infarction and other common diseases9. To our knowledge, no integrated assessment of SNPs and CNVs in the same samples has been reported for myocardial infarction or any other trait. Several technological developments make such systematic surveys now possi- ble, including hybrid oligonucleotide microarrays10 and analytical methods11 to simultaneously assess SNPs and CNVs genome-wide in each sample. �� 2009 Nature America, Inc. All rights reserved. Stage Samples DNA sequence variants Stage 1 Stage 2 Stage 3 Stage 4 2,967 cases of early-onset MI 3,075 controls from six studies ~2.5 million directly genotyped and imputed SNPs, common CNVs rare CNVs Symmetric effective sample size 3,922 cases of early-onset MI 3,922 controls from four studies 1,433 top SNPs associated with MI in stage 1 + SNPs from eight previously studied loci Symmetric effective sample size 4,321 cases of MI 4,321 controls from six studies 25 top SNPs after combined analysis of stages 1 and 2 + SNPs from eight previously studied loci Symmetric effective sample size 1,503 cases of early-onset MI 1,503 controls from one study 5 top SNPs after combined analysis of stages 1, 2, and 3 + SNPs from eight previously studied loci Figure 1 Study design. The GWAS consisted of four stages with an evaluation of common SNPs, common CNPs and rare CNVs in stage 1. The design called for all variants with a P o 0.001 to be taken forward to stage 2. As only SNPs met this criterion, 1,441 SNPs were taken forward to stage 2. Thirty-three SNPs were tested in stage 3. Thirteen SNPs were tested in stage 4. Statistical evidence for association was combined across stages 1���4 using meta-analysis. Received 13 November 2008 accepted 16 January 2009 published online 8 February 2009 corrected after print 27 May 2009 doi:10.1038/ng.327 *The complete list of participants and affiliations is provided at the end of this article. Correspondence should be addressed to S.K. (skathiresan1@partners.org). 334 VOLUME 41 [ NUMBER 3 [ MARCH 2009 NATURE GENETICS L E T T E R S

We designed a four-stage GWAS of early-onset myocardial infarc- tion with SNPs, common copy number polymorphisms (CNPs) and rare CNVs (Fig. 1). Stage 1 consisted of the Myocardial Infarction Genetics Consortium (MIGen), a collection of 2,967 cases of early- onset myocardial infarction (in men r50 y old or women r60 y old) and 3,075 age- and sex-matched controls free of myocardial infarction from six international sites: Boston and Seattle in the United States, as well as Sweden, Finland, Spain and Italy (Table 1 and Supplementary Methods online). The mean age at the time of myocardial infarction was 41 y among males and 47 y among females. Variants with P o 0.001 were advanced through three stages of replication (Fig. 1 see Methods for power calculations). Descriptions of the replication studies are provided in Supplementary Methods and Supplementary Tables 1 and 2 online. After stages 1���4, we observed that SNPs at nine loci were associated with myocardial infarction at a pre-specified threshold for genome- wide significance of P o 5 10 8 (corresponding to P o 0.05 after adjusting for B1 million independent tests12) (Tables 2 and 3). Of these nine, four represent confirmation of associations previously reported by Samani et al.3 (Table 2). These four genetic association signals map to 9p21, 1p13 near CELSR2-PSRC1-SORT1, 10q11 near CXCL12 and 1q41 in MIA3. In samples fully independent of the two original discovery studies (Wellcome Trust Case Control Consortium and German MI Family Study I), the statistical evidence for these four variants was robust, with the same allele associated in the same direction as the original report (replication P ranging from 3 10 5 to 1 10 30 Table 2). Three of the loci previously suggested by Samani et al. did not replicate (Table 2). In samples independent of the two original discovery studies, the statistical evidence for these loci was the following: rs6922269 in MTHFD1L (OR �� 1.04, 95% CI �� 0.99��� 1.09, P �� 0.08) rs17228212 in SMAD3 (OR �� 1.01, 95% CI �� 0.96���1.05, P �� 0.69) and rs2943634 on 2q36 (OR �� 0.94, 95% CI �� 0.90���0.98, P �� 0.01). Three previously unreported associations were observed with gen- ome-wide significance (Table 3): (i) in an intergenic region between MRPS6 (mitochondrial ribosomal protein S6), SLC5A3 (solute carrier family 5 (inositol transporters) member 3) and KCNE2 (potassium voltage-gated channel, Isk-related family, member 2) on chromosome 21q22 (rs9982601, OR �� 1.19, P �� 6 10 11) (ii) in an intron of PHACTR1 (phophastase and actin regulator 1) on chromosome 6p24 (rs12526453, OR �� 1.13, P �� 1 10 9) and (iii) in an intron of WDR12 (WD repeat domain 12) on chromosome 2q33 (rs6725887, OR �� 1.17, P �� 1 10 8). MRPS6 encodes a subunit of the mitochondrial ribosomal protein 28S13. SLC5A3 is a gene embedded within MRPS6 and encodes a protein that transports sodium and myo-inositol in response to hypertonic stress14. KCNE2 encodes a subunit of a potassium channel, and mutations in this gene cause inherited arrhythmias15. PHACTR1 is an inhibitor of protein phosphatase 1, an enzyme that depho- sphorylates serine and threonine residues on a range of proteins16. WDR12 has been shown to complex with several proteins to enable ribosome biogenesis and cell proliferation17. The mechanisms by which gene(s) at these three genomic regions confer increased risk of myocardial infarction remain to be defined. Of note, the PHACTR1 locus may lead to myocardial infarction by directly promoting the development of atherosclerosis in the coronary arteries. In an independent GWAS for coronary artery calcification in 410,000 participants from six prospective cohort studies, PHACTR1 SNPs (along with chromosome 9p21 SNPs) are associated with coronary artery calcification at genome-wide signifi- cance (C.J. O���Donnell, National Heart, Lung and Blood Institute, personal communication). Of the nine loci with convincing association evidence, the remain- ing two (19p13 near LDLR and 1p32 near PCSK9) relate to a causal risk factor for myocardial infarction: low-density lipoprotein (LDL) cholesterol. Common, low-frequency and/or rare mutations at LDLR and PCSK9 have previously been shown to influence LDL cholesterol and consequently affect risk for myocardial infarction18���22. We con- firm that common variants near LDLR and PCSK9 are associated with risk for myocardial infarction. The specific alleles (LDLR rs1122608 and PCSK9 rs11206510) corresponding to higher risk for myocardial �� 2009 Nature America, Inc. All rights reserved. Table 1 Participant characteristics of case and control subjects in stage 1 of the GWAS Study Italian ATVB Study Heart Attack Risk in Puget Sound REGICOR MGH Premature Coronary Artery Disease Study FINRISK Malmo �� Diet and Cancer Study Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls n 1,693 1,668 505 559 312 317 204 260 167 172 86 99 Ascertainment scheme Hospital- based Hospital- based Community- based Community- based Hospital- based Drawn from community- based cohort Hospital- based Hospital- based Drawn from population- based cohort Nested case- cohort Drawn from population- based cohort Nested case- cohort Myocardial infarction age criterion Men or women r45 ��� Men r50 or women r60 ��� Men r50 or women r60 ��� Men r50 or women r60 ��� Men r50 or women r60 ��� Men r50 or women r60 ��� Country of origina Italy Italy US US Spain Spain US US Finland Finland Sweden Sweden Mean age (y)b 39.4 �� 4.9 39.3 �� 5.0 46.0 �� 6.9 45.2 �� 7.3 45.9 �� 5.8 46.0 �� 5.6 47.0 �� 6.1 53.8 �� 11.1 47.1 �� 6.2 47.1 �� 6.0 48.5 �� 4.4 48.7 �� 4.6 Female gender (%) 11.4 11.6 51.1 55.5 20.2 21.5 29.9 33.5 33.5 31.4 41.9 42.4 Ever cigarette smoking (%) 87.0 49.3 73.9 41.7 82.8 61.9 74.9 57.3 74.4 58.2 87.2 61.6 Hypertension (%)c 32.6 11.9 50.5 30.8 38.0 31.5 33.5 25.3 72.5 68.0 81.4 62.6 Diabetes mellitus (%)d 7.8 0.8 14.9 3.0 14.8 6.1 19.2 0.4 17.7 5.9 4.7 1.0 Hypercholestero- lemia (%)e 60.4 44.4 43.7 26.0 48.9 33.1 79.0 31.3 75.2 48.2 37.2 1.0 Body mass index (kg/m2) 26.7 �� 4.2 25.0 �� 3.3 29.2 �� 6.8 26.9 �� 5.7 27.5 �� 4.2 27.0 �� 3.9 30.0 �� 7.0 27.9 �� 6.5 29.6 �� 5.0 27.7 �� 4.0 26.9 �� 4.2 25.7 �� 4.3 Values with �������� are means �� s.d. The body-mass index is the weight in kilograms divided by the square of the height in meters. aAll cases and controls were of European ancestry. bMean age at myocardial infarction for cases and at age of recruitment for controls. cHypertension was defined as a previous diagnosis of hypertension, on antihypertensive therapy or with recorded systolic blood pressure Z 140 mmHg or diastolic blood pressure Z 90 mmHg. dDiabetes mellitus was defined as a previous diagnosis of diabetes or treatment with antidiabetic medications. eHypercholesterolemia was defined as a previous diagnosis of hypercholesterolemia or treatment with lipid-lowering therapy. NATURE GENETICS VOLUME 41 [ NUMBER 3 [ MARCH 2009 335 L E T T E R S