Curr Atheroscler Rep (2015) 17:14 DOI 10.1007/s11883-015-0498-5

GENETICS (AJ MARIAN, SECTION EDITOR)

Shared Genetic Aetiology of Coronary Artery Disease and Atherosclerotic Stroke—2015 Thorsten Kessler & Jeanette Erdmann & Martin Dichgans & Heribert Schunkert

# Springer Science+Business Media New York 2015

Abstract In the last years, genome-wide association studies have allowed to identify multiple genetic variants associated with atherosclerosis. In this review, we highlight the identification of genomic variants associated with coronary artery disease and myocardial infarction as well as large-vessel stroke. We will focus on genetic variants that displayed overlap for these atherosclerotic diseases. Current research is focusing on the identification of the functional mechanisms underlying these associations. As frequent variants are often only associated with This article is part of the Topical Collection on Genetics T. Kessler : H. Schunkert (*) Klinik für Herz- und Kreislauferkrankungen, Deutsches Herzzentrum München, Technische Universität München, Lazarettstr. 36, 80636 Munich, Germany e-mail: [email protected] T. Kessler e-mail: [email protected] J. Erdmann Institut für Integrative und Experimentelle Genomik, Universität zu Lübeck, Maria-Goeppert-Str. 1, 23562 Lübeck, Germany e-mail: [email protected] J. Erdmann Deutsches Zentrum für Herz-Kreislauf-Forschung (DZHK) e.V., partner site Hamburg/Kiel/Lübeck, Lübeck, Germany M. Dichgans Institut für Schlaganfall- und Demenzforschung, Klinikum der Universität München, Ludwig-Maximilians-Universität München, Feodor-Lynen-Straße 17, 81377 Munich, Germany e-mail: [email protected] M. Dichgans Munich Cluster for Systems Neurology (SyNergy), Munich, Germany H. Schunkert Deutsches Zentrum für Herz-Kreislauf-Forschung (DZHK) e.V., partner site Munich Heart Alliance, Munich, Germany

small increases in risk, the search for the identification of rare variants with large increases in risk is ongoing. Whole-exome sequencing recently revealed rare variants dramatically increasing cardiovascular risk. Taken together, the developments of the past few years light the vision of improved prevention and therapy of coronary artery disease and stroke. Keywords Atherosclerosis . Coronary artery disease . Myocardial infarction . Stroke . Genome-wide association study . GWAS . Next-generation sequencing

Introduction Cardiovascular diseases on the basis of atherosclerosis, i.e. coronary artery disease (CAD) and myocardial infarction (MI) as well as atherosclerotic stroke, are the leading causes of disability and death in the industrialised world [1]. Both CAD/MI and stroke share distinct mechanisms: 1. modifiable risk factors [2], e.g. arterial hypertension, hyperlipidaemia, obesity, smoking, and diabetes; and 2. a non-modifiable genetic predisposition [3, 4]. This genetic predisposition becomes obvious in individuals with a positive family history for either CAD or stroke [5]. It has also been shown that family history for CAD increases the risk for stroke and vice versa [6]. Whereas previously, it was not possible to determine the genetic factors leading to such increase of risk, recent advances in molecular biology, i.e. high-throughput genotyping, and statistics have allowed identifying genomic loci increasing as well as decreasing the cardiovascular risk. In this review, we summarise recent discoveries in genetics of atherosclerosis and highlight the raised possibilities in prevention and therapy.

14

Page 2 of 10

Large-Scale Association Analyses in CAD and Atherosclerotic Stroke Starting from results from epidemiological studies, candidate gene analyses (for a review, see [7]) were carried out to determine genetic factors associated with the risk of atherosclerosis but had often only limited success. After the development of large-scale genotyping methods as well as the identification of the full human genome sequence with the annotation of common single nucleotide polymorphisms, it was possible to investigate large cohorts of controls and patients suffering from CAD and stroke in genome-wide association studies (GWAS) (for a review, see [8]). In 2007, three independent genomewide association studies identified a variant on chromosome 9p21 to be genome-wide significantly associated with CAD [9–11]. Remarkably, the same locus was associated with the risk of large-vessel stroke [12, 13] and arterial aneurysms [14]. In the following years, increasing the number of cases and controls, especially by the formation of large international consortia, has allowed to identify several further loci [15]. In 2013, the CARDIoGRAMplusC4D consortium, consisting of the CARDIoGRAM and the Coronary Artery Disease (C4D) Genetics consortia, conducted the thus far largest analysis in CAD/MI with more than 60,000 cases and more than 130,000 controls [16••]. In this analysis, 31 previously identified loci were replicated, and 15 loci were found to be genome-wide significantly associated with CAD/MI for the first time (Fig. 1, Tables 1 and 2). A number of GWAS aimed to detect genetic variants associated with ischemic stroke [13, 21, 28, 38] with the hassle to

Curr Atheroscler Rep (2015) 17:14

include several etiological sub-phenotypes. Cardioembolic stroke for example in most of the cases occurs secondary to atrial fibrillation, which is in contrast to large-vessel stroke. Furthermore, there are several monogenic disorders with a Mendelian pattern of inheritance including stroke in their manifestations (for a review, see [39]). In 2012, the META STROKE consortium conducted a meta-analysis on ischemic stroke including more than 12,000 patients and 60,000 controls. The authors replicated two known loci for cardioembolic stroke, both of which had previously been shown to be associated with atrial fibrillation (PITX2 [22–25], ZFHX3 [22]) and three loci for large-vessel stroke (Chr. 9p21 [12, 28], HDAC9 [28], SUPT3H/CDC5L [21]), whereas the AB0 locus [32] is rather associated with both cardioembolic and largevessel stroke [13, 40]. Furthermore, 12 potential novel loci were detected but lacked replication [13]. Recently, an extended GWAS using the Immunochip array followed by a metaanalysis identified a novel locus on chromosome 12 in high LD with a variant tagging the SH2B3 gene [35], a locus also known to be associated with CAD [16••, 34]. Variants in the genes MMP12 [33] and COL4A2 [36] have lately also been found to be associated with large-vessel stroke and smallvessel stroke, respectively. In summary, to date, 9 loci are known to be associated with cardioembolic and atherosclerotic stroke (Fig. 1, Table 2). Further fine-mapping of the genetic variability is aimed to be reached via the enhancement of SNP coverage: arrays with genome-wide coverage investigate variants throughout the whole genome with smaller density. The imputation of 1000 Genomes data in GWAS analyses will, by increasing the

Fig. 1 Genetic loci associated with CAD/MI (red), ischemic stroke (green), and both (yellow), and CAD variants also showing study-wide association with atherosclerotic stroke (grey; b associated with large-vessel stroke, c associated with ischemic stroke [17••])

Curr Atheroscler Rep (2015) 17:14

Page 3 of 10 14

Table 1 Association of top CAD risk variants with stroke phenotypes and association of top stroke risk variants with CAD SNP

Chr.

Gene

Risk allele

OR CAD

OR IS OR LVS

rs2023938 rs1333049 rs1333047 rs579459 rs12413409 rs3184504 rs7696736 rs12936587

7p21 9p21 9p21 9q34 10q24 12q24 12q24 17p11

HDAC9 CDKN2BA CDKN2BA AB0 CYP17A1 SH2B3 SH2B3 RAI1

C C T C G T G T

1.08a,c 1.24a,c 1.24c 1.10a,c 1.11a,c 1.07a,c 1.07c 1.07a,c

1.14b 1.05b 1.05b 1.08c 1.05b 1.08c 1.10a,c 1.05b

1.38c 1.19c 1.20a,c 1.13b 1.20c 1.14c 1.11b 1.14c

From [17••] SNP single nucleotide polymorphism, Chr. chromosome, OR odds ratio, CAD coronary artery disease, IS ischemic stroke, LVS large-vessel stroke a

Primary association

b

Nominally significant

c

Study-wide significant

number of SNPs available for analysis from roughly 500 k to about 15 million, enhance the density and allow to identify further loci associated with CAD and stroke. The 1000 Gen o m e s i m p u t a t i o n o f 4 8 G WA S i n t h e C A R D IoGRAMplusC4D consortium led to the identification of 38 novel variants mapping to ten novel genomic loci further reducing the missing heritability in CAD (presented at the Scientific Sessions 2014 of the American Heart Association [41]). Figure 2 shows how the density of genomic maps and the number of subjects investigated affected the number of loci with genome-wide significance of CAD risk. Arrays with exome-wide coverage [42] investigate SNPs thought to be functionally relevant, but thereby missing the intronic variants. Finally, arrays focusing on SNPs in distinct pathways, e.g. metabolic traits, are missing other relevant parts of the genome [43]. Further options to identify risk-modulating genetic variants include the methods of next-generation sequencing, e.g. coding DNA (whole-exome sequencing) or whole genome (whole-genome sequencing).

Shared Genetics of CAD/MI and Atherosclerotic Stroke As shown in Fig. 1, several genes have been shown to be associated with CAD/MI or atherosclerotic stroke. To analyse shared genetic risk factors, we performed a meta-analysis investigating the loci identified to be associated with both diseases as well as loci associated with either one disease including data from the CARDIoGRAM [15], C4D [29], and META STROKE [13] consortia [17••]. Six CAD risk loci were associated with either ischemic or large artery stroke: CYP17A1, RAI1, SH2B3, HDAC9, AB0, and chr. 9p21 (Table 2).

Interestingly, the effects for the risk alleles pointed towards the same direction for loci discovered for stroke and loci associated with CAD in the vice versa analyses. Furthermore, the stroke loci tagging SH2B3 and chr. 9p21 reached studywide significance in the CAD analysis; three (SH2B3, HDAC9, AB0) and four (CYP17A1, RAI1, SH2B3, HDAC9) of the investigated CAD loci also reached study-wide significance for ischemic stroke and large artery stroke, respectively (Table 2) [17••]. In a second analysis investigating combined phenotypes of CAD with ischemic stroke or large-artery stroke, several loci previously not identified to be associated with ischemic stroke or large artery stroke, respectively, showed a strong association signal. SH2B3 displayed a strong association signal for ischemic stroke and CAD, whereas, e.g. RAI1 showed a prominent signal for both large-artery stroke and CAD. Aside, other loci seem to be associated with CAD, e.g. SORT1, PHACTR1, WDR12, or TCF21, whilst there was no signal for ischemic stroke or large-vessel stroke [17••]. Figure 3 gives examples for a locus that shows a similar architecture for MI and stroke, i.e. the same SNPs increase the risk likewise for both diseases (HDAC9), and a locus that exclusively affects MI risk (TCF21, SORT1). The signal for SH2B3 as a major genetic risk factor for several cardiovascular diseases, including both stroke phenotypes and CAD, is, from a genetic point of view, very interesting. As a matter of fact, the SH2B3 locus has been associated not only with these diseases but also with different risk factors for cardiovascular diseases, e.g. lipids [44], type-1 diabetes mellitus [45], and blood pressure [46, 47]. Furthermore, the SH2B3 locus was also found to be associated with platelet count [48]. In line, the investigation of thrombocyte function in Lnk−/− (=Sh2b3−/−) mice has revealed elevated platelet numbers but also impaired thrombus stability [49]. However, the mechanism involving SH2B3 in atherosclerosis thus far remains elusive and is the object of the current research. In summary, these results indicate a common genetic basis of CAD and stroke but significant differences in the contribution of common variants to each phenotype. The elucidation of the molecular mechanisms underlying the association will probably at least partly allow explaining these observations. Recently, functional studies on the HDAC9 locus, that has been clearly identified to be associated with CAD and stroke, gave first insight into the possible mechanisms [50•].

Novel Mechanisms: SORT1, HDAC9, ADAMTS7 In the first GWAS meta-analysis, a locus on chromosome 1 had been identified to be genome wide significantly associated with CAD [9] as well as low-density lipoprotein (LDL) cholesterol levels [51]. The causal gene remained unknown until Musunuru et al. carried out a comprehensive analysis of genes

14 Table 2 Chr.

1

2

3 4

5 6

7

8 9

10

11

12 13 14 15 16

Curr Atheroscler Rep (2015) 17:14

Page 4 of 10 Currently known loci associated with CAD and ischemic stroke Locus

CAD/MI

Ischemic stroke

SNP

AF

OR

Ref.

PCSK9 PPAP2B MIA3 SORT1 IL6R ABCG5/8 WDR12 APOB ZEB2 VAMP5/8 MRAS GUCY1A3 EDNRA PITX2

rs11206510 rs17114036 rs17465637 rs599839 rs4845625 rs6544713 rs6725887 rs515135 rs2252641 rs1561198 rs2306374 rs7692387 rs1878406

T (0.82) A (0.91) C (0.74) A (0.78) T (0.47) T (0.30) C (0.15) G (0.83) G (0.46) A (0.45) C (0.18) G (0.81) T (0.15)

1.08 1.17 1.14 1.11 1.06 1.06 1.14 1.07 1.06 1.06 1.12 1.08 1.10

[18] [15] [9, 15] [9, 15] [16••] [16••, 19] [15, 18] [16••] [16••] [16••] [15, 20] [16••] [16••]

IL5 SLC22A4/5 SUPT3H/CDC5L C6orf105 PHACTR1 ANKS1A TCF21 LPA KCNK5 PLG HDAC9 BCAP29 ZC3HC1 TRIB1 LPL 9p21 AB0

rs2706399 rs273909 rs6903956* rs12526453 rs17609940 rs12190287 rs3798220 rs10947789 rs4252120 rs2023938 rs10953541 rs11556924 rs2954029 rs264 rs4977574 rs579459

A (0.07) C (0.67) G (0.75) C (0.62) C (0.02) T (0.76) T (0.73) G (0.10) C (0.80) C (0.62) A (0.55) G (0.86) G (0.46) C (0.21)

1.51 1.10 1.07 1.08 1.51 1.07 1.07 1.08 1.08 1.09 1.06 1.11 1.29 1.10

[26] [15, 18] [15] [15] [15, 27] [16••] [16••] [16••] [29] [15] [16••, 19] [16••] [9–11, 15, 30] [15, 31]

KIAA1462 CXCL12 LIPA CYP17A1

rs2505083 rs1746048 rs1412444 rs12413409

C (0.38) C (0.87) T (0.42) G (0.89)

1.07 1.09 1.09 1.12

[20, 29] [9, 15] [29] [15]

PDGFD APOA1-C3-A4-A5 MMP12 SH2B3 COL4A1/2 FLT1 HHIPL1 ADAMTS7 FURIN-FES ZFHX3

rs974819 rs964184

T (0.32) G (0.13)

1.07 1.13

[29] [15]

rs3184504 rs4773144 rs9319428 rs2895811 rs3825807 rs17514846

G (0.51) C (0.14)

T (0.44) G (0.44) A (0.32) C (0.43) A (0.57) A (0.44)

1.07 1.07

1.07 1.07 1.06 1.07 1.08 1.07

SNP

AF

OR

Ref.

rs6843082

G (0.21)

1.36a

[21–25]

rs556621

A (0.33)

1.21b

[13, 21]

rs2107595

A (0.16)

1.39b

[13, 28]

rs2383207** rs505922

G (0.52) C (0.39)

1.15b 1.13a 1.23b

[13] [13] [32]

See left

See left

1.20b,e 1.05c,e

[17••] [17••]

rs660599 rs10744777 rs9521732

A (0.19) T (0.66) A (0.41)

1.18b 1.10c 1.29d

[33] [35] [36]

rs879324

A (0.19)

1.25a

[13, 37]

[19] [16••]

[15, 34] [15] [16••] [15] [15, 29, 31] [16••]

Curr Atheroscler Rep (2015) 17:14

Page 5 of 10 14

Table 2 (continued) Chr.

17

19 21

Locus

CAD/MI

Ischemic stroke

SNP

AF

OR

Ref.

SNP

AF

OR

Ref.

SMG6 RAI1-PEMT-RASD1

rs216172 rs12936587

C (0.37) G (0.56)

1.07 1.07

[15] [15]

See left

See left

1.14b,e 1.05c,e

[17••] [17••]

UBE2Z LDLR APOE MRPS6

rs46522 rs1122608 rs2075650 rs9982601

T (0.53) G (0.77) G (0.14) T (0.15)

1.06 1.14 1.14 1.18

[15] [15, 18] [19] [18]

Chr. chromosome, CAD coronary artery disease, MI myocardial infarction, SNP single nucleotide polymorphism, AF allele frequency, OR odds ratio, Ref. reference(s); only in Han Chinese a

Cardioembolic stroke

b

Large-vessel stroke

c

Ischemic stroke

d

Small-vessel stroke

e

Study-wide significantly associated

which expression was differentially regulated in risk allele carriers particularly in the liver, which is known to play a major role in lipid metabolism. A small number of genes were associated with the polymorphism and the analysis of further variants at the locus associated with LDL cholesterol levels pointed to one variant, rs12740374, which indeed affected binding of the transcription factor C/EBP. Further analysis Fig. 2 Relationship between density of genomic maps and number of investigated individuals with the number of loci identified by genetic analyses. The red boxes display the respective studies

revealed SORT1, which encodes for sortilin 1, to be most strongly affected by the variant regarding its expression. The overexpression of sortilin 1 led to a reduction of LDL cholesterol and vice versa [52•]. Previous studies had already pointed to a reduced secretion of very-low-density lipoprotein (VLDL) cholesterol [53]. Another study in line demonstrated increased endocytosis of LDL cholesterol secondary to

14

Page 6 of 10

Curr Atheroscler Rep (2015) 17:14

Fig. 3 Architecture of genetics in CAD and stroke. The SNPs at the HDAC9 loci (a) show the same effects for stroke and CAD, whereas TCF21 (b) and SORT1 (c) seem to be exclusively associated with CAD (left regional association plots, right corresponding Spearman correlation plots [17••])

sortilin 1 overexpression in vitro [54]. However, the subcellular mechanism thus far remains unknown. An example for a locus associated with both CAD and stroke tags HDAC9, encoding histone deacetylase 9 [13, 16••, 28]. As the locus is also associated with carotidintima thickness [55], a link to the formation of atherosclerotic lesions is strong. This locus is also remarkable since the analysis of coronary sub-phenotypes - CAD and MI revealed that HDAC9 is the only of more than 60 loci to give a significantly stronger signal for coronary atherosclerosis rather than MI. By contrast, the risk increase was similar for these sub-phenotypes at almost all other loci. Azghandi et al. were able to show that the risk allele at the lead SNP associated with stroke at this locus, rs2107595, is indeed associated with the increased expression of the HDAC9 gene in peripheral blood mononuclear cells. In further experiments using mice lacking Hdac9 expression on a proatherogenic background, i.e. Hdac9−/−ApoE−/− mice,

atherosclerotic lesion sizes were significantly reduced compared to ApoE−/− mice carrying the Hdac9 wild-type gene [50•]. Histone deacetylases are critical in several cellular processes as they, together with histone acetylases, mediate modification of histone and nonhistone proteins to influence, e.g. gene transcription and posttranslational protein modification. Therefore, facing the promising results regarding atherosclerotic lesions in mice lacking Hdac9, it is not yet possible to definitively evaluate the therapeutic potential of a HDAC9 inhibition in humans to prevent or treat atherosclerosis. This is especially important, as there is thus far no clue to the molecular mechanism linking Hdac9 deficiency and reduced atherosclerosis despite results from Cao et al. pointing to a putative effect of macrophages via reduced cholesterol efflux [56]. As a matter of fact, an association of a plaque phenotype with the investigated risk allele could not be proven examining subjects included in the AtheroExpress Study [50•].

Curr Atheroscler Rep (2015) 17:14

A promising therapeutic target is the extracellular matrix protease ADAMTS7, encoded by the CAD risk gene ADAMTS7. ADAMTS7 had previously been mainly linked to rheumatoid arthritis where its expression had been shown to be increased in synovium and cartilage from affected individuals [57]. Two studies linked balloon-mediated vascular injury to increased expression of Adamts7 in rats [58] and depletion of its thus far only known substrate cartilage oligomeric matrix protein (COMP, thrombospondin-5) to dedifferentiation of smooth muscle cells [59] until genome-wide association studies identified SNPs tagging ADAMTS7 to be genome wide significantly associated with CAD [15, 29, 31]. In a sub-phenotype analysis of CAD and stroke, ADAMTS7 was also associated with ischemic and large-vessel stroke, but with different directions of the risk allele [17••]. Interestingly, ADAMTS7 was rather associated with CAD than MI [31], implicating a role in the vascular processes leading to the formation of atherosclerotic plaques. Whilst Pu et al. described an influence of the risk allele with increased degradation of COMP suggesting a detrimental effect on vascular smooth muscle cells [60]; until recently, the molecular mechanism remained unknown. Mice lacking expression of functional Adamts7 were resistant to neointima formation secondary to a wire-mediated vascular injury. Further analysis revealed re-endothelialisation to be the critical step in this scenario. Adamts7 − / − mice displayed accelerated reendothelialisation compared to wild-type animals due to increased proliferation as well as migration of endothelial cells. Interestingly, this effect was independent of COMP. On the contrary, thrombospondin-1 (TSP-1), another member of the thrombospondin family, was identified as a putative target with a potential inhibitory effect of TSP-1 fragments generated by ADAMTS7-mediated cleavage on endothelial cell proliferation and migration [61••]. Thus, ADAMTS7 seems to play a major role in vascular remodelling. Whether other processes contribute to its pathophysiological role in atherosclerosis is under investigation.

Exome Sequencing/Rare Variants: GUCY1A3, NPC1L1 As reported previously, frequent variants tagging the GUCY1A3 gene are genome-wide significantly associated with coronary artery disease [16••]. A method to identify rare variants presumably associated with a stronger increase in cardiovascular risk is to analyse extended families with high prevalence of CAD/MI. Our group assembled a large collection of such families and, with the rise of next-generation sequencing techniques, analysed one of these families by exome sequencing. After stringent filtering, we identified four mutations. Studying these in the entire family revealed LOD scores of about 1.0, thereby not giving reason for already knowing the causal mutation. One of the four mutations was a frameshift

Page 7 of 10 14

mutation in the GUCY1A3 gene, and a second mutation also leading to a loss of function was located in the CCT7 gene [62], which encodes a chaperone protein (CCTη) needed to ensure proper function of the soluble guanylyl cyclase (sGC) [63]. Subsequently, we were able to demonstrate that individuals carrying both mutations are at a 100 % risk of suffering from CAD/MI, whereas family members carrying either one of the mutations were affected in half of the cases. As the sGC forms a functional unit consisting of a β-subunit in addition to the αsubunit encoded by the GUCY1A3 gene, lack of functional αsubunit went along with reduced levels of the β-subunit and decreased levels of cyclic guanosine monophosphate (cGMP). The second messenger cGMP plays a major role in vasodilatation and inhibition of platelet aggregation. Therefore, an increased risk in family members affected by the mutation sounded reasonable to explain the genetic association. Investigating transgenic mice lacking the sGC’s α-subunit, we then demonstrated that the lack of this protein accelerates time to thrombus formation after vascular laser injury [62]. However, whether this private mutation can serve as a role model for the investigation of common variants in the GUCY1A3 gene and, even much more important, whether measures to inhibit platelet function, e.g. by aspirin intake in primary prevention, are able to protect from CAD/MI remains object of research. A different approach also utilising exome sequencing was used by the Myocardial Infarction Genetics Consortium Investigators. The authors performed exome sequencing in more than 7000 CAD cases and almost 15,000 controls and subsequently searched for inactivating mutations in the NPC1L1 gene, encoding for the Niemann-Pick C1-like 1 protein (NPC1L1) [64]. These mutations were further genotyped in more than 20,000 patients and almost 70,000 controls. Thereby, the authors found out that these inactivating mutations affected LDL cholesterol or triglyceride levels and subsequently relative risk to suffer from CAD [64–66]. Recently, the results on the NPC1L1 gene were corroborated by a clinical trial with pharmacological proof to the genetic findings: the IMPROVE IT trial analysed simvastatin ± ezetimibe in patients with acute coronary syndrome. Ezetimibe is known to target NPC1L1 and thereby lower LDL cholesterol. The authors found a further reduction of LDL cholesterol adding ezetimibe to simvastatin and, interestingly, a significant reduction of a combined endpoint including death for cardiovascular reasons, stroke, and hospitalisation as well as revascularisation due to angina pectoris (presented at the Scientific Sessions 2014 of the American Heart Association [67]).

Clinical Application: Prevention/Therapy and Risk Prediction The previously mentioned loci GUCY1A3 and NPC1L1 are more or less in the pipeline from bench to bedside. The

14

Curr Atheroscler Rep (2015) 17:14

Page 8 of 10

PCSK9 locus can be regarded as a role model for the translation from a genetic association over the identification of the pathophysiological mechanism to therapeutic application. PCSK9 was identified to be associated with lipid traits [18, 44, 68] and CAD [18]. The encoded protein, proprotein convertase subtilisin/kexin type 9 (PCSK9), targets the LDL cholesterol receptor leading to its degradation. Interestingly, this happens in the cell where intracellular PCSK9 binds to its target as well as in the extracellular space where secreted PCSK9 binds to LDL cholesterol receptors. In the latter case, the receptors are internalised and, together with the intracellularly captured receptor molecules, degraded (for a review, see [69]). This was proven by the investigation of gain-offunction mutations in the PCSK9 gene, which were associated with increased LDL cholesterol and accelerated atherosclerosis [68, 70]. As a pharmacological approach, an antibody against PCSK9 (evolocumab) was developed and tested successfully in phase II trials with a promising 40–59 % reduction in LDL cholesterol and acceptable rates of adverse events [71] without evidence for safety concerns after long-term analysis [72]. The FOURIER trial (NCT01764633) will now investigate, whether the treatment with evolocumab also reduces the incidences of clinical relevant endpoints as cardiovascular death and myocardial infarction. With alirocumab, there is already a second monoclonal PCSK9 antibody in the pipeline (ODYSSEY trial, NCT01954394). Several studies assessed the predictive value of risk scores including genetic markers compared to established risk scores in CAD. In these studies, a set of genomewide significantly associated SNPs was added to traditional risk scores to assess measures of risk stratification improvement compared to traditional risk scores alone (for a review, see [73]). The additional value of genetic information was very limited. Hughes et al. published a promising result where inclusion of genetic variants substantially improved risk prediction in middle-aged men [74].

Conclusion and Outlook The past years led to the identification of several genetic variants associated with CAD and stroke. Most of the genes tagged by these variants were previously linked neither to these diseases directly nor to intermediate phenotypes, e.g. lipid metabolism or arterial hypertension. The functional analysis of these genes and the encoded proteins led to the discovery of novel mechanisms. Nevertheless, it has been shown that several variants do not necessarily impact the nearby genes [75]. Both the identification of risk-associated variants and the functional analysis are just in the beginnings. However, examples as NPC1L1 and, even more impressive, PCSK9 exhibit the potential usage of the novel knowledge for prevention and therapy of atherosclerosis and the related diseases.

Investigation of gene-gene and gene-environment interactions will hopefully further elucidate the inheritance of the diseases. Comprehensive analysis of different OMICs data sets will open the door to system medicine as a cornerstone of individualised medicine approaches. Compliance with Ethics Guidelines Conflict of Interest Thorsten Kessler, Jeanette Erdmann, Martin Dichgans, and Heribert Schunkert do not have any conflicts of interest in connection with this work. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors. Funding This work has been supported by the German Federal Ministry of Education and Research (BMBF) in the context of the e:Med programme (e:AtheroSysMed, to Jeanette Erdmann, Martin Dichgans, and Heribert Schunkert), the FP7 European Union project CVgenes@target (261123, to Jeanette Erdmann, Martin Dichgans, and Heribert Schunkert), and the Foundation Leducq (CADgenomics: Understanding Coronary Artery Disease Genes, 12CVD02, to Jeanette Erdmann and Heribert Schunkert).

References Papers of particular interest, published recently, have been highlighted as: • Of importance •• Of major importance 1.

Lozano R, Naghavi M, Foreman K, et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2013;380(9859):2095–128. doi:10.1016/S0140-6736(12)61728-0. 2. Yusuf S, Hawken S, Ounpuu S, et al. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case–control study. Lancet. 2004;364(9438):937–52. doi:10.1016/S0140-6736(04)17018-9. 3. Myers RH, Kiely DK, Cupples LA, Kannel WB. Parental history is an independent risk factor for coronary artery disease: the Framingham Study. Am Heart J. 1990;120(4):963–9. 4. Brass LM, Isaacsohn JL, Merikangas KR, Robinette CD. A study of twins and stroke. Stroke. 1992;23(2):221–3. 5. Murabito JM, Pencina MJ, Nam B-H, et al. Sibling cardiovascular disease as a risk factor for cardiovascular disease in middle-aged adults. JAMA. 2005;294(24):3117–23. doi:10.1001/jama.294.24.3117. 6. Banerjee A, Lim CCS, Silver LE, Welch SJV, Banning AP, Rothwell PM. Familial history of stroke is associated with acute coronary syndromes in women. Circ: Cardiovasc Genet. 2011;4(1): 9–15. doi:10.1161/CIRCGENETICS.110.957688. 7. Mayer B, Erdmann J, Schunkert H. Genetics and heritability of coronary artery disease and myocardial infarction. Clin Res Cardiol. 2007;96(1):1–7. doi:10.1007/s00392-006-0447-y. 8. Manolio TA. Genomewide association studies and assessment of the risk of disease. N Engl J Med. 2010;363(2):166–76. doi:10.1056/NEJMra0905980.

Curr Atheroscler Rep (2015) 17:14 9.

10.

11.

12.

13.

14.

15.

16.••

17.••

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

Samani NJ, Erdmann J, Hall AS, et al. Genomewide association analysis of coronary artery disease. N Engl J Med. 2007;357(5): 443–53. doi:10.1056/NEJMoa072366. McPherson R, Pertsemlidis A, Kavaslar N, et al. A common allele on chromosome 9 associated with coronary heart disease. Science. 2007;316(5830):1488–91. doi:10.1126/science.1142447. Helgadottir A, Thorleifsson G, Manolescu A, et al. A common variant on chromosome 9p21 affects the risk of myocardial infarction. Science. 2007;316(5830):1491–3. doi:10.1126/science.1142842. Gschwendtner A, Bevan S, Cole JW, et al. Sequence variants on chromosome 9p21.3 confer risk for atherosclerotic stroke. Ann Neurol. 2009;65(5):531–9. doi:10.1002/ana.21590. Traylor M, Farrall M, Holliday EG, et al. Genetic risk factors for ischaemic stroke and its subtypes (the METASTROKE collaboration): a meta-analysis of genome-wide association studies. Lancet Neurol. 2012;11(11):951–62. doi:10.1016/S1474-4422(12)70234-X. Helgadottir A, Thorleifsson G, Magnusson KP, et al. The same sequence variant on 9p21 associates with myocardial infarction, abdominal aortic aneurysm and intracranial aneurysm. Nat Genet. 2008;40(2):217–24. doi:10.1038/ng.72. Schunkert H, König IR, Kathiresan S, et al. Large-scale association analysis identifies 13 new susceptibility loci for coronary artery disease. Nat Genet. 2011;43(4):333–8. doi:10.1038/ng.784. CARDIoGRAMplusC4D Consortium, Deloukas P, Kanoni S, et al. Large-scale association analysis identifies new risk loci for coronary artery disease. Nat Genet. 2013;45(1):25–33. doi:10.1038/ng.2480. This study was the thus far largest meta-analysis of genome-wide association studies in coronary artery disease. Dichgans M, Malik R, König IR, et al. Shared genetic susceptibility to ischemic stroke and coronary artery disease: a genome-wide analysis of common variants. Stroke. 2014;45(1):24–36. doi:10. 1161/STROKEAHA.113.002707. This study investigated the shared genetics of coronary artery disease and stroke. Myocardial Infarction Genetics Consortium, Kathiresan S, Voight BF, et al. Genome-wide association of early-onset myocardial infarction with single nucleotide polymorphisms and copy number variants. Nat Genet. 2009;41(3):334–41. doi:10.1038/ng.327. The IBC 50K CAD Consortium. Large-scale gene-centric analysis identifies novel variants for coronary artery disease. Visscher PM, ed. PLoS Genet. 2011;7(9):e1002260. doi:10.1371/journal.pgen. 1002260.t001 Erdmann J, Grosshennig A, Braund PS, et al. New susceptibility locus for coronary artery disease on chromosome 3q22.3. Nat Genet. 2009;41(3):280–2. doi:10.1038/ng.307. Holliday EG, Maguire JM, Evans T-J, et al. Common variants at 6p21.1 are associated with large artery atherosclerotic stroke. Nat Genet. 2012;44(10):1147–51. doi:10.1038/ng.2397. Gudbjartsson DF, Arnar DO, Helgadottir A, et al. Variants conferring risk of atrial fibrillation on chromosome 4q25. Nature. 2007;448(7151):353–7. doi:10.1038/nature06007. Gretarsdottir S, Thorleifsson G, Manolescu A, et al. Risk variants for atrial fibrillation on chromosome 4q25 associate with ischemic stroke. Ann Neurol. 2008;64(4):402–9. doi:10.1002/ana.21480. Lemmens R, Buysschaert I, Geelen V, et al. The association of the 4q25 susceptibility variant for atrial fibrillation with stroke is limited to stroke of cardioembolic etiology. Stroke. 2010;41(9):1850–7. doi:10.1161/STROKEAHA.110.587980. Lubitz SA, Sinner MF, Lunetta KL, et al. Independent susceptibility markers for atrial fibrillation on chromosome 4q25. Circulation. 2010;122(10):976–84. doi:10.1161/CIRCULATIONAHA.109. 886440. Wang F, Xu C-Q, He Q, et al. Genome-wide association identifies a susceptibility locus for coronary artery disease in the Chinese Han population. Nat Genet. 2011;43(4):345–9. doi:10.1038/ng.783. Tregouet D-A, König IR, Erdmann J, et al. Genome-wide haplotype association study identifies the SLC22A3-LPAL2-LPA gene cluster

Page 9 of 10 14 as a risk locus for coronary artery disease. Nat Genet. 2009;41(3): 283–5. doi:10.1038/ng.314. 28. International Stroke Genetics Consortium ISGC, Wellcome Trust Case Control Consortium 2 (WTCCC2), Bellenguez C, et al. Genome-wide association study identifies a variant in HDAC9 associated with large vessel ischemic stroke. Nat Genet. 2012;44(3): 328–33. doi:10.1038/ng.1081. 29. Coronary Artery Disease (C4D) Genetics Consortium. A genomewide association study in Europeans and South Asians identifies five new loci for coronary artery disease. Nat Genet. 2011;43(4): 339–44. doi:10.1038/ng.782. 30. WTCC Consortium. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature. 2007;447(7145):661–78. doi:10.1038/nature05911. 31. Reilly MP, Li M, He J, et al. Identification of ADAMTS7 as a novel locus for coronary atherosclerosis and association of ABO with myocardial infarction in the presence of coronary atherosclerosis: two genome-wide association studies. Lancet. 2011;377(9763): 383–92. doi:10.1016/S0140-6736(10)61996-4. 32. Wu O, Bayoumi N, Vickers MA, Clark P. ABO(H) blood groups and vascular disease: a systematic review and meta-analysis. J Thromb Haemost. 2008;6(1):62–9. doi:10.1111/j.1538-7836.2007.02818.x. 33. Traylor M, Mäkelä K-M, Kilarski LL, et al. A novel MMP12 locus is associated with large artery atherosclerotic stroke using a genome-wide age-at-onset informed approach. PLoS Genet. 2014;10(7):e1004469. doi:10.1371/journal.pgen.1004469. 34. Gudbjartsson DF, Bjornsdottir US, Halapi E, et al. Sequence variants affecting eosinophil numbers associate with asthma and myocardial infarction. Nat Genet. 2009;41(3):342–7. doi:10.1038/ng.323. 35. Kilarski LL, Achterberg S, Devan WJ, et al. Meta-analysis in more than 17,900 cases of ischemic stroke reveals a novel association at 12q24.12. Neurology. 2014;83(8):678–85. doi:10.1212/WNL. 0000000000000707. 36. Rannikmäe K, Davies G, Thomson PA, et al. Common variation in COL4A1/COL4A2 is associated with sporadic cerebral small vessel disease. Neurology. 2015;(accepted for publication). 37. Gudbjartsson DF, Holm H, Gretarsdottir S, et al. A sequence variant in ZFHX3 on 16q22 associates with atrial fibrillation and ischemic stroke. Nat Genet. 2009;41(8):876–8. doi:10.1038/ng.417. 38. Ikram MA, Seshadri S, Bis JC, et al. Genomewide association studies of stroke. N Engl J Med. 2009;360(17):1718–28. doi:10.1056/ NEJMoa0900094. 39. Falcone GJ, Malik R, Dichgans M, Rosand J. Current concepts and clinical applications of stroke genetics. Lancet Neurol. 2014;13(4): 405–18. doi:10.1016/S1474-4422(14)70029-8. 40. Williams FMK, Carter AM, Hysi PG, et al. Ischemic stroke is associated with the ABO locus: the EuroCLOT study. Ann Neurol. 2013;73(1):16–31. doi:10.1002/ana.23838. 41. Nikpay M, Goel A, Won H-H, CARDIoGRAMplusC4D Consortium. Identification of novel CAD genetic loci by 1000 genomes-based imputation and a non-additive discovery screen. Circulation. 2014;130(22, Suppl 2):A16274. 42. Grove ML, Yu B, Cochran BJ, et al. Best practices and joint calling of the HumanExome BeadChip: the CHARGE Consortium. PLoS One. 2013;8(7):e68095. doi:10.1371/journal.pone.0068095. 43. Voight BF, Kang HM, Ding J, et al. The metabochip, a custom genotyping array for genetic studies of metabolic, cardiovascular, and anthropometric traits. PLoS Genet. 2012;8(8):e1002793. doi: 10.1371/journal.pgen.1002793. 44. Teslovich TM, Musunuru K, Smith AV, et al. Biological, clinical and population relevance of 95 loci for blood lipids. Nature. 2010;466(7307):707–13. doi:10.1038/nature09270. 45. Barrett JC, Clayton DG, Concannon P, et al. Genome-wide association study and meta-analysis find that over 40 loci affect risk of type 1 diabetes. Nat Genet. 2009;41(6):703–7. doi:10.1038/ng.381.

14 46.

47.

48.

49.

50.•

51.

52.•

53.

54.

55.

56.

57.

58.

59.

60.

Page 10 of 10 International Consortium for Blood Pressure Genome-Wide Association Studies, Ehret GB, Munroe PB, et al. Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk. Nature. 2011;478(7367):103–9. doi:10.1038/ nature10405. Wain LV, Verwoert GC, O’reilly PF, et al. Genome-wide association study identifies six new loci influencing pulse pressure and mean arterial pressure. Nat Genet. 2011;43(10):1005–11. doi:10.1038/ng.922. Soranzo N, Spector TD, Mangino M, et al. A genome-wide metaanalysis identifies 22 loci associated with eight hematological parameters in the HaemGen consortium. Nat Genet. 2009;41(11): 1182–90. doi:10.1038/ng.467. Takizawa H, Nishimura S, Takayama N, et al. Lnk regulates integrin αIIbβ3 outside-in signaling in mouse platelets, leading to stabilization of thrombus development in vivo. J Clin Invest. 2010;120(1):179–90. doi:10.1172/JCI39503DS1. Azghandi S, Prell C, van der Laan SW, et al. Deficiency of the stroke relevant HDAC9 gene attenuates atherosclerosis in accord with allele-specific effects at 7p21.1. Stroke. 2014. doi:10.1161/ STROKEAHA.114.007213. This study investigated the impact of Hdac9, a gene associated with both CAD and stroke, on atherosclerosis in mice with proatherogenic background. Kathiresan S, Melander O, Guiducci C, et al. Six new loci associated with blood low-density lipoprotein cholesterol, high-density lipoprotein cholesterol or triglycerides in humans. Nat Genet. 2008;40(2):189–97. doi:10.1038/ng.75. Musunuru K, Strong A, Frank-Kamenetsky M, et al. From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus. Nature. 2010;466(7307):714–9. doi:10.1038/nature09266. This study elucidated the mechanism involving frequent variants at the SORT1 locus in LDL cholesterol metabolism. Kjolby M, Andersen OM, Breiderhoff T, et al. Sort1, encoded by the cardiovascular risk locus 1p13.3, is a regulator of hepatic lipoprotein export. Cell Metab. 2010;12(3):213–23. doi:10.1016/j. cmet.2010.08.006. Diemert P, Heeren J, Aherrahrou Z, et al. Genetic variation at chromosome 1p13.3 affects sortilin mRNA expression, cellular LDLuptake and serum LDL levels which translates to the risk of coronary artery disease. Atherosclerosis. 2010;208(1):183–9. doi:10. 1016/j.atherosclerosis.2009.06.034. Markus HS, Mäkelä K-M, Bevan S, et al. Evidence HDAC9 genetic variant associated with ischemic stroke increases risk via promoting carotid atherosclerosis. Stroke. 2013;44(5):1220–5. doi:10. 1161/STROKEAHA.111.000217. Cao Q, Rong S, Repa JJ, St Clair R, Parks JS, Mishra N. Histone deacetylase 9 represses cholesterol efflux and alternatively activated macrophages in atherosclerosis development. Arterioscler Thromb Vasc Biol. 2014;34(9):1871–9. doi:10.1161/ATVBAHA.114.303393. Liu C-J, Kong W, Ilalov K, et al. ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein. FASEB J. 2006;20(7):988–90. doi:10.1096/fj.05-3877fje. Wang L, Zheng J, Bai X, et al. ADAMTS-7 mediates vascular smooth muscle cell migration and neointima formation in balloon-injured rat arteries. Circ Res. 2009;104(5):688–98. doi: 10.1161/CIRCRESAHA.108.188425. Wang L, Zheng J, Du Y, et al. Cartilage oligomeric matrix protein maintains the contractile phenotype of vascular smooth muscle cells by interacting with alpha(7)beta(1) integrin. Circ Res. 2010;106(3): 514–25. doi:10.1161/CIRCRESAHA.109.202762. Pu X, Xiao Q, Kiechl S, et al. ADAMTS7 cleavage and vascular smooth muscle cell migration is affected by a coronary-artery-

Curr Atheroscler Rep (2015) 17:14 disease-associated variant. Am J Hum Genet. 2013;92(3):366–74. doi:10.1016/j.ajhg.2013.01.012. 61.•• Kessler T, Zhang L, Liu Z, et al. ADAMTS-7 inhibits reendothelialization of injured arteries and promotes vascular remodeling via cleavage of thrombospondin-1. Circulation. 2015 in press. This study investigates the effect of Adamts7 deficiency in a mouse model showing that Adamts7 promotes neointima formation secondary to vascular injury due to retarded re-endothelialisation. 62. Erdmann J, Stark K, Esslinger UB, et al. Dysfunctional nitric oxide signalling increases risk of myocardial infarction. Nature. 2013;504(7480):432–6. doi:10.1038/nature12722. 63. Hanafy KA, Martin E, Murad F. CCTeta, a novel soluble guanylyl cyclase-interacting protein. J Biol Chem. 2004;279(45):46946–53. doi:10.1074/jbc.M404134200. 64. Myocardial Infarction Genetics Consortium Investigators, Stitziel NO, Won H-H, et al. Inactivating mutations in NPC1L1 and protection from coronary heart disease. N Engl J Med. 2014;371(22): 2072–82. doi:10.1056/NEJMoa1405386. 65. Do R, Stitziel NO, Won H-H, et al. Exome sequencing identifies rare LDLR and APOA5 alleles conferring risk for myocardial infarction. Nature. 2014. doi:10.1038/nature13917. 66. The TG and HDL Working Group of the Exome Sequencing Project, National Heart, Lung, and Blood Institute. Loss-offunction mutations in APOC3, triglycerides, and coronary disease. N Engl J Med. 2014;371(1):22–31. doi:10.1056/NEJMoa1307095. 67. Investigators II. IMPROVE-IT Trial: a comparison of ezetimibe/ simvastatin versus simvastatin monotherapy on cardiovascular outcomes after acute coronary syndromes. Circulation. 2014;130(22, Suppl2):2105–26. 68. Abifadel M, Varret M, Rabès J-P, et al. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet. 2003;34(2): 154–6. doi:10.1038/ng1161. 69. Brautbar A, Ballantyne CM. Pharmacological strategies for lowering LDL cholesterol: statins and beyond. Nat Rev Cardiol. 2011;8(5):253–65. doi:10.1038/nrcardio.2011.2. 70. Cameron J, Holla ØL, Ranheim T, Kulseth MA, Berge KE, Leren TP. Effect of mutations in the PCSK9 gene on the cell surface LDL receptors. Hum Mol Genet. 2006;15(9):1551–8. doi:10.1093/hmg/ddl077. 71. Stein EA, Giugliano RP, Koren MJ, et al. Efficacy and safety of evolocumab (AMG 145), a fully human monoclonal antibody to PCSK9, in hyperlipidaemic patients on various background lipid therapies: pooled analysis of 1359 patients in four phase 2 trials. Eur Heart J. 2014;35(33):2249–59. doi:10.1093/eurheartj/ehu085. 72. Koren MJ, Giugliano RP, Raal FJ, et al. Efficacy and safety of longer-term administration of evolocumab (AMG 145) in patients with hypercholesterolemia: 52-week results from the Open-Label Study of Long-Term Evaluation Against LDL-C (OSLER) randomized trial. Circulation. 2014;129(2):234–43. doi:10.1161/ CIRCULATIONAHA.113.007012. 73. Kessler T, Schunkert H. Clinical validation of genetic markers for improved risk estimation. Eur J Prev Cardiol. 2012;19(2 Suppl):25– 32. doi:10.1177/2047487312448993. 74. Hughes M, Saarela O, Stritzke J, et al. Genetic markers enhance coronary risk prediction in men: The MORGAM Prospective Cohorts. Schäfer A, ed. PLoS ONE. 2012;7(7):e40922. doi:10. 1371/journal.pone.0040922.t005. 75. Maurano MT, Humbert R, Rynes E, et al. Systematic localization of common disease-associated variation in regulatory DNA. Science. 2012;337(6099):1190–5. doi:10.1126/science.1222794.