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Lipoprotein (a) as a cause of cardiovascular disease: insights from epidemiology, genetics, and biology

Open AccessPublished:September 27, 2016DOI:https://doi.org/10.1194/jlr.R071233
      Human epidemiologic and genetic evidence using the Mendelian randomization approach in large-scale studies now strongly supports that elevated lipoprotein (a) [Lp(a)] is a causal risk factor for cardiovascular disease, that is, for myocardial infarction, atherosclerotic stenosis, and aortic valve stenosis. The Mendelian randomization approach used to infer causality is generally not affected by confounding and reverse causation, the major problems of observational epidemiology. This approach is particularly valuable to study causality of Lp(a), as single genetic variants exist that explain 27–28% of all variation in plasma Lp(a). The most important genetic variant likely is the kringle IV type 2 (KIV-2) copy number variant, as the apo(a) product of this variant influences fibrinolysis and thereby thrombosis, as opposed to the Lp(a) particle per se. We speculate that the physiological role of KIV-2 in Lp(a) could be through wound healing during childbirth, infections, and injury, a role that, in addition, could lead to more blood clots promoting stenosis of arteries and the aortic valve, and myocardial infarction. Randomized placebo-controlled trials of Lp(a) reduction in individuals with very high concentrations to reduce cardiovascular disease are awaited. Recent genetic evidence documents elevated Lp(a) as a cause of myocardial infarction, atherosclerotic stenosis, and aortic valve stenosis.
      The first articles on lipoprotein (a) [Lp(a)] were published in 1963 by Kåre Berg from Norway, describing Lp(a) in human plasma as a heritable trait (
      • Berg K.
      A new serum type system in man–the LP system.
      ,
      • Berg K.
      • Mohr J.
      Genetics of the LP system.
      ,
      • Mohr J.
      • Berg K.
      Genetics of the LP serum types: associations and linkage relations.
      ). Over the next more than 20 years, the scientific interest in this lipoprotein was modest. Then in 1987, Richard Lawn, Angelo Scanu, and colleagues cloned and sequenced the LPA gene coding for apo(a) (
      • McLean J.W.
      • Tomlinson J.E.
      • Kuang W.J.
      • Eaton D.L.
      • Chen E.Y.
      • Fless G.M.
      • Scanu A.M.
      • Lawn R.M.
      cDNA sequence of human apolipoprotein(a) is homologous to plasminogen.
      ,
      • Lawn R.M.
      Lipoprotein(a) in heart disease.
      ), a protein with homology to plasminogen, which accounts for the difference between an Lp(a) particle and an LDL particle. These observations generated a huge scientific interest in Lp(a), leading to an exponential growth in the number of articles published, which then later declined again.
      Renewed interested in Lp(a) came in 2009 from Pia Kamstrup et al. (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Steffensen R.
      • Nordestgaard B.G.
      Genetically elevated lipoprotein(a) and increased risk of myocardial infarction.
      ) with genetic evidence from Mendelian randomization that high Lp(a) is causally associated with cardiovascular disease, from the Emerging Risk Factors Collaboration led by John Danesh that Lp(a) epidemiologically is continuously and independently associated with cardiovascular disease risk (
      • Erqou S.
      • Kaptoge S.
      • Perry P.L.
      • Di A.E.
      • Thompson A.
      • White I.R.
      • Marcovina S.M.
      • Collins R.
      • Thompson S.G.
      • Danesh J.
      Lipoprotein(a) concentration and the risk of coronary heart disease, stroke, and nonvascular mortality.
      ), and from Robert Clarke et al. (
      • Clarke R.
      • Peden J.F.
      • Hopewell J.C.
      • Kyriakou T.
      • Goel A.
      • Heath S.C.
      • Parish S.
      • Barlera S.
      • Franzosi M.G.
      • Rust S.
      • et al.
      Genetic variants associated with Lp(a) lipoprotein level and coronary disease.
      ) that of 2,100 candidate genes for cardiovascular disease, genetic variation in the LPA gene was the strongest genetic cardiovascular risk factor. Following the publication of these studies, high concentrations of Lp(a) have been considered to be a direct cause of cardiovascular disease, just like high LDL cholesterol concentrations. Genetic evidence largely free of confounding and fully free of reverse causation, two major problems of observational epidemiology, is what has cemented the understanding of causality of Lp(a) for cardiovascular disease.
      These novel and important findings led the European Atherosclerosis Society to publish a 2010 consensus panel statement recommending screening for elevated Lp(a) in individuals at intermediate, high, or very high cardiovascular risk, and suggested a desirable plasma concentration of less than 50 mg/dl (
      • Nordestgaard B.G.
      • Chapman M.J.
      • Ray K.
      • Boren J.
      • Andreotti F.
      • Watts G.F.
      • Ginsberg H.
      • Amarenco P.
      • Catapano A.
      • Descamps O.S.
      • et al.
      Lipoprotein(a) as a cardiovascular risk factor: current status.
      ). Of note, we naturally were fully aware that the risk of cardiovascular disease increases already at Lp(a) levels above 30 mg/dl, but to get the attention of the clinical community, we believed it was the best strategy to start focusing on individuals with the highest cardiovascular risk, that is, the 20% of individuals with concentrations above 50 mg/dl.
      Although some cardiologists suspected for many years, as far back as the mid-1990s, that Lp(a) was a risk factor for aortic valve stenosis, a further novel development was the documentation by George Thanassoulis et al. (
      • Thanassoulis G.
      • Campbell C.Y.
      • Owens D.S.
      • Smith J.G.
      • Smith A.V.
      • Peloso G.M.
      • Kerr K.F.
      • Pechlivanis S.
      • Budoff M.J.
      • Harris T.B.
      • et al.
      Genetic associations with valvular calcification and aortic stenosis.
      ) in 2013 that genetic variation in the LPA gene is strongly associated with aortic valve calcification and stenosis. It was later shown that high plasma Lp(a) concentrations are likewise causally associated with high risk of aortic valve stenosis (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Elevated lipoprotein(a) and risk of aortic valve stenosis in the general population.
      ). Taken together therefore, the genetic evidence now firmly demonstrates that high plasma Lp(a) concentration is a direct cause of cardiovascular disease, that is, myocardial infarction, atherosclerotic stenosis, and aortic valve stenosis (Fig. 1).
      Figure thumbnail gr1
      Fig. 1Summary of the strongest causal genetic evidence linking high Lp(a) concentrations with corresponding small apo(a) size due to low number of KIV-2 repeats to risk of disease.
      Although mainly recent genetic research has firmly established the claim for causality for high Lp(a) concentrations to cardiovascular disease, these findings have only been possible due to the many other excellent scientific works on Lp(a) published from 1963 until today, produced by a large number of dedicated researchers within this field. In this review, it will not be possible to do justice to all the important scientific discoveries on Lp(a) in relation to cardiovascular disease published during the 50 years. That said, with our own personal touch to the story and with focus on evidence from human studies, we will try our best, in a historical perspective, to cover insights in epidemiology, genetics, and biology that led to the understanding of Lp(a) as a cause of cardiovascular disease. Many classic and more recent reviews, viewpoints, and meta-analyses cover related areas (
      • Lawn R.M.
      Lipoprotein(a) in heart disease.
      ,
      • Erqou S.
      • Kaptoge S.
      • Perry P.L.
      • Di A.E.
      • Thompson A.
      • White I.R.
      • Marcovina S.M.
      • Collins R.
      • Thompson S.G.
      • Danesh J.
      Lipoprotein(a) concentration and the risk of coronary heart disease, stroke, and nonvascular mortality.
      ,
      • Nordestgaard B.G.
      • Chapman M.J.
      • Ray K.
      • Boren J.
      • Andreotti F.
      • Watts G.F.
      • Ginsberg H.
      • Amarenco P.
      • Catapano A.
      • Descamps O.S.
      • et al.
      Lipoprotein(a) as a cardiovascular risk factor: current status.
      ,
      • Brown M.S.
      • Goldstein J.L.
      Plasma lipoproteins: teaching old dogmas new tricks.
      ,
      • Utermann G.
      The mysteries of lipoprotein(a).
      ,
      • Utermann G.
      Lipoprotein(a).
      ,
      • Scanu A.M.
      • Fless G.M.
      Lipoprotein (a). Heterogeneity and biological relevance.
      ,
      • Scanu A.M.
      ,
      • Dahlén G.H.
      Lp(a) lipoprotein in cardiovascular disease.
      ,
      • Rader D.J.
      • Hoeg J.M.
      • Brewer Jr, H.B.
      Quantitation of plasma apolipoproteins in the primary and secondary prevention of coronary artery disease.
      ,
      • Howard G.C.
      • Pizzo S.V.
      Lipoprotein(a) and its role in atherothrombotic disease.
      ,
      • MBewu A.D.
      • Durrington P.N.
      Lipoprotein (a): structure, properties and possible involvement in thrombogenesis and atherogenesis.
      ,
      • Nielsen L.B.
      Atherogenecity of lipoprotein(a) and oxidized low density lipoprotein: insight from in vivo studies of arterial wall influx, degradation and efflux.
      ,
      • Kamstrup P.R.
      Lipoprotein(a) and ischemic heart disease-A causal association? A review.
      ,
      • Danesh J.
      • Collins R.
      • Peto R.
      Lipoprotein(a) and coronary heart disease. Meta-analysis of prospective studies.
      ,
      • Erqou S.
      • Thompson A.
      • Di A.E.
      • Saleheen D.
      • Kaptoge S.
      • Marcovina S.
      • Danesh J.
      Apolipoprotein(a) isoforms and the risk of vascular disease: systematic review of 40 studies involving 58,000 participants.
      ,
      • Nordestgaard B.G.
      • Tybjaerg-Hansen A.
      Genetic determinants of LDL, lipoprotein(a), triglyceride-rich lipoproteins and HDL: concordance and discordance with cardiovascular disease risk.
      ,
      • Dubé J.B.
      • Boffa M.B.
      • Hegele R.A.
      • Koschinsky M.L.
      Lipoprotein(a): more interesting than ever after 50 years.
      ,
      • Kronenberg F.
      • Utermann G.
      Lipoprotein(a): resurrected by genetics.
      ,
      • Kostner K.M.
      • Marz W.
      • Kostner G.M.
      When should we measure lipoprotein (a)?.
      ,
      • Jacobson T.A.
      Lipoprotein(a), cardiovascular disease, and contemporary management.
      ,
      • Jansen H.
      • Samani N.J.
      • Schunkert H.
      Mendelian randomization studies in coronary artery disease.
      ,
      • Nave A.H.
      • Lange K.S.
      • Leonards C.O.
      • Siegerink B.
      • Doehner W.
      • Landmesser U.
      • Steinhagen-Thiessen E.
      • Endres M.
      • Ebinger M.
      Lipoprotein (a) as a risk factor for ischemic stroke: a meta-analysis.
      ,
      • Orho-Melander M.
      Genetics of coronary heart disease: towards causal mechanisms, novel drug targets and more personalized prevention.
      ,
      • Witztum J.L.
      • Ginsberg H.N.
      Lipoprotein (a): coming of age at last.
      ,
      • Boffa M.B.
      • Koschinsky M.L.
      Lipoprotein (a): truly a direct prothrombotic factor in cardiovascular disease?.
      ,
      • Thanassoulis G.
      Lipoprotein (a) in calcific aortic valve disease: from genomics to novel drug target for aortic stenosis.
      ,
      • Tsimikas S.
      Lipoprotein(a): novel target and emergence of novel therapies to lower cardiovascular disease risk.
      ,
      • Stender S.
      • Tybjaerg-Hansen A.
      Using human genetics to predict the effects and side-effects of drugs.
      ,
      • Musunuru K.
      • Kathiresan S.
      Surprises from genetic analyses of lipid risk factors for atherosclerosis.
      ,
      • Schmidt K.
      • Noureen A.
      • Kronenberg F.
      • Utermann G.
      Structure, function, and genetics of lipoprotein(a).
      ,
      • Kronenberg F.
      Human genetics and the causal role of lipoprotein(a) for various diseases.
      ), include even more references, and, together with the present review, provide a comprehensive coverage of Lp(a) as a cause of cardiovascular disease.

      Lp(a) IN NORMAL, ATHEROSCLEROTIC, AND INJURED INTIMA

      A necessary condition for Lp(a) to cause cardiovascular disease is its ability to enter into and accumulate in the intima of arteries and aortic valve leaflets. Lp(a) or apo(a) is indeed found within the lesioned intima of human arteries and coronary artery bypass vein grafts removed at reoperation (
      • Rath M.
      • Niendorf A.
      • Reblin T.
      • Dietel M.
      • Krebber H.J.
      • Beisiegel U.
      Detection and quantification of lipoprotein(a) in the arterial wall of 107 coronary bypass patients.
      ,
      • Niendorf A.
      • Rath M.
      • Wolf K.
      • Peters S.
      • Arps H.
      • Beisiegel U.
      • Dietel M.
      Morphological detection and quantification of lipoprotein(a) deposition in atheromatous lesions of human aorta and coronary arteries.
      ,
      • Cushing G.L.
      • Gaubatz J.W.
      • Nava M.L.
      • Burdick B.J.
      • Bocan T.M.
      • Guyton J.R.
      • Weilbaecher D.
      • DeBakey M.E.
      • Lawrie G.M.
      • Morrisett J.D.
      Quantitation and localization of apolipoproteins [a] and B in coronary artery bypass vein grafts resected at re-operation.
      ,
      • Smith E.B.
      • Cochran S.
      Factors influencing the accumulation in fibrous plaques of lipid derived from low density lipoprotein. II. Preferential immobilization of lipoprotein (a) (Lp(a)).
      ,
      • Hoff H.F.
      • O'Neil J.
      • Yashiro A.
      Partial characterization of lipoproteins containing apo[a] in human atherosclerotic lesions.
      ,
      • Reblin T.
      • Meyer N.
      • Labeur C.
      • Henne-Bruns D.
      • Beisiegel U.
      Extraction of lipoprotein(a), apo B, and apo E from fresh human arterial wall and atherosclerotic plaques.
      ), as well as within the lesioned intima of monkey arteries (
      • Nachman R.L.
      • Gavish D.
      • Azrolan N.
      • Clarkson T.B.
      Lipoprotein(a) in diet-induced atherosclerosis in nonhuman primates.
      ) and arteries in rabbits and mice after intravenous injection of human Lp(a) (
      • Kreuzer J.
      • Lloyd M.B.
      • Bok D.
      • Fless G.M.
      • Scanu A.M.
      • Lusis A.J.
      • Haberland M.E.
      Lipoprotein (a) displays increased accumulation compared with low-density lipoprotein in the murine arterial wall.
      ,
      • Nielsen L.B.
      • Nordestgaard B.G.
      • Stender S.
      • Niendorf A.
      • Kjeldsen K.
      Transfer of lipoprotein(a) and LDL into aortic intima in normal and in cholesterol-fed rabbits.
      ) or after transgenic modification with human Lp(a) (
      • Yeang C.
      • Cotter B.
      • Tsimikas S.
      Experimental animal models evaluating the causal role of lipoprotein(a) in atherosclerosis and aortic stenosis.
      ). Likewise, apo(a) is found within early to end-stage lesions of human aortic valve stenosis (
      • O'Brien K.D.
      • Reichenbach D.D.
      • Marcovina S.M.
      • Kuusisto J.
      • Alpers C.E.
      • Otto C.M.
      Apolipoproteins B, (a), and E accumulate in the morphologically early lesion of ‘degenerative’ valvular aortic stenosis.
      ).
      In vivo kinetic studies show that radiolabeled human Lp(a) enters the intima at similar rates to LDL in normal and atherosclerotic arteries in humans and rabbits (
      • Nielsen L.B.
      • Nordestgaard B.G.
      • Stender S.
      • Niendorf A.
      • Kjeldsen K.
      Transfer of lipoprotein(a) and LDL into aortic intima in normal and in cholesterol-fed rabbits.
      ,
      • Nielsen L.B.
      • Gronholdt M.L.
      • Schroeder T.V.
      • Stender S.
      • Nordestgaard B.G.
      In vivo transfer of lipoprotein(a) into human atherosclerotic carotid arterial intima.
      ,
      • Nielsen L.B.
      • Stender S.
      • Jauhiainen M.
      • Nordestgaard B.G.
      Preferential influx and decreased fractional loss of lipoprotein(a) in atherosclerotic compared with nonlesioned rabbit aorta.
      ), which appears to be like other lipoproteins, through a simple molecular sieving not involving any receptors, but dependent on lipoprotein plasma concentrations and on lipoprotein particle size, blood pressure, and on arterial wall permeability (
      • Nordestgaard B.G.
      • Nielsen L.B.
      Atherosclerosis and arterial influx of lipoproteins.
      ). Importantly, however, as the plasma concentration of LDL is much higher than that of Lp(a) in most individuals, the mass intimal influx of LDL was 15-fold that of Lp(a) in humans (
      • Nielsen L.B.
      • Gronholdt M.L.
      • Schroeder T.V.
      • Stender S.
      • Nordestgaard B.G.
      In vivo transfer of lipoprotein(a) into human atherosclerotic carotid arterial intima.
      ). Again using kinetic studies in vivo, the rates of relative removal of Lp(a) and LDL from the arterial intima were similar, while the trapping of both lipoproteins was higher in lesioned compared with normal intima (
      • Nielsen L.B.
      • Stender S.
      • Jauhiainen M.
      • Nordestgaard B.G.
      Preferential influx and decreased fractional loss of lipoprotein(a) in atherosclerotic compared with nonlesioned rabbit aorta.
      ); it cannot entirely be excluded that there may be preferential trapping of Lp(a) in settings where lesion formation is ongoing and/or more advanced. Also, degradation in vivo of human Lp(a) was higher in atherosclerotic compared with nonlesioned rabbit intima (
      • Nielsen L.B.
      • Juul K.
      • Nordestgaard B.G.
      Increased degradation of lipoprotein(a) in atherosclerotic compared with nonlesioned aortic intima-inner media of rabbits: in vivo evidence that lipoprotein(a) may contribute to foam cell formation.
      ).
      Surprisingly, when the arterial intima in a rabbit model was subjected to a balloon injury, radiolabeled human Lp(a) accumulated in vivo 2- to 3-fold greater than that of radiolabeled human LDL in the balloon-injured aortic intima, but not in the adjacent uninjured arterial intima (
      • Nielsen L.B.
      • Stender S.
      • Kjeldsen K.
      • Nordestgaard B.G.
      Specific accumulation of lipoprotein(a) in balloon-injured rabbit aorta in vivo.
      ). As removal of intact endothelial cells would not explain why Lp(a) preferentially accumulates, because entry into the intima would be enhanced for LDL and Lp(a) equally, this can most likely occur because of prolonged residence time, e.g., enhanced binding of Lp(a) selectively to the matrix in the intima. A simple explanation for this observation could be that the balloon-injury removes the endothelial cells and thus exposes the intima directly to flowing blood, favoring deposition of fibrin. Thereby Lp(a), unlike LDL, can bind to small blood clots rapidly forming at the injured sites, or to exposed glycosaminoglycans (
      • Nielsen L.B.
      Atherogenecity of lipoprotein(a) and oxidized low density lipoprotein: insight from in vivo studies of arterial wall influx, degradation and efflux.
      ,
      • Nielsen L.B.
      • Stender S.
      • Kjeldsen K.
      • Nordestgaard B.G.
      Specific accumulation of lipoprotein(a) in balloon-injured rabbit aorta in vivo.
      ,
      • Nordestgaard B.G.
      The vascular endothelial barrier–selective retention of lipoproteins.
      ). Preferential accumulation of Lp(a) compared with LDL at injured sites could thus be related to the greater capacity of Lp(a) than LDL to bind to fibrin (
      • Fless G.M.
      • Snyder M.L.
      Polymorphic forms of Lp(a) with different structural and functional properties: cold-induced self-association and binding to fibrin and lysine-Sepharose.
      ) or glycosaminoglycans (
      • Bihari-Varga M.
      • Gruber E.
      • Rotheneder M.
      • Zechner R.
      • Kostner G.M.
      Interaction of lipoprotein Lp(a) and low density lipoprotein with glycosaminoglycans from human aorta.
      ). Indeed, Lp(a) compared with LDL appeared to be preferentially immobilized via fibrin binding in human arterial tissue (
      • Smith E.B.
      • Cochran S.
      Factors influencing the accumulation in fibrous plaques of lipid derived from low density lipoprotein. II. Preferential immobilization of lipoprotein (a) (Lp(a)).
      ).
      Taken together, these data suggest that Lp(a) accumulation at sites of injury could be a primary mechanism by which elevated Lp(a) causes cardiovascular disease. That said, there is also evidence that Lp(a) can be taken up by macrophages to produce foam cells (
      • Bottalico L.A.
      • Keesler G.A.
      • Fless G.M.
      • Tabas I.
      Cholesterol loading of macrophages leads to marked enhancement of native lipoprotein(a) and apoprotein(a) internalization and degradation.
      ,
      • Skiba P.J.
      • Keesler G.A.
      • Tabas I.
      Interferon-gamma down-regulates the lipoprotein(a)/apoprotein(a) receptor activity on macrophage foam cells. Evidence for disruption of ligand-induced receptor recycling by interferon-gamma.
      ,
      • Keesler G.A.
      • Li Y.
      • Skiba P.J.
      • Fless G.M.
      • Tabas I.
      Macrophage foam cell lipoprotein(a)/apoprotein(a) receptor. Cell-surface localization, dependence of induction on new protein synthesis, and ligand specificity.
      ), a mechanism by which LDL and triglyceride-rich lipoproteins are believed to cause the development of atherosclerosis (
      • Stary H.C.
      • Chandler A.B.
      • Glagov S.
      • Guyton J.R.
      • Insull Jr., W.
      • Rosenfeld M.E.
      • Schaffer S.A.
      • Schwartz C.J.
      • Wagner W.D.
      • Wissler R.W.
      A definition of initial, fatty streak, and intermediate lesions of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association.
      ,
      • Nordestgaard B.G.
      • Varbo A.
      Triglycerides and cardiovascular disease.
      ,
      • Nordestgaard B.G.
      Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: new insights from epidemiology, genetics, and biology.
      ). Interestingly, in coronary artery bypass vein grafts and relative to plasma concentrations, Lp(a) accumulated 2.4-fold more than all apoB-containing lipoproteins combined, that is, LDL, Lp(a), and triglyceride-rich lipoproteins, and unlike apoB that was found mainly at atherosclerotic core regions, this was not the case for Lp(a) (
      • Cushing G.L.
      • Gaubatz J.W.
      • Nava M.L.
      • Burdick B.J.
      • Bocan T.M.
      • Guyton J.R.
      • Weilbaecher D.
      • DeBakey M.E.
      • Lawrie G.M.
      • Morrisett J.D.
      Quantitation and localization of apolipoproteins [a] and B in coronary artery bypass vein grafts resected at re-operation.
      ). The latter suggests that the intimal accumulation of Lp(a) is by a different mechanism than that of other apoB-containing lipoproteins, and that Lp(a) accumulation is throughout the intima, while other apoB-containing lipoproteins mainly are found at atherosclerotic lesions.

      PLASMA CONCENTRATIONS AND GENETICS

      The fact that plasma concentrations of Lp(a) are mainly genetically determined (
      • Utermann G.
      Lipoprotein(a).
      ,
      • Schmidt K.
      • Noureen A.
      • Kronenberg F.
      • Utermann G.
      Structure, function, and genetics of lipoprotein(a).
      ) has been instrumental in the study of causality of Lp(a) for cardiovascular disease. Because of this, the genetic evidence for causality in cardiovascular disease is much stronger for Lp(a) than for most other cardiovascular risk factors (
      • Clarke R.
      • Peden J.F.
      • Hopewell J.C.
      • Kyriakou T.
      • Goel A.
      • Heath S.C.
      • Parish S.
      • Barlera S.
      • Franzosi M.G.
      • Rust S.
      • et al.
      Genetic variants associated with Lp(a) lipoprotein level and coronary disease.
      ,
      • Jansen H.
      • Samani N.J.
      • Schunkert H.
      Mendelian randomization studies in coronary artery disease.
      ,
      • Schunkert H.
      • Konig I.R.
      • Kathiresan S.
      • Reilly M.P.
      • Assimes T.L.
      • Holm H.
      • Preuss M.
      • Stewart A.F.
      • Barbalic M.
      • Gieger C.
      • et al.
      Large-scale association analysis identifies 13 new susceptibility loci for coronary artery disease.
      ). It is also of huge importance for Mendelian randomization studies that single genetic variants exist that explain 27–28% of all variation in plasma Lp(a), and that these variants can be genotyped in large-scale studies (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Steffensen R.
      • Nordestgaard B.G.
      Genetically elevated lipoprotein(a) and increased risk of myocardial infarction.
      ,
      • Clarke R.
      • Peden J.F.
      • Hopewell J.C.
      • Kyriakou T.
      • Goel A.
      • Heath S.C.
      • Parish S.
      • Barlera S.
      • Franzosi M.G.
      • Rust S.
      • et al.
      Genetic variants associated with Lp(a) lipoprotein level and coronary disease.
      ,
      • Thanassoulis G.
      • Campbell C.Y.
      • Owens D.S.
      • Smith J.G.
      • Smith A.V.
      • Peloso G.M.
      • Kerr K.F.
      • Pechlivanis S.
      • Budoff M.J.
      • Harris T.B.
      • et al.
      Genetic associations with valvular calcification and aortic stenosis.
      ,
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Elevated lipoprotein(a) and risk of aortic valve stenosis in the general population.
      ,
      • Kamstrup P.R.
      • Nordestgaard B.G.
      Lipoprotein(a) concentrations, isoform size, and risk of type 2 diabetes: a Mendelian randomisation study.
      ). Therefore, a brief discussion of plasma Lp(a) concentrations and the genetic principles used in Mendelian randomization studies is given below. An in depth review of this topic is presented elsewhere in this Thematic Review Series (
      • Schmidt K.
      • Noureen A.
      • Kronenberg F.
      • Utermann G.
      Structure, function, and genetics of lipoprotein(a).
      ,
      • Enkhmaa B.
      • Anuurad E.
      • Berglund L.
      Lipoprotein (a): impact by ethnicity and environmental and medical conditions.
      ).
      In the general population, plasma concentrations of Lp(a) vary to a great extent among individuals (
      • Utermann G.
      Lipoprotein(a).
      ). Concentrations also differ between different ethnicities, with higher concentrations in individuals of African compared with European and Asian descent (
      • Utermann G.
      Lipoprotein(a).
      ,
      • Enkhmaa B.
      • Anuurad E.
      • Berglund L.
      Lipoprotein (a): impact by ethnicity and environmental and medical conditions.
      ,
      • Sandholzer C.
      • Hallman D.M.
      • Saha N.
      • Sigurdsson G.
      • Lackner C.
      • Csaszar A.
      • Boerwinkle E.
      • Utermann G.
      Effects of the apolipoprotein(a) size polymorphism on the lipoprotein(a) concentration in 7 ethnic groups.
      ,
      • Kraft H.G.
      • Lingenhel A.
      • Pang R.W.
      • Delport R.
      • Trommsdorff M.
      • Vermaak H.
      • Janus E.D.
      • Utermann G.
      Frequency distributions of apolipoprotein(a) kringle IV repeat alleles and their effects on lipoprotein(a) levels in Caucasian, Asian, and African populations: the distribution of null alleles is non-random.
      ,
      • Gaw A.
      • Boerwinkle E.
      • Cohen J.C.
      • Hobbs H.H.
      Comparative analysis of the apo(a) gene, apo(a) glycoprotein, and plasma concentrations of Lp(a) in three ethnic groups. Evidence for no common “null” allele at the apo(a) locus.
      ,
      • Matthews K.A.
      • Sowers M.F.
      • Derby C.A.
      • Stein E.
      • Miracle-McMahill H.
      • Crawford S.L.
      • Pasternak R.C.
      Ethnic differences in cardiovascular risk factor burden among middle-aged women: Study of Women's Health Across the Nation (SWAN).
      ,
      • Marcovina S.M.
      • Albers J.J.
      • Wijsman E.
      • Zhang Z.
      • Chapman N.H.
      • Kennedy H.
      Differences in Lp[a] concentrations and apo[a] polymorphs between black and white Americans.
      ,
      • Helmhold M.
      • Bigge J.
      • Muche R.
      • Mainoo J.
      • Thiery J.
      • Seidel D.
      • Armstrong V.W.
      Contribution of the apo[a] phenotype to plasma Lp[a] concentrations shows considerable ethnic variation.
      ). In Europeans and Asians, Lp(a) concentrations are highly skewed with a tail toward higher concentrations (Fig. 2, top panel), and in the Copenhagen General Population Study, we observed concentrations as high as 387 mg/dl. Concentrations of other lipoproteins are often affected by life-style and physiological factors, whereas Lp(a) concentrations remain stable inter-individually throughout life, indirectly suggesting that Lp(a) concentrations are mostly genetically determined.
      Figure thumbnail gr2
      Fig. 2Distribution of and correlation between plasma Lp(a) concentrations and KIV-2 number of repeats in the Danish general population.
      In very early studies, Lp(a) was suggested to be an inherited trait with autosomal dominant inheritance (
      • Berg K.
      • Mohr J.
      Genetics of the LP system.
      ,
      • Mohr J.
      • Berg K.
      Genetics of the LP serum types: associations and linkage relations.
      ,
      • Schultz J.S.
      • Schreffler D.C.
      • Sing C.F.
      • Harvie N.R.
      The genetics of the Lp antigen. I. Its quantitation and distribution in a sample population.
      ,
      • Sing C.F.
      • Schultz J.S.
      • Shreffler D.C.
      The genetics of the Lp antigen. II. A family study and proposed models of genetic control.
      ,
      • Iselius L.
      • Dahlen G.
      • de Faire U.
      • Lundman T.
      Complex segregation analysis of the Lp(a)/pre-beta 1-lipoprotein trait.
      ) and a major gene and polygenic factors were proposed as contributors to the variation in Lp(a) concentrations (
      • Hasstedt S.J.
      • Wilson D.E.
      • Edwards C.Q.
      • Cannon W.N.
      • Carmelli D.
      • Williams R.R.
      The genetics of quantitative plasma Lp(a): analysis of a large pedigree.
      ,
      • Morton N.E.
      • Berg K.
      • Dahlen G.
      • Ferrell R.E.
      • Rhoads G.G.
      Genetics of the Lp lipoprotein in Japanese-Americans.
      ,
      • Hasstedt S.J.
      • Williams R.R.
      Three alleles for quantitative Lp(a).
      ). Hasstedt et al. (
      • Hasstedt S.J.
      • Wilson D.E.
      • Edwards C.Q.
      • Cannon W.N.
      • Carmelli D.
      • Williams R.R.
      The genetics of quantitative plasma Lp(a): analysis of a large pedigree.
      ) found that the major gene and the polygenic factors accounted for 95% of the variation in plasma Lp(a) concentrations. Following this, a number of studies primarily led by Gerd Utermann and colleagues revealed that apo(a) varied in size due to kringle IV type 2 (KIV-2) copy number variation, and that the number of KIV-2 copies were inversely correlated to plasma Lp(a) concentrations (
      • Fless G.M.
      • ZumMallen M.E.
      • Scanu A.M.
      Physicochemical properties of apolipoprotein(a) and lipoprotein(a-) derived from the dissociation of human plasma lipoprotein (a).
      ,
      • Utermann G.
      • Menzel H.J.
      • Kraft H.G.
      • Duba H.C.
      • Kemmler H.G.
      • Seitz C.
      Lp(a) glycoprotein phenotypes. Inheritance and relation to Lp(a)-lipoprotein concentrations in plasma.
      ,
      • Grinstead G.F.
      • Ellefson R.D.
      Heterogeneity of lipoprotein Lp(a) and apolipoprotein(a).
      ,
      • Utermann G.
      • Duba C.
      • Menzel H.J.
      Genetics of the quantitative Lp(a) lipoprotein trait. II. Inheritance of Lp(a) glycoprotein phenotypes.
      ,
      • Utermann G.
      • Kraft H.G.
      • Menzel H.J.
      • Hopferwieser T.
      • Seitz C.
      Genetics of the quantitative Lp(a) lipoprotein trait. I. Relation of LP(a) glycoprotein phenotypes to Lp(a) lipoprotein concentrations in plasma.
      ,
      • Boerwinkle E.
      • Menzel H.J.
      • Kraft H.G.
      • Utermann G.
      Genetics of the quantitative Lp(a) lipoprotein trait. III. Contribution of Lp(a) glycoprotein phenotypes to normal lipid variation.
      ,
      • Kraft H.G.
      • Sandholzer C.
      • Menzel H.J.
      • Utermann G.
      Apolipoprotein (a) alleles determine lipoprotein (a) particle density and concentration in plasma.
      ) (Fig. 2, middle and lower panel). However, the correlation between the apo(a) size polymorphism and Lp(a) plasma concentrations was found to vary greatly among individuals of different ethnicity, as do plasma Lp(a) concentrations (
      • Sandholzer C.
      • Hallman D.M.
      • Saha N.
      • Sigurdsson G.
      • Lackner C.
      • Csaszar A.
      • Boerwinkle E.
      • Utermann G.
      Effects of the apolipoprotein(a) size polymorphism on the lipoprotein(a) concentration in 7 ethnic groups.
      ,
      • Kraft H.G.
      • Lingenhel A.
      • Pang R.W.
      • Delport R.
      • Trommsdorff M.
      • Vermaak H.
      • Janus E.D.
      • Utermann G.
      Frequency distributions of apolipoprotein(a) kringle IV repeat alleles and their effects on lipoprotein(a) levels in Caucasian, Asian, and African populations: the distribution of null alleles is non-random.
      ,
      • Marcovina S.M.
      • Albers J.J.
      • Wijsman E.
      • Zhang Z.
      • Chapman N.H.
      • Kennedy H.
      Differences in Lp[a] concentrations and apo[a] polymorphs between black and white Americans.
      ,
      • Helmhold M.
      • Bigge J.
      • Muche R.
      • Mainoo J.
      • Thiery J.
      • Seidel D.
      • Armstrong V.W.
      Contribution of the apo[a] phenotype to plasma Lp[a] concentrations shows considerable ethnic variation.
      ,
      • Hallman D.M.
      • Boerwinkle E.
      • Saha N.
      • Sandholzer C.
      • Menzel H.J.
      • Csazar A.
      • Utermann G.
      The apolipoprotein E polymorphism: a comparison of allele frequencies and effects in nine populations.
      ). One study found that the apo(a) allele frequencies were different among different populations (
      • Sandholzer C.
      • Hallman D.M.
      • Saha N.
      • Sigurdsson G.
      • Lackner C.
      • Csaszar A.
      • Boerwinkle E.
      • Utermann G.
      Effects of the apolipoprotein(a) size polymorphism on the lipoprotein(a) concentration in 7 ethnic groups.
      ). In that study, the size variation of apo(a) explained from 19% in Sudanese to 77% in Malays of the variability in plasma Lp(a) concentrations.
      In 49,615 individuals from the Copenhagen General Population Study, the KIV-2 number of repeats in the LPA gene coding for the apo(a) size polymorphism explained 27% of the variation in plasma Lp(a) concentrations (Fig. 2, lower panel); however, the KIV-2 number of repeats was measured as both alleles combined and does not take different expression of alleles into account, suggesting that the 27% should be viewed as a minimal estimate for Danes. Therefore, the measurements of KIV-2 in these studies do not necessarily reflect the dominant isoform that is associated with the highest Lp(a) concentrations. For Mendelian randomization studies, the most important genetic variant is likely the KIV-2 copy number variation, or corresponding apo(a) size polymorphism, as this causal variant possibly is the direct cause of cardiovascular disease (
      • Schmidt K.
      • Noureen A.
      • Kronenberg F.
      • Utermann G.
      Structure, function, and genetics of lipoprotein(a).
      ). However, although genotyping of this variant can be done in large-scale-studies, it requires, at present, quantitative polymerase chain reaction (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Steffensen R.
      • Nordestgaard B.G.
      Genetically elevated lipoprotein(a) and increased risk of myocardial infarction.
      ) or even more complicated techniques (
      • Schmidt K.
      • Noureen A.
      • Kronenberg F.
      • Utermann G.
      Structure, function, and genetics of lipoprotein(a).
      ). Importantly, for now, it is not possible to determine for sure whether it is the KIV-2 copy number variation or the associated plasma Lp(a) concentration that is causally associated with cardiovascular disease.
      In 2009, Robert Clarke et al. (
      • Clarke R.
      • Peden J.F.
      • Hopewell J.C.
      • Kyriakou T.
      • Goel A.
      • Heath S.C.
      • Parish S.
      • Barlera S.
      • Franzosi M.G.
      • Rust S.
      • et al.
      Genetic variants associated with Lp(a) lipoprotein level and coronary disease.
      ) published an article using a gene chip with 48,742 SNPs in 2,100 candidate genes examining individuals with coronary disease and controls. They found that the rs10455872 and rs3798220 SNPs in the LPA gene explained 25% and 8% of the variation in plasma Lp(a) concentrations, and each was associated with high risk of coronary heart disease. Importantly, in numerous other studies, including genome-wide linkage and association studies, multiple genetic variants in or around LPA on chromosome 6q27 were also found to be major determinants of plasma Lp(a) concentrations (
      • Schmidt K.
      • Noureen A.
      • Kronenberg F.
      • Utermann G.
      Structure, function, and genetics of lipoprotein(a).
      ,
      • Barlera S.
      • Specchia C.
      • Farrall M.
      • Chiodini B.D.
      • Franzosi M.G.
      • Rust S.
      • Green F.
      • Nicolis E.B.
      • Peden J.
      • Assmann G.
      • et al.
      Multiple QTL influence the serum Lp(a) concentration: a genome-wide linkage screen in the PROCARDIS study.
      ,
      • López S.
      • Buil A.
      • Ordoñez J.
      • Souto J.C.
      • Almasy L.
      • Lathrop M.
      • Blangero J.
      • Blanco-Vaca F.
      • Fontcuberta J.
      • Soria J.M.
      Genome-wide linkage analysis for identifying quantitative trait loci involved in the regulation of lipoprotein a (Lpa) levels.
      ,
      • Ober C.
      • Nord A.S.
      • Thompson E.E.
      • Pan L.
      • Tan Z.
      • Cusanovich D.
      • Sun Y.
      • Nicolae R.
      • Edelstein C.
      • Schneider D.H.
      • et al.
      Genome-wide association study of plasma lipoprotein(a) levels identifies multiple genes on chromosome 6q.
      ,
      • Zabaneh D.
      • Kumari M.
      • Sandhu M.
      • Wareham N.
      • Wainwright N.
      • Papamarkou T.
      • Hopewell J.
      • Clarke R.
      • Li K.
      • Palmen J.
      • et al.
      Meta analysis of candidate gene variants outside the LPA locus with Lp(a) plasma levels in 14,500 participants of six White European cohorts.
      ,
      • Qi Q.
      • Workalemahu T.
      • Zhang C.
      • Hu F.B.
      • Qi L.
      Genetic variants, plasma lipoprotein(a) levels, and risk of cardiovascular morbidity and mortality among two prospective cohorts of type 2 diabetes.
      ). For Mendelian randomization studies, however, the most important SNP is LPA rs10455872, as this variant explains up to 28% of variation in plasma Lp(a) concentrations in the general population of Whites (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Elevated lipoprotein(a) and risk of aortic valve stenosis in the general population.
      ,
      • Kamstrup P.R.
      • Nordestgaard B.G.
      Lipoprotein(a) concentrations, isoform size, and risk of type 2 diabetes: a Mendelian randomisation study.
      ), and this variant, unlike the KIV-2 copy number variation, is very easy to genotype in large-scale studies. Even though this SNP is not causal for high Lp(a) (
      • Schmidt K.
      • Noureen A.
      • Kronenberg F.
      • Utermann G.
      Structure, function, and genetics of lipoprotein(a).
      ), it can still be an excellent instrument to examine causality of high Lp(a) concentrations per se.
      Taken together, the LPA KIV-2 copy number variation and the LPA rs10455872 SNP have so far been the best genetic instruments in large-scale Mendelian randomization studies, each explaining 27–28% variation in plasma Lp(a) concentrations. Because KIV-2 copy number variation directly measures apo(a) size differences as opposed to rs10455872, which marks both apo(a) size differences and Lp(a) concentrations unrelated to this genetic variation (
      • Kamstrup P.R.
      • Nordestgaard B.G.
      Lipoprotein(a) concentrations, isoform size, and risk of type 2 diabetes: a Mendelian randomisation study.
      ), the combined use of both has the potential to explore whether it is the KIV-2 copy number variation or the Lp(a) particle, per se, that is the direct cause of cardiovascular disease.
      While KIV-2 number of repeats above 40 and rs10455872 noncarrier state both mark very low Lp(a) concentrations, heterozygosity and homozygosity for rs10455872 mark very different plasma Lp(a) concentration profiles than intermediate or low number of KIV-2 number of repeats (Fig. 3). Using these two genetic variants simultaneously has previously been used by us to suggest that the well-documented slightly higher risk of type 2 diabetes in those with the lowest Lp(a) concentration (
      • Kamstrup P.R.
      • Nordestgaard B.G.
      Lipoprotein(a) concentrations, isoform size, and risk of type 2 diabetes: a Mendelian randomisation study.
      ,
      • Mora S.
      • Kamstrup P.R.
      • Rifai N.
      • Nordestgaard B.G.
      • Buring J.E.
      • Ridker P.M.
      Lipoprotein(a) and risk of type 2 diabetes.
      ,
      • Ye Z.
      • Haycock P.C.
      • Gurdasani D.
      • Pomilla C.
      • Boekholdt S.M.
      • Tsimikas S.
      • Khaw K.T.
      • Wareham N.J.
      • Sandhu M.S.
      • Forouhi N.G.
      The association between circulating lipoprotein(a) and type 2 diabetes: is it causal?.
      ,
      • Ding L.
      • Song A.
      • Dai M.
      • Xu M.
      • Sun W.
      • Xu B.
      • Sun J.
      • Wang T.
      • Xu Y.
      • Lu J.
      • et al.
      Serum lipoprotein (a) concentrations are inversely associated with T2D, prediabetes, and insulin resistance in a middle-aged and elderly Chinese population.
      ) is possibly explained by the KIV-2 copy number variation rather than the Lp(a) particle per se (
      • Kamstrup P.R.
      • Nordestgaard B.G.
      Lipoprotein(a) concentrations, isoform size, and risk of type 2 diabetes: a Mendelian randomisation study.
      ). In other words, these data indirectly suggest that lowering of Lp(a) pharmacologically is unlikely to lead to increased risk of diabetes. Importantly however, more evidence using even better genetic instruments is needed before this can be concluded definitively (
      • Kamstrup P.R.
      • Nordestgaard B.G.
      Lipoprotein(a) concentrations, isoform size, and risk of type 2 diabetes: a Mendelian randomisation study.
      ,
      • Lamina C.
      • Kronenberg F.
      The mysterious lipoprotein(a) is still good for a surprise.
      ).
      Figure thumbnail gr3
      Fig. 3Distribution of plasma Lp(a) concentrations as a function of LPA KIV-2 number of repeats and of LPA rs10455872 in the Copenhagen General Population Study. Green and red parts correspond to the bottom 80% and top 20% of the entire population distribution of plasma Lp(a) concentrations (see ).

      GENETIC STUDIES TO INFER CAUSALITY: THE MENDELIAN RANDOMIZATION APPROACH

      Genetic studies, like randomized intervention trials, are completely free of reverse causation and largely free of confounding. Therefore, if a genetic variant or a drug leads to higher or lower concentrations of a lipoprotein and this further leads to higher or lower risk of cardiovascular disease, then it is quite likely that it is the lipoprotein that causes the effect on cardiovascular disease. In essence, this is the principle of the Mendelian randomization approach. In contrast, results from observational epidemiology can mislead through confounding and reverse causation. Confounding is when a third factor influences both lipoprotein concentrations and cardiovascular disease risk, while reverse causation implies that cardiovascular disease leads to changes in lipoprotein concentrations, rather than vice versa.
      There are several early examples of studies that suggest the idea that if a risk factor is elevated or reduced due to genetic variation, and if such genetic variation is or is not associated with a disease of interest, then it would be possible to infer or exclude causality of the risk factor (
      • Katan M.B.
      Apolipoprotein E isoforms, serum cholesterol, and cancer.
      ,
      • Gray R.
      • Wheatley K.
      How to avoid bias when comparing bone marrow transplantation with chemotherapy.
      ,
      • Tybjaerg-Hansen A.
      • Agerholm-Larsen B.
      • Humphries S.E.
      • Abildgaard S.
      • Schnohr P.
      • Nordestgaard B.G.
      A common mutation (G-455→ A) in the beta-fibrinogen promoter is an independent predictor of plasma fibrinogen, but not of ischemic heart disease. A study of 9,127 individuals based on the Copenhagen City Heart Study.
      ). This idea involving the causal genetic influence of high Lp(a) concentrations on risk of coronary heart disease was already presented in 1992 by Gerd Utermann and colleagues (
      • Sandholzer C.
      • Boerwinkle E.
      • Saha N.
      • Tong M.C.
      • Utermann G.
      Apolipoprotein(a) phenotypes, Lp(a) concentration and plasma lipid levels in relation to coronary heart disease in a Chinese population: evidence for the role of the apo(a) gene in coronary heart disease.
      ,
      • Sandholzer C.
      • Saha N.
      • Kark J.D.
      • Rees A.
      • Jaross W.
      • Dieplinger H.
      • Hoppichler F.
      • Boerwinkle E.
      • Utermann G.
      Apo(a) isoforms predict risk for coronary heart disease. A study in six populations.
      ). However, the concept of the Mendelian randomization approach, including in-depth discussion of strengths and limitations, mainly crystallized due to many insightful publications from George Davey Smith and colleagues (
      • Smith G.D.
      • Ebrahim S.
      ‘Mendelian randomization’: can genetic epidemiology contribute to understanding environmental determinants of disease?.
      ,
      • Smith G.D.
      • Ebrahim S.
      Mendelian randomization: prospects, potentials, and limitations.
      ,
      • Davey Smith G.
      • Ebrahim S.
      • Lewis S.
      • Hansell A.L.
      • Palmer L.J.
      • Burton P.R.
      Genetic epidemiology and public health: hope, hype, and future prospects.
      ,
      • Lawlor D.A.
      • Harbord R.M.
      • Sterne J.A.
      • Timpson N.
      • Davey Smith G.
      Mendelian randomization: using genes as instruments for making causal inferences in epidemiology.
      ,
      • Palmer T.M.
      • Sterne J.A.
      • Harbord R.M.
      • Lawlor D.A.
      • Sheehan N.A.
      • Meng S.
      • Granell R.
      • Davey Smith G.
      • Didelez V.
      Instrumental variable estimation of causal risk ratios and causal odds ratios in Mendelian randomization analyses.
      ,
      • Davey Smith G.
      • Hemani G.
      Mendelian randomization: genetic anchors for causal inference in epidemiological studies.
      ), publications that can be used as a “starter's kit” to understand the Mendelian randomization approach. Growing out of the awareness of the limitations of observational epidemiology, it was suggested that Mendelian randomization, that is, the random assortment of genes from parents to offspring that occurs during gamete formation and conception, would provide a method for assessing the causal nature of risk factors on disease. The clear formulation of these ideas has substantially influenced thinking on how to understand disease causality, especially in cardiovascular medicine and most importantly for the role of Lp(a) as a cause of cardiovascular disease.
      Epidemiology alone cannot determine causality due to potential problems with confounding and reverse causation (Fig. 4, double-pointed arrow #1). Thus, potential confounders, including life-style factors, may be unevenly distributed between those with high and low Lp(a) concentrations, and such confounders may be the real explanation for the high risk of cardiovascular disease in those with high Lp(a) (Fig. 5, left panel). In contrast, in the Mendelian randomization study design, such confounders are always evenly distributed between those with high and low Lp(a), and therefore, cannot explain the high cardiovascular risk in those with genetically high Lp(a) concentrations (Fig. 5, middle and right panels). The other major potential limitation of observational studies is reverse causation, that is, the possibility that cardiovascular disease leads to high Lp(a) concentrations, rather than vice versa (Fig. 5, left panel). In the Mendelian randomization study design, reverse causation is simply not possible, as cardiovascular disease cannot change your genes (Fig. 5, middle and right panels). In other words, the Mendelian randomization study design can be used to infer causality just like a randomized double-blind placebo-controlled Lp(a)-reducing trial and these two types of studies share many advantages and have similar limitations [see Fig. 5 in (
      • Nordestgaard B.G.
      Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: new insights from epidemiology, genetics, and biology.
      )]. Unfortunately however, so far no randomized double-blind placebo-controlled Lp(a)-reducing trials to prevent cardiovascular disease have been published or even initiated. Therefore, for now, the human evidence to suggest that high Lp(a) causes cardiovascular disease has to depend on genetics and the Mendelian randomization approach. While these approaches are powerful, it is naturally the totality of evidence that counts in understanding causality.
      Figure thumbnail gr4
      Fig. 4The four different statistical parts of a complete Mendelian randomization study design to examine causality from high plasma Lp(a) concentrations to high risk of cardiovascular disease. Potential limitations are shown with question marks.
      Figure thumbnail gr5
      Fig. 5Comparison of observational studies and Mendelian randomization studies to help understand causality from high plasma Lp(a) concentrations to high risk of cardiovascular disease.
      Another limitation of observational studies is the problem of regression dilution bias (
      • Clarke R.
      • Shipley M.
      • Lewington S.
      • Youngman L.
      • Collins R.
      • Marmot M.
      • Peto R.
      Underestimation of risk associations due to regression dilution in long-term follow-up of prospective studies.
      ,
      • MacMahon S.
      • Peto R.
      • Cutler J.
      • Collins R.
      • Sorlie P.
      • Neaton J.
      • Abbott R.
      • Godwin J.
      • Dyer A.
      • Stamler J.
      Blood pressure, stroke, and coronary heart disease. Part 1, Prolonged differences in blood pressure: prospective observational studies corrected for the regression dilution bias.
      ) because risk factors typically are only measured once, and therefore the association observed will only represent a single point estimate (Fig. 5, left panel). Regression dilution bias means that the effect size of the risk estimate is underestimated, although this bias does not influence statistical significance. In contrast, SNPs used in the Mendelian randomization design generally are measured precisely and have less of a problem with regression dilution bias (Fig. 5, right panel). The exception here is KIV-2 number of repeats measured using quantitative polymerase chain reaction, as this measurement will vary due to measurement error just like plasma Lp(a) concentrations and likewise is affected by regression dilution bias (Fig. 5, middle panel), suggesting that the effect sizes for KIV-2 number of repeats for causal associations with cardiovascular disease should be viewed as minimal estimates (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Steffensen R.
      • Nordestgaard B.G.
      Genetically elevated lipoprotein(a) and increased risk of myocardial infarction.
      ,
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Elevated lipoprotein(a) and risk of aortic valve stenosis in the general population.
      ,
      • Kamstrup P.R.
      • Nordestgaard B.G.
      Lipoprotein(a) concentrations, isoform size, and risk of type 2 diabetes: a Mendelian randomisation study.
      ,
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Genetic evidence that lipoprotein(a) associates with atherosclerotic stenosis rather than venous thrombosis.
      ,
      • Langsted A.
      • Varbo A.
      • Kamstrup P.R.
      • Nordestgaard B.G.
      Elevated lipoprotein(a) does not cause low-grade inflammation despite causal association with aortic valve stenosis and myocardial infarction: a study of 100,578 individuals from the general population.
      ,
      • Langsted A.
      • Kamstrup P.R.
      • Benn M.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      High lipoprotein(a) as a possible cause of clinical familial hypercholesterolaemia: a prospective cohort study.
      ).
      Mendelian randomization studies using human genetics have many similarities with randomized double-blind placebo-controlled trials and, thus, advantages over traditional observational studies (Fig. 5). Like in randomized trials, Mendelian randomization studies are double-blind and confounding and reserve causation are circumvented due to nature's own randomization method during distribution of alleles at meiosis. Mendelian randomization studies have the additional advantage over conventional epidemiology that genetics typically capture a life-long effect (Fig. 5, middle and right panels), while observational studies only include the time between risk factor assessment and end of follow-up. Like a drug in randomized trials, genetics can have the problem of pleiotropic effects; however, while this can be a problem with SNPs as markers of high Lp(a) concentrations, direct measurement of KIV-2 number of repeats or corresponding apo(a) size polymorphism will not have this problem (Fig. 5, right and middle panels).
      Another potential problem of Mendelian randomization studies includes linkage disequilibrium with other causative genetic variants in other genes; however, this is not a problem for KIV-2, although it can be a problem for SNPs associated with high Lp(a) concentrations. Also, population admixture can be a major problem if both genotype and the disease studied are found preferentially in certain subpopulations; however, this potential problem can be largely circumvented by studying ethnically homogeneous populations or by adjusting for different ethnicity using genetic information. Finally, it is essential to use genetic variation with sufficiently large effect sizes, which has been done in Lp(a) causality research more than for any other cardiovascular risk factor: the LPA KIV-2 copy number variation and the LPA rs10455872 SNP, which can be used for genotyping large-scale studies, each explain 27–28% of the variation in plasma Lp(a) concentrations.
      Technically, what is done in a complete Mendelian randomization study is depicted in Fig. 4. Published examples of this complete design that are easy to follow for the nonspecialist include that low concentrations of vitamin D are causally associated with high all-cause mortality (
      • Afzal S.
      • Brondum-Jacobsen P.
      • Bojesen S.E.
      • Nordestgaard B.G.
      Genetically low vitamin D concentrations and increased mortality: Mendelian randomisation analysis in three large cohorts.
      ), and that low concentrations of nonfasting triglycerides are causally associated with low all-cause mortality (
      • Thomsen M.
      • Varbo A.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Low nonfasting triglycerides and reduced all-cause mortality: a Mendelian randomization study.
      ).
      First, the well-known observational association is shown in the study population (Fig. 4, double-pointed arrow #1). Second, the causal association between LPA genotype (KIV-2 or a SNP) on Lp(a) concentration is documented and the extent of variation on plasma concentrations determined by genotype is quantified. This allows assessment of the value of the genotype as an instrument in the Mendelian randomization study design (Fig. 4, single-pointed arrow #2). At this stage, it is also important to exclude pleiotropic effects, that is, genotype should not be associated with any other factor that might cause cardiovascular disease (Table 1). Third, genotype is then directly associated with cardiovascular disease, using a study with sufficient statistical power to document or reject the causal association (Fig. 4, single-pointed arrow #3). Fourth, the formal test of causality involves running an instrumental variable analysis (Fig. 4, single-pointed arrows #4) that integrates the effect of genotype on Lp(a) concentrations (arrow #2) with the effect of genotype on cardiovascular disease risk (arrow #3). Observational and genetic causal risk estimates can then be compared directly, as arrows #1 and #4 will be on the same scale, that is, for Lp(a) per, for example, a 30 mg/dl higher concentration either observationally or genetically.
      TABLE 1Baseline characteristic in individuals from the Copenhagen General Population and the Copenhagen City Heart Study combined
      CharacteristicObservational (N = 58,232), Plasma Lp(a)Genetic (N = 98,941), LPA KIV-2 Number of RepeatsGenetic (N = 104,366), LPA rs10455872 SNP
      High 80%Low 20%Low 20%High 80%Non-carrier 86%Carrier 14%
      Age, years58 (49–68)59 (50–68)58 (48–67)58 (48–67)58 (48–67)58 (48–67)
      Women, %545755555555
      Hypertension, %676866666666
      Diabetes, %444444
      Smoking, %171717191717
      Cholesterol-lowering therapy, %111713111114
      Body Mass Index, kg/m226 (23–28)26 (23–28)26 (23–28)26 (23–29)26 (23–28)26 (23–28)
      Total cholesterol, mmol/l5.5 (4.8–6.2)5.7 (5.0–6.4)5.6 (4.9–6.3)5.5 (4.8–6.3)5.5 (4.8–6.3)5.6 (4.9–6.4)
      Triglycerides, mmol/l1.4 (1.0–2.0)1.4 (1.0–2.1)1.4 (1.0–2.1)1.4 (1.0–2.1)1.4 (1.0–2.1)1.4 (1.0–2.1)
      Values are median (interquartile range).
      For research on Lp(a) as a cause of cardiovascular disease, it can be argued that even conventional epidemiology will suffice in understanding causality of Lp(a), as the concentrations of this lipoprotein are largely genetically determined (
      • Utermann G.
      Lipoprotein(a).
      ,
      • Schmidt K.
      • Noureen A.
      • Kronenberg F.
      • Utermann G.
      Structure, function, and genetics of lipoprotein(a).
      ) and minimally confounded by environmental variables (Table 1). Although this seems to be a valid statement, experience has shown that it was mainly the large-scale genetic Mendelian randomization studies published from 2009 and onwards that paved the path for a general understanding that high concentrations of Lp(a) are a direct cause of myocardial infarction, atherosclerotic stenosis, and aortic valve stenosis (Fig. 1).

      MYOCARDIAL INFARCTION AND ISCHEMIC HEART DISEASE

      Early retrospective case-control studies found that Lp(a) concentrations were higher in patients with myocardial infarction and ischemic heart disease (coronary heart disease), than in individuals without these diseases (
      • Dahlén G.
      • Ericson C.
      • Furberg C.
      • Lundkvist L.
      • Svardsudd K.
      Studies on an extra pre-beta lipoprotein fraction.
      ,
      • Berg K.
      • Dahlén G.
      • Frick M.H.
      Lp(a) lipoprotein and pre-beta1-lipoprotein in patients with coronary heart disease.
      ,
      • Dahlén G.
      • Berg K.
      • Gillnas T.
      • Ericson C.
      Lp(a) lipoprotein/pre-beta1-lipoprotein in Swedish middle-aged males and in patients with coronary heart disease.
      ). Later on, many similar studies followed (
      • Sandholzer C.
      • Saha N.
      • Kark J.D.
      • Rees A.
      • Jaross W.
      • Dieplinger H.
      • Hoppichler F.
      • Boerwinkle E.
      • Utermann G.
      Apo(a) isoforms predict risk for coronary heart disease. A study in six populations.
      ,
      • Rhoads G.G.
      • Morton N.E.
      • Gulbrandsen C.L.
      • Kagan A.
      Sinking pre-beta lipoprotein and coronary heart disease in Japanese-American men in Hawaii.
      ,
      • Kostner G.M.
      • Avogaro P.
      • Cazzolato G.
      • Marth E.
      • Bittolo-Bon G.
      • Qunici G.B.
      Lipoprotein Lp(a) and the risk for myocardial infarction.
      ,
      • Dahlen G.H.
      • Guyton J.R.
      • Attar M.
      • Farmer J.A.
      • Kautz J.A.
      • Gotto Jr, A.M.
      Association of levels of lipoprotein Lp(a), plasma lipids, and other lipoproteins with coronary artery disease documented by angiography.
      ,
      • Wiklund O.
      • Angelin B.
      • Olofsson S.O.
      • Eriksson M.
      • Fager G.
      • Berglund L.
      • Bondjers G.
      Apolipoprotein(a) and ischaemic heart disease in familial hypercholesterolaemia.
      ,
      • Rhoads G.G.
      • Dahlén G.
      • Berg K.
      • Morton N.E.
      • Dannenberg A.L.
      Lp(a) lipoprotein as a risk factor for myocardial infarction.
      ,
      • Durrington P.N.
      • Ishola M.
      • Hunt L.
      • Arrol S.
      • Bhatnagar D.
      Apolipoproteins (a), AI, and B and parental history in men with early onset ischaemic heart disease.
      ,
      • Seed M.
      • Hoppichler F.
      • Reaveley D.
      • McCarthy S.
      • Thompson G.R.
      • Boerwinkle E.
      • Utermann G.
      Relation of serum lipoprotein(a) concentration and apolipoprotein(a) phenotype to coronary heart disease in patients with familial hypercholesterolemia.
      ,
      • Sandkamp M.
      • Funke H.
      • Schulte H.
      • Kohler E.
      • Assmann G.
      Lipoprotein(a) is an independent risk factor for myocardial infarction at a young age.
      ,
      • Kark J.D.
      • Sandholzer C.
      • Friedlander Y.
      • Utermann G.
      Plasma Lp(a), apolipoprotein(a) isoforms and acute myocardial infarction in men and women: a case-control study in the Jerusalem population.
      ,
      • Simons L.
      • Friedlander Y.
      • Simons J.
      • McCallum J.
      Lipoprotein(a) is not associated with coronary heart disease in the elderly: cross-sectional data from the Dubbo study.
      ,
      • Zhuang Y.Y.
      • Wang J.J.
      • Xu P.
      Increased lipoprotein (a) as an independent risk factor for cardiovascular and cerebrovascular diseases.
      ,
      • Orth-Gomér K.
      • Mittleman M.A.
      • Schenck-Gustafsson K.
      • Wamala S.P.
      • Eriksson M.
      • Belkic K.
      • Kirkeeide R.
      • Svane B.
      • Rydén L.
      Lipoprotein(a) as a determinant of coronary heart disease in young women.
      ), the majority with the same conclusion that Lp(a) was higher in those with than in those without myocardial infarction and ischemic heart disease. On hindsight, with the current understanding of the Mendelian randomization approach, this is an impressive set of studies; however, at the time of publication of these studies evidence from prospective population-based studies was needed.
      Early population-based prospective cohort and nested case-control studies (
      • Rosengren A.
      • Wilhelmsen L.
      • Eriksson E.
      • Risberg B.
      • Wedel H.
      Lipoprotein (a) and coronary heart disease: a prospective case-control study in a general population sample of middle aged men.
      ,
      • Jauhiainen M.
      • Koskinen P.
      • Ehnholm C.
      • Frick M.H.
      • Manttari M.
      • Manninen V.
      • Huttunen J.K.
      Lipoprotein (a) and coronary heart disease risk: a nested case-control study of the Helsinki Heart Study participants.
      ,
      • Coleman M.P.
      • Key T.J.
      • Wang D.Y.
      • Hermon C.
      • Fentiman I.S.
      • Allen D.S.
      • Jarvis M.
      • Pike M.C.
      • Sanders T.A.
      A prospective study of obesity, lipids, apolipoproteins and ischaemic heart disease in women.
      ,
      • Ridker P.M.
      • Hennekens C.H.
      • Stampfer M.J.
      A prospective study of lipoprotein(a) and the risk of myocardial infarction.
      ,
      • Cremer P.
      • Nagel D.
      • Labrot B.
      • Mann H.
      • Muche R.
      • Elster H.
      • Seidel D.
      Lipoprotein Lp(a) as predictor of myocardial infarction in comparison to fibrinogen, LDL cholesterol and other risk factors: results from the prospective Gottingen Risk Incidence and Prevalence Study (GRIPS).
      ,
      • Alfthan G.
      • Pekkanen J.
      • Jauhiainen M.
      • Pitkaniemi J.
      • Karvonen M.
      • Tuomilehto J.
      • Salonen J.T.
      • Ehnholm C.
      Relation of serum homocysteine and lipoprotein(a) concentrations to atherosclerotic disease in a prospective Finnish population based study.
      ,
      • Wald N.J.
      • Law M.
      • Watt H.C.
      • Wu T.
      • Bailey A.
      • Johnson A.M.
      • Craig W.Y.
      • Ledue T.B.
      • Haddow J.E.
      Apolipoproteins and ischaemic heart disease: implications for screening.
      ,
      • Assmann G.
      • Schulte H.
      • von Eckardstein A.
      Hyper­triglyceridemia and elevated lipoprotein(a) are risk factors for major coronary events in middle-aged men.
      ,
      • Klausen I.C.
      • Sjol A.
      • Hansen P.S.
      • Gerdes L.U.
      • Moller L.
      • Lemming L.
      • Schroll M.
      • Faergeman O.
      Apolipoprotein(a) isoforms and coronary heart disease in men: a nested case-control study.
      ,
      • Wild S.H.
      • Fortmann S.P.
      • Marcovina S.M.
      A prospective case-control study of lipoprotein(a) levels and apo(a) size and risk of coronary heart disease in Stanford Five-City Project participants.
      ) were summarized in a meta-analysis by Wendy Craig et al. (
      • Craig W.Y.
      • Neveux L.M.
      • Palomaki G.E.
      • Cleveland M.M.
      • Haddow J.E.
      Lipoprotein(a) as a risk factor for ischemic heart disease: metaanalysis of prospective studies.
      ) in 1998. In these studies dominated by White participants, 12 of 14 prospective studies found that Lp(a) concentrations were higher in subjects who later developed ischemic heart disease than in those who did not. After addition of six more prospective population-based studies (
      • Dahlén G.
      Lipoprotein (a) as a risk factor for atherosclerotic diseases.
      ,
      • Dahlén G.H.
      • Weinehall L.
      • Stenlund H.
      • Jansson J.H.
      • Hallmans G.
      • Huhtasaari F.
      • Wall S.
      Lipoprotein(a) and cholesterol levels act synergistically and apolipoprotein A-I is protective for the incidence of primary acute myocardial infarction in middle-aged males. An incident case-control study from Sweden.
      ,
      • Cantin B.
      • Gagnon F.
      • Moorjani S.
      • Despres J.P.
      • Lamarche B.
      • Lupien P.J.
      • Dagenais G.R.
      Is lipoprotein(a) an independent risk factor for ischemic heart disease in men? The Quebec Cardiovascular Study.
      ,
      • Bostom A.G.
      • Cupples L.A.
      • Jenner J.L.
      • Ordovas J.M.
      • Seman L.J.
      • Wilson P.W.
      • Schaefer E.J.
      • Castelli W.P.
      Elevated plasma lipoprotein(a) and coronary heart disease in men aged 55 years and younger. A prospective study.
      ,
      • Bostom A.G.
      • Gagnon D.R.
      • Cupples L.A.
      • Wilson P.W.
      • Jenner J.L.
      • Ordovas J.M.
      • Schaefer E.J.
      • Castelli W.P.
      A prospective investigation of elevated lipoprotein (a) detected by electrophoresis and cardiovascular disease in women. The Framingham Heart Study.
      ,
      • Nguyen T.T.
      • Ellefson R.D.
      • Hodge D.O.
      • Bailey K.R.
      • Kottke T.E.
      • Abu-Lebdeh H.S.
      Predictive value of electrophoretically detected lipoprotein(a) for coronary heart disease and cerebrovascular disease in a community-based cohort of 9936 men and women.
      ), John Danesh and colleagues updated the meta-analysis in 2000 to show that after including 4,044 deaths from coronary heart disease or nonfatal myocardial infarction during a mean follow-up of 10 years in 18 studies, individuals in the top versus bottom third of the Lp(a) concentration distribution had a combined risk ratio of 1.7 [95% confidence interval (CI): 1.4–1.9] (
      • Danesh J.
      • Collins R.
      • Peto R.
      Lipoprotein(a) and coronary heart disease. Meta-analysis of prospective studies.
      ).
      Different results in some of the early studies could be because measurement of Lp(a) is complicated by the varying isoform sizes of apo(a), that is, assays that are not isoform independent might overestimate Lp(a) plasma concentrations when large isoforms are present and underestimate concentrations when small isoforms are present (
      • Marcovina S.M.
      • Albers J.J.
      • Gabel B.
      • Koschinsky M.L.
      • Gaur V.P.
      Effect of the number of apolipoprotein(a) kringle 4 domains on immunochemical measurements of lipoprotein(a).
      ,
      • Tate J.R.
      • Rifai N.
      • Berg K.
      • Couderc R.
      • Dati F.
      • Kostner G.M.
      • Sakurabayashi I.
      • Steinmetz A.
      International Federation of Clinical Chemistry standardization project for the measurement of lipoprotein(a). Phase I. Evaluation of the analytical performance of lipoprotein(a) assay systems and commercial calibrators.
      ,
      • Marcovina S.M.
      • Koschinsky M.L.
      • Albers J.J.
      • Skarlatos S.
      Report of the National Heart, Lung, and Blood Institute Workshop on Lipoprotein(a) and Cardiovascular Disease: recent advances and future directions.
      ). Further, many of these early studies used Lp(a) samples that had been frozen for several years, which might affect the structure of Lp(a) and thereby lead to incorrect measurement of plasma Lp(a) concentrations (
      • Kronenberg F.
      • Trenkwalder E.
      • Dieplinger H.
      • Utermann G.
      Lipoprotein(a) in stored plasma samples and the ravages of time. Why epidemiological studies might fail.
      ). Also, most previous studies did not estimate risk in individuals with extremely high Lp(a) concentrations, the concentrations that would seem most relevant clinically. Finally, many early studies did not correct for regression dilution bias (
      • Clarke R.
      • Shipley M.
      • Lewington S.
      • Youngman L.
      • Collins R.
      • Marmot M.
      • Peto R.
      Underestimation of risk associations due to regression dilution in long-term follow-up of prospective studies.
      ,
      • MacMahon S.
      • Peto R.
      • Cutler J.
      • Collins R.
      • Sorlie P.
      • Neaton J.
      • Abbott R.
      • Godwin J.
      • Dyer A.
      • Stamler J.
      Blood pressure, stroke, and coronary heart disease. Part 1, Prolonged differences in blood pressure: prospective observational studies corrected for the regression dilution bias.
      ) and, therefore, effect sizes for risk of myocardial infarction and ischemic heart disease likely were underestimated.
      After publication of yet another six prospective population-based studies (
      • von Eckardstein A.
      • Schulte H.
      • Cullen P.
      • Assmann G.
      Lipoprotein(a) further increases the risk of coronary events in men with high global cardiovascular risk.
      ,
      • Sharrett A.R.
      • Ballantyne C.M.
      • Coady S.A.
      • Heiss G.
      • Sorlie P.D.
      • Catellier D.
      • Patsch W.
      Coronary heart disease prediction from lipoprotein cholesterol levels, triglycerides, lipoprotein(a), apolipoproteins A-I and B, and HDL density subfractions: The Atherosclerosis Risk in Communities (ARIC) Study.
      ,
      • Luc G.
      • Bard J.M.
      • Arveiler D.
      • Ferrieres J.
      • Evans A.
      • Amouyel P.
      • Fruchart J.C.
      • Ducimetiere P.
      Lipoprotein (a) as a predictor of coronary heart disease: the PRIME Study.
      ,
      • Ariyo A.A.
      • Thach C.
      • Tracy R.
      Lp(a) lipoprotein, vascular disease, and mortality in the elderly.
      ,
      • Rifai N.
      • Ma J.
      • Sacks F.M.
      • Ridker P.M.
      • Hernandez W.J.
      • Stampfer M.J.
      • Marcovina S.M.
      Apolipoprotein(a) size and lipoprotein(a) concentration and future risk of angina pectoris with evidence of severe coronary atherosclerosis in men: The Physicians' Health Study.
      ,
      • Suk Danik J.
      • Rifai N.
      • Buring J.E.
      • Ridker P.M.
      Lipoprotein(a), measured with an assay independent of apolipoprotein(a) isoform size, and risk of future cardiovascular events among initially healthy women.
      ), we published in 2008 the results of the Copenhagen City Heart Study with 9,330 individuals followed for 10 years during which time 498 developed a myocardial infarction (
      • Kamstrup P.R.
      • Benn M.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Extreme lipoprotein(a) levels and risk of myocardial infarction in the general population: the Copenhagen City Heart Study.
      ). We measured Lp(a) concentrations in 1991–1994 shortly after sampling using an apo(a) isoform-insensitive assay. Risk estimates were corrected for regression dilution bias, and we focused on the risk of myocardial infarction in those with the extremely high concentrations. Specifically, we chose myocardial infarction as the endpoint, as this disease is registered correctly 99.5% of the time in the Danish registries and the follow-up in this population is 100% complete (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Steffensen R.
      • Nordestgaard B.G.
      Genetically elevated lipoprotein(a) and increased risk of myocardial infarction.
      ). In women, multifactorially adjusted hazard ratios for myocardial infarction for high Lp(a) concentrations were 1.1 (95% CI: 0.6–1.9) for 5–29 mg/dl, 1.7 (95% CI: 1.0–3.1) for 30–84 mg/dl, 2.6 (95% CI: 1.2–5.9) for 85–119 mg/dl, and 3.6 (95% CI: 1.7–7.7) for above 120 mg/dl versus concentrations below 5 mg/dl (
      • Kamstrup P.R.
      • Benn M.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Extreme lipoprotein(a) levels and risk of myocardial infarction in the general population: the Copenhagen City Heart Study.
      ). Corresponding hazard ratios in men were 1.5 (95% CI: 0.9–2.3), 1.6 (95% CI: 1.0–2.6), 2.6 (95% CI: 1.2–5.5), and 3.7 (95% CI: 1.7–8.0), respectively. Also, absolute 10 year risks of myocardial infarction were 10 and 20% in smoking hypertensive women aged above 60 years with Lp(a) concentrations of below 5 mg/dl and above 120 mg/dl, respectively, with corresponding values in men of 19 and 35%.
      The Emerging Risk Factors Collaboration included individual records for 126,634 participants from 36 prospective studies, recorded 9,318 myocardial infarctions and coronary deaths, corrected for regression dilution bias, and also focused on the risk of myocardial infarction and coronary death in those with the extremely high Lp(a) concentrations (
      • Erqou S.
      • Kaptoge S.
      • Perry P.L.
      • Di A.E.
      • Thompson A.
      • White I.R.
      • Marcovina S.M.
      • Collins R.
      • Thompson S.G.
      • Danesh J.
      Lipoprotein(a) concentration and the risk of coronary heart disease, stroke, and nonvascular mortality.
      ) (Fig. 6, right panel). In that study, risk was higher after approximately 30 mg/dl and the age- and sex-adjusted risk ratio was 1.5-fold in those with Lp(a) above 100 mg/dl versus below 4 mg/dl. There were continuous, independent, and modest associations of Lp(a) concentration with risk of coronary heart disease that appeared exclusive to vascular outcomes. Importantly however, this meta-analysis included many former studies using frozen samples and nonoptimal Lp(a) assays, likely partly explaining the relatively modest overall effect sizes observed. Also, this may partly be explained by nondifferential misclassification of events from study to study, as myocardial infarction and coronary death events were classified according to the International Classification of Diseases or, where this was not available, on study-specific classification systems.
      Figure thumbnail gr6
      Fig. 6Observational associations between high plasma Lp(a) concentrations and risk of cardiovascular disease in the Copenhagen City Heart Study and Copenhagen General Population Study combined (left panel) and in the Emerging Risk Factors Collaboration (right panel). Hazard ratios in the left panel were estimated by Cox proportional hazard regression models and were adjusted for age and sex and corrected for regression dilution bias. Right panel was adapted from (
      • Erqou S.
      • Kaptoge S.
      • Perry P.L.
      • Di A.E.
      • Thompson A.
      • White I.R.
      • Marcovina S.M.
      • Collins R.
      • Thompson S.G.
      • Danesh J.
      Lipoprotein(a) concentration and the risk of coronary heart disease, stroke, and nonvascular mortality.
      ).
      For use specifically in this review, we updated our former epidemiological studies (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Steffensen R.
      • Nordestgaard B.G.
      Genetically elevated lipoprotein(a) and increased risk of myocardial infarction.
      ,
      • Langsted A.
      • Varbo A.
      • Kamstrup P.R.
      • Nordestgaard B.G.
      Elevated lipoprotein(a) does not cause low-grade inflammation despite causal association with aortic valve stenosis and myocardial infarction: a study of 100,578 individuals from the general population.
      ,
      • Kamstrup P.R.
      • Benn M.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Extreme lipoprotein(a) levels and risk of myocardial infarction in the general population: the Copenhagen City Heart Study.
      ) based on the Copenhagen City Heart Study and the Copenhagen General Population Study combined to achieve maximal statistical power (Fig. 6, left panel). We included 58,340 individuals, measured Lp(a) concentrations in fresh samples using apo(a)-insensitive assays, corrected for regression dilution bias, recorded 1,897 validated myocardial infarctions, and also focused on the risk in those with the extremely high Lp(a) concentrations. In this new analysis, the risk was again higher after approximately 30 mg/dl and the age- and sex-adjusted hazard ratio for myocardial infarction was 2.4-fold in those with Lp(a) above 100 mg/dl versus below 5 mg/dl, and thus more pronounced than in the Emerging Risk Factors Collaboration (compare Fig. 6, left and right panels).
      A complete large-scale Mendelian randomization study was first published by us in 2009 (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Steffensen R.
      • Nordestgaard B.G.
      Genetically elevated lipoprotein(a) and increased risk of myocardial infarction.
      ), although the basic idea had been suggested previously (
      • Sandholzer C.
      • Boerwinkle E.
      • Saha N.
      • Tong M.C.
      • Utermann G.
      Apolipoprotein(a) phenotypes, Lp(a) concentration and plasma lipid levels in relation to coronary heart disease in a Chinese population: evidence for the role of the apo(a) gene in coronary heart disease.
      ,
      • Sandholzer C.
      • Saha N.
      • Kark J.D.
      • Rees A.
      • Jaross W.
      • Dieplinger H.
      • Hoppichler F.
      • Boerwinkle E.
      • Utermann G.
      Apo(a) isoforms predict risk for coronary heart disease. A study in six populations.
      ). For use specifically in this review, we updated our former Mendelian randomization studies (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Steffensen R.
      • Nordestgaard B.G.
      Genetically elevated lipoprotein(a) and increased risk of myocardial infarction.
      ,
      • Kamstrup P.R.
      • Nordestgaard B.G.
      Lipoprotein(a) concentrations, isoform size, and risk of type 2 diabetes: a Mendelian randomisation study.
      ,
      • Langsted A.
      • Varbo A.
      • Kamstrup P.R.
      • Nordestgaard B.G.
      Elevated lipoprotein(a) does not cause low-grade inflammation despite causal association with aortic valve stenosis and myocardial infarction: a study of 100,578 individuals from the general population.
      ) based on the Copenhagen City Heart Study and the Copenhagen General Population Study combined to achieve maximal statistical power (Fig. 7, top panel). In instrumental variable analyses, a doubling in plasma Lp(a) concentrations caused a 15% (95% CI: 11–20%) higher risk ratio for myocardial infarction using LPA KIV-2 number of repeats and a 10% (95% CI: 6–13%) higher risk ratio using LPA rs10455872 SNP. This should be compared with the corresponding observational estimate of 9% (95% CI: 7–12%). Importantly, as Lp(a) concentrations vary up to a 1,000-fold between individuals (
      • Utermann G.
      Lipoprotein(a).
      ), then Lp(a) can double many times. LPA genotypes were largely unconfounded by conventional cardiovascular risk factors (Table 1), which was also close to being the case for plasma Lp(a) concentrations.
      Figure thumbnail gr7
      Fig. 7Observational and causal genetic associations between high plasma Lp(a) concentrations and risk of cardiovascular disease in the Copenhagen City Heart Study and Copenhagen General Population Study combined. Hazard ratios for observational analyses of plasma Lp(a) concentrations were estimated by Cox proportional hazard regression models and were adjusted for age and sex. Causal risk ratios for analyses of genetically determined plasma Lp(a) concentrations were estimated by instrumental variable analyses and were adjusted for age and sex.
      Many recent large-scale genetic studies also strongly supported high Lp(a) concentrations as a cause of myocardial infarction and ischemic heart disease (
      • Clarke R.
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      • et al.
      Genetic variants associated with Lp(a) lipoprotein level and coronary disease.
      ,
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      Large-scale association analysis identifies 13 new susceptibility loci for coronary artery disease.
      ,
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      • et al.
      Genome-wide haplotype association study identifies the SLC22A3-LPAL2-LPA gene cluster as a risk locus for coronary artery disease.
      ,
      • Helgadottir A.
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      • Thorleifsson G.
      • Holm H.
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      • Gudnason T.
      • Jones G.T.
      • van Rij A.M.
      • Eapen D.J.
      • Baas A.F.
      • et al.
      Apolipoprotein(a) genetic sequence variants associated with systemic atherosclerosis and coronary atherosclerotic burden but not with venous thromboembolism.
      ); however, these publications did not include a complete Mendelian randomization approach, but mainly provided data for the direct association between genotypes and risk of disease (Fig. 4, arrow #3). Most importantly, Robert Clarke et al. (
      • Clarke R.
      • Peden J.F.
      • Hopewell J.C.
      • Kyriakou T.
      • Goel A.
      • Heath S.C.
      • Parish S.
      • Barlera S.
      • Franzosi M.G.
      • Rust S.
      • et al.
      Genetic variants associated with Lp(a) lipoprotein level and coronary disease.
      ) identified two common LPA variants, rs10455872 with a per allele odds ratio for coronary heart disease of 1.70 (95% CI: 1.49–1.95; allele frequency 0.07) and rs3798220 with a per allele odds ratio of 1.92 (1.48–2.49; allele frequency 0.02).
      Also important was a 2010 meta-analysis on apo(a) isoforms and risk of coronary heart disease (
      • Erqou S.
      • Thompson A.
      • Di A.E.
      • Saleheen D.
      • Kaptoge S.
      • Marcovina S.
      • Danesh J.
      Apolipoprotein(a) isoforms and the risk of vascular disease: systematic review of 40 studies involving 58,000 participants.
      ). The 34 included studies of mainly White and Asian individuals used either phenotyping (
      • Sandholzer C.
      • Hallman D.M.
      • Saha N.
      • Sigurdsson G.
      • Lackner C.
      • Csaszar A.
      • Boerwinkle E.
      • Utermann G.
      Effects of the apolipoprotein(a) size polymorphism on the lipoprotein(a) concentration in 7 ethnic groups.
      ,
      • Kraft H.G.
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      • Pang R.W.
      • Delport R.
      • Trommsdorff M.
      • Vermaak H.
      • Janus E.D.
      • Utermann G.
      Frequency distributions of apolipoprotein(a) kringle IV repeat alleles and their effects on lipoprotein(a) levels in Caucasian, Asian, and African populations: the distribution of null alleles is non-random.
      ,
      • Sandholzer C.
      • Saha N.
      • Kark J.D.
      • Rees A.
      • Jaross W.
      • Dieplinger H.
      • Hoppichler F.
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      • Utermann G.
      Apo(a) isoforms predict risk for coronary heart disease. A study in six populations.
      ,
      • Kark J.D.
      • Sandholzer C.
      • Friedlander Y.
      • Utermann G.
      Plasma Lp(a), apolipoprotein(a) isoforms and acute myocardial infarction in men and women: a case-control study in the Jerusalem population.
      ,
      • Klausen I.C.
      • Sjol A.
      • Hansen P.S.
      • Gerdes L.U.
      • Moller L.
      • Lemming L.
      • Schroll M.
      • Faergeman O.
      Apolipoprotein(a) isoforms and coronary heart disease in men: a nested case-control study.
      ,
      • Rifai N.
      • Ma J.
      • Sacks F.M.
      • Ridker P.M.
      • Hernandez W.J.
      • Stampfer M.J.
      • Marcovina S.M.
      Apolipoprotein(a) size and lipoprotein(a) concentration and future risk of angina pectoris with evidence of severe coronary atherosclerosis in men: The Physicians' Health Study.
      ,
      • Gazzaruso C.
      • Garzaniti A.
      • Buscaglia P.
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      • Fratino P.
      • Finardi G.
      • Geroldi D.
      Apolipoprotein(a) phenotypes and their predictive value for coronary heart disease: identification of an operative cut-off of apolipoprotein(a) polymorphism.
      ,
      • Qin S.
      • Wang S.
      • Li C.
      Apolipoprotein (a) polymorphism in relation to coronary heart disease in Chinese Han nationality [article in Chinese].
      ,
      • Emanuele E.
      • Peros E.
      • Minoretti P.
      • D'Angelo A.
      • Piccinni M.N.
      • Montagna L.
      • Geroldi D.
      Apolipoprotein(a) size polymorphism is associated with coronary heart disease in polygenic hypercholesterolemia.
      ,
      • Emanuele E.
      • Peros E.
      • Minoretti P.
      • D'Angelo A.
      • Montagna L.
      • Falcone C.
      • Geroldi D.
      Significance of apolipoprotein(a) phenotypes in acute coronary syndromes: relation with clinical presentation.
      ,
      • Emanuele E.
      • Peros E.
      • Minoretti P.
      • Falcone C.
      • D'Angelo A.
      • Montagna L.
      • Geroldi D.
      Relationship between apolipoprotein(a) size polymorphism and coronary heart disease in overweight subjects.
      ,
      • Parlavecchia M.
      • Pancaldi A.
      • Taramelli R.
      • Valsania P.
      • Galli L.
      • Pozza G.
      • Chierchia S.
      • Ruotolo G.
      Evidence that apolipoprotein(a) phenotype is a risk factor for coronary artery disease in men < 55 years of age.
      ,
      • Martín S.
      • Pedro-Botet J.
      • Joven J.
      • Simó J.M.
      • Ladona M.G.
      • Pavesi M.
      • Rubiés-Prat J.
      Heterozygous apolipoprotein (a) status and protein expression as a risk factor for premature coronary heart disease.
      ,
      • Simó J.M.
      • Joven J.
      • Vilella E.
      • Ribas M.
      • Pujana M.A.
      • Sundaram I.M.
      • Hammel J.P.
      • Hoover-Plow J.L.
      Impact of apolipoprotein(a) isoform size heterogeneity on the lysine binding function of lipoprotein(a) in early onset coronary artery disease.
      ,
      • Geethanjali F.S.
      • Jose V.J.
      • Kanagasabapathy A.S.
      Lipoprotein (a) phenotypes in south Indian patients with coronary artery disease.
      ,
      • Zeljkovic A.
      • Bogavac-Stanojevic N.
      • Jelic-Ivanovic Z.
      • Spasojevic-Kalimanovska V.
      • Vekic J.
      • Spasic S.
      Combined effects of small apolipoprotein (a) isoforms and small, dense LDL on coronary artery disease risk.
      ,
      • Calmarza P.
      • Cordero J.
      • Santos V.
      • Vella J.C.
      Apolipoprotein(a) isoforms in infarcted men under 60 years old.
      ,
      • Akanji A.O.
      Apo(a) isoforms do not predict risk for coronary heart disease in a Gulf Arab population.
      ,
      • Katsouras C.S.
      • Karabina S.A.
      • Tambaki A.P.
      • Goudevenos J.A.
      • Michalis L.K.
      • Tsironis L.D.
      • Stroumbis C.S.
      • Elisaf M.S.
      • Sideris D.A.
      • Tselepis A.D.
      Serum lipoprotein(a) concentrations and apolipoprotein(a) isoforms: association with the severity of clinical presentation in patients with coronary heart disease.
      ,
      • Gazzaruso C.
      • Garzaniti A.
      • Falcone C.
      • Geroldi D.
      • Finardi G.
      • Fratino P.
      Association of lipoprotein(a) levels and apolipoprotein(a) phenotypes with coronary artery disease in type 2 diabetic patients and in non-diabetic subjects.
      ,
      • Gambhir J.K.
      • Kaur H.
      • Prabhu K.M.
      • Morrisett J.D.
      • Gambhir D.S.
      Association between lipoprotein(a) levels, apo(a) isoforms and family history of premature CAD in young Asian Indians.
      ,
      • Zorio E.
      • Falco C.
      • Arnau M.A.
      • Espana F.
      • Osa A.
      • Ramon L.A.
      • Castello R.
      • Almenar L.
      • Palencia M.A.
      • Estelles A.
      Lipoprotein (a) in young individuals as a marker of the presence of ischemic heart disease and the severity of coronary lesions.
      ,
      • Kalina A.
      • Csaszar A.
      • Fust G.
      • Nagy B.
      • Szalai C.
      • Karadi I.
      • Duba J.
      • Prohaszka Z.
      • Horvath L.
      • Dieplinger H.
      The association of serum lipoprotein(a) levels, apolipoprotein(a) size and (TTTTA)(n) polymorphism with coronary heart disease.
      ,
      • Bigot E.
      • Robert B.
      • Bard J.M.
      • Mainard F.
      Lipoprotein (a) phenotype distribution in a population of bypass patients and its influence on lipoprotein (a) concentration.
      ,
      • Paultre F.
      • Pearson T.A.
      • Weil H.F.
      • Tuck C.H.
      • Myerson M.
      • Rubin J.
      • Francis C.K.
      • Marx H.F.
      • Philbin E.F.
      • Reed R.G.
      • et al.
      High levels of Lp(a) with a small apo(a) isoform are associated with coronary artery disease in African American and white men.
      ,
      • Abe A.
      • Noma A.
      • Lee Y.J.
      • Yamaguchi H.
      Studies on apolipoprotein(a) phenotypes. Part 2. Phenotype frequencies and Lp(a) concentrations in different phenotypes in patients with angiographically defined coronary artery diseases.
      ,
      • Brazier L.
      • Tiret L.
      • Luc G.
      • Arveiler D.
      • Ruidavets J.B.
      • Evans A.
      • Chapman J.
      • Cambien F.
      • Thillet J.
      Sequence polymorphisms in the apolipoprotein(a) gene and their association with lipoprotein(a) levels and myocardial infarction. The ECTIM Study.
      ,
      • Holmer S.R.
      • Hengstenberg C.
      • Kraft H.G.
      • Mayer B.
      • Poll M.
      • Kurzinger S.
      • Fischer M.
      • Lowel H.
      • Klein G.
      • Riegger G.A.
      • et al.
      Association of polymorphisms of the apolipoprotein(a) gene with lipoprotein(a) levels and myocardial infarction.
      ), that is, gel migration speed of plasma Lp(a) and molecular weight determination of apo(a), or genotyping of KIV-2 number of repeats (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Steffensen R.
      • Nordestgaard B.G.
      Genetically elevated lipoprotein(a) and increased risk of myocardial infarction.
      ,
      • Geethanjali F.S.
      • Luthra K.
      • Lingenhel A.
      • Kanagasaba-Pathy A.S.
      • Jacob J.
      • Srivastava L.M.
      • Vasisht S.
      • Kraft H.G.
      • Utermann G.
      Analysis of the apo(a) size polymorphism in Asian Indian populations: association with Lp(a) concentration and coronary heart disease.
      ). For the 30 studies using phenotyping and including 7,382 cases and 8,514 controls, relative risk of coronary heart disease for smaller versus larger apo(a) isoforms was 2.08 (95% CI: 1.67–2.58). The genotyping studies were dominated by our own study mentioned above in the complete Mendelian randomization study design (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Steffensen R.
      • Nordestgaard B.G.
      Genetically elevated lipoprotein(a) and increased risk of myocardial infarction.
      ). It is also important to note that individuals carrying LPA loss-of-function alleles with low Lp(a) concentrations have low risk of cardiovascular disease (
      • Lim E.T.
      • Wurtz P.
      • Havulinna A.S.
      • Palta P.
      • Tukiainen T.
      • Rehnstrom K.
      • Esko T.
      • Magi R.
      • Inouye M.
      • Lappalainen T.
      • et al.
      Distribution and medical impact of loss-of-function variants in the Finnish founder population.
      ,
      • Kyriakou T.
      • Seedorf U.
      • Goel A.
      • Hopewell J.C.
      • Clarke R.
      • Watkins H.
      • Farrall M.
      A common LPA null allele associates with lower lipoprotein(a) levels and coronary artery disease risk.
      ). Although relatively few Blacks have been studied in the past, a study in 2012 documented that risk of coronary heart disease is also higher in Black individuals with high plasma Lp(a) concentrations (
      • Virani S.S.
      • Brautbar A.
      • Davis B.C.
      • Nambi V.
      • Hoogeveen R.C.
      • Sharrett A.R.
      • Coresh J.
      • Mosley T.H.
      • Morrisett J.D.
      • Catellier D.J.
      • et al.
      Associations between lipoprotein(a) levels and cardiovascular outcomes in black and white subjects: the Atherosclerosis Risk in Communities (ARIC) Study.
      ).
      Taken together, there is now overwhelming evidence from epidemiology and genetics that high Lp(a) concentrations cause high risk of myocardial infarction. It could be argued that the same is true for ischemic and coronary heart disease, but the findings for these disease endpoints could be driven largely by that for myocardial infarction and for atherosclerotic stenosis as described below.

      ATHEROSCLEROTIC STENOSIS

      When high Lp(a) concentrations lead to high risk of myocardial infarction and ischemic heart disease, then automatically almost everybody will think that this must be because Lp(a) is atherogenic. However, there are other possible explanations and one could be that high Lp(a) leads to thrombosis causing myocardial infarction and to atherosclerotic stenosis causing angina pectoris, rather than Lp(a) causing atherosclerosis per se (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Genetic evidence that lipoprotein(a) associates with atherosclerotic stenosis rather than venous thrombosis.
      ,
      • Spence J.D.
      Lipoprotein(a): involved in events, but not burden of atherosclerotic disease?.
      ,
      • Spence J.D.
      • Koschinsky M.
      Mechanisms of lipoprotein(a) pathogenicity: prothrombotic, proatherosclerotic, or both?.
      ). To us, the latter idea became more plausible when it was suddenly documented that high Lp(a) concentrations also cause aortic valve stenosis (
      • Thanassoulis G.
      • Campbell C.Y.
      • Owens D.S.
      • Smith J.G.
      • Smith A.V.
      • Peloso G.M.
      • Kerr K.F.
      • Pechlivanis S.
      • Budoff M.J.
      • Harris T.B.
      • et al.
      Genetic associations with valvular calcification and aortic stenosis.
      ,
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Elevated lipoprotein(a) and risk of aortic valve stenosis in the general population.
      ). However, it can be argued that this is a poor analogy. Indeed, the pathophysiology of “stenosis” in a coronary artery is complex and due to atherosclerotic mediated narrowing of the arterial lumen, some smooth muscle cell proliferation, and likely some element of thrombosis. Thus, the process of atherosclerotic stenosis is very complex and possibly very different than what goes on in aortic valve stenosis. Nevertheless, let us review the human evidence for this idea.
      In a study by Kamstrup, Tybjærg-Hansen, and Nordestgaard (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Genetic evidence that lipoprotein(a) associates with atherosclerotic stenosis rather than venous thrombosis.
      ) in the Copenhagen Ischemic Heart Disease Study, the Copenhagen Carotid Stroke Study, and the Copenhagen City Heart Study, the highest versus lowest tertile of Lp(a) was associated with high risk of coronary, carotid, and femoral atherosclerotic stenosis; that the findings were similar for the lowest versus highest tertile of KIV-2 number of repeats supports that these findings represent causal relationships. For risk of coronary atherosclerotic stenosis, the age- and sex-adjusted odds ratio for highest versus lowest tertile of Lp(a) was 5.0 (95% CI: 3.9–6.5) (Fig. 8, top panel). Corresponding odd ratios were 1.7 (95% CI: 1.2–2.5) for carotid atherosclerotic stenosis (Fig. 8, middle panel) and 1.6 (95% CI: 1.3–2.0) for femoral atherosclerotic stenosis (Fig. 8, bottom panel).
      Figure thumbnail gr8
      Fig. 8Observational associations between high plasma Lp(a) concentrations and risk of coronary, carotid, and femoral atherosclerotic stenosis in the Copenhagen Ischemic Heart Disease Study, Copenhagen Carotid Stroke Study, and Copenhagen City Heart Study, respectively. Odds ratios were estimated by logistic regression models and were adjusted for age and sex. CIHDS, Copenhagen Ischemic Heart Disease Study; CCSS, Copenhagen Carotid Stroke Study; CCHS, Copenhagen City Heart Study. Adapted from (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Genetic evidence that lipoprotein(a) associates with atherosclerotic stenosis rather than venous thrombosis.
      ).
      Many other studies have also found an association between high concentrations of Lp(a) or LPA risk genotypes and high risk of coronary, carotid, and femoral atherosclerotic stenosis (
      • Sandholzer C.
      • Boerwinkle E.
      • Saha N.
      • Tong M.C.
      • Utermann G.
      Apolipoprotein(a) phenotypes, Lp(a) concentration and plasma lipid levels in relation to coronary heart disease in a Chinese population: evidence for the role of the apo(a) gene in coronary heart disease.
      ,
      • Sandholzer C.
      • Saha N.
      • Kark J.D.
      • Rees A.
      • Jaross W.
      • Dieplinger H.
      • Hoppichler F.
      • Boerwinkle E.
      • Utermann G.
      Apo(a) isoforms predict risk for coronary heart disease. A study in six populations.
      ,
      • Dahlen G.H.
      • Guyton J.R.
      • Attar M.
      • Farmer J.A.
      • Kautz J.A.
      • Gotto Jr, A.M.
      Association of levels of lipoprotein Lp(a), plasma lipids, and other lipoproteins with coronary artery disease documented by angiography.
      ,
      • Helgadottir A.
      • Gretarsdottir S.
      • Thorleifsson G.
      • Holm H.
      • Patel R.S.
      • Gudnason T.
      • Jones G.T.
      • van Rij A.M.
      • Eapen D.J.
      • Baas A.F.
      • et al.
      Apolipoprotein(a) genetic sequence variants associated with systemic atherosclerosis and coronary atherosclerotic burden but not with venous thromboembolism.
      ,
      • Frick M.H.
      • Dahlen G.
      • Berg K.
      • Valle M.
      • Hekali P.
      Serum lipids in angiographically assessed coronary atherosclerosis.
      ,
      • Tsimikas S.
      • Brilakis E.S.
      • Miller E.R.
      • McConnell J.P.
      • Lennon R.J.
      • Kornman K.S.
      • Witztum J.L.
      • Berger P.B.
      Oxidized phospholipids, Lp(a) lipoprotein, and coronary artery disease.
      ,
      • Klein J.H.
      • Hegele R.A.
      • Hackam D.G.
      • Koschinsky M.L.
      • Huff M.W.
      • Spence J.D.
      Lipoprotein(a) is associated differentially with carotid stenosis, occlusion, and total plaque area.
      ,
      • Volpato S.
      • Vigna G.B.
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      • Guralnik J.M.
      • Fellin R.
      • et al.
      Lipoprotein(a), inflammation, and peripheral arterial disease in a community-based sample of older men and women (the InCHIANTI study).
      ,
      • Ronald J.
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      • Cerrato F.
      • Nord A.S.
      • Hatsukami T.
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      • Marcovina S.
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      Genetic variation in LPAL2, LPA, and PLG predicts plasma lipoprotein(a) level and carotid artery disease risk.
      ,
      • Armstrong V.W.
      • Cremer P.
      • Eberle E.
      • Manke A.
      • Schulze F.
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      • Seidel D.
      The association between serum Lp(a) concentrations and angiographically assessed coronary atherosclerosis. Dependence on serum LDL levels.
      ,
      • Groves P.
      • Rees A.
      • Bishop A.
      • Morgan R.
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      • Lewis N.
      • Lane I.
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      Apolipoprotein (a) concentrations and susceptibility to coronary artery disease in patients with peripheral vascular disease.
      ,
      • Budde T.
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      • Bosenberg E.
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      • Enbergs A.
      • Schulte H.
      • Assmann G.
      • Breithardt G.
      Plasma Lp(a) levels correlate with number, severity, and length-extension of coronary lesions in male patients undergoing coronary arteriography for clinically suspected coronary atherosclerosis.
      ,
      • Wang X.L.
      • Tam C.
      • McCredie R.M.
      • Wilcken D.E.
      Determinants of severity of coronary artery disease in Australian men and women.
      ,
      • Zenker G.
      • Koltringer P.
      • Bone G.
      • Niederkorn K.
      • Pfeiffer K.
      • Jurgens G.
      Lipoprotein(a) as a strong indicator for cerebrovascular disease.
      ,
      • Cambillau M.
      • Simon A.
      • Amar J.
      • Giral P.
      • Atger V.
      • Segond P.
      • Levenson J.
      • Merli I.
      • Megnien J.L.
      • Plainfosse M.C.
      Serum Lp(a) as a discriminant marker of early atherosclerotic plaque at three extracoronary sites in hypercholesterolemic men. The PCVMETRA Group.
      ,
      • Brown S.A.
      • Morrisett J.D.
      • Boerwinkle E.
      • Hutchinson R.
      • Patsch W.
      The relation of lipoprotein[a] concentrations and apolipoprotein[a] phenotypes with asymptomatic atherosclerosis in subjects of the Atherosclerosis Risk in Communities (ARIC) Study.
      ,
      • Baldassarre D.
      • Tremoli E.
      • Franceschini G.
      • Michelagnoli S.
      • Sirtori C.R.
      Plasma lipoprotein(a) is an independent factor associated with carotid wall thickening in severely but not moderately hypercholesterolemic patients.
      ,
      • Norrgård O.
      • Angquist K.A.
      • Dahlen G.
      Lp(a) lipoprotein in patients with arterial insufficiency of the lower extremities.
      ,
      • Mölgaard J.
      • Klausen I.C.
      • Lassvik C.
      • Faergeman O.
      • Gerdes L.U.
      • Olsson A.G.
      Significant association between low-molecular-weight apolipoprotein(a) isoforms and intermittent claudication.
      ,
      • Widmann M.D.
      • Sumpio B.E.
      Lipoprotein (a): a risk factor for peripheral vascular disease.
      ,
      • Pedro-Botet J.
      • Senti M.
      • Auguet T.
      • Nogues X.
      • Rubies-Prat J.
      • Aubo C.
      • Vidal-Barraquer F.
      Apolipoprotein(a) genetic polymorphism and serum lipoprotein(a) concentration in patients with peripheral vascular disease.
      ,
      • Valentine R.J.
      • Grayburn P.A.
      • Vega G.L.
      • Grundy S.M.
      Lp(a) lipoprotein is an independent, discriminating risk factor for premature peripheral atherosclerosis among white men.
      ,
      • Schreiner P.J.
      • Morrisett J.D.
      • Sharrett A.R.
      • Patsch W.
      • Tyroler H.A.
      • Wu K.
      • Heiss G.
      Lipoprotein[a] as a risk factor for preclinical atherosclerosis.
      ). For example, in a cohort of 504 patients, Sam Tsimikas, Joe Witztum, and colleagues showed a strong association between high oxidized phospholipids and high Lp(a) concentrations with the presence and extent of coronary artery disease, detected by coronary angiography as the number of vessels with a stenosis of more than 50% of the luminal diameter (
      • Tsimikas S.
      • Brilakis E.S.
      • Miller E.R.
      • McConnell J.P.
      • Lennon R.J.
      • Kornman K.S.
      • Witztum J.L.
      • Berger P.B.
      Oxidized phospholipids, Lp(a) lipoprotein, and coronary artery disease.
      ). Also, in the InCHIANTI Study, 1,002 individuals aged above 60 years had their ankle-brachial index assessed over a 6 year period, and the authors found that high Lp(a) was an independent risk factor for peripheral arterial disease of the lower limbs (
      • Volpato S.
      • Vigna G.B.
      • McDermott M.M.
      • Cavalieri M.
      • Maraldi C.
      • Lauretani F.
      • Bandinelli S.
      • Zuliani G.
      • Guralnik J.M.
      • Fellin R.
      • et al.
      Lipoprotein(a), inflammation, and peripheral arterial disease in a community-based sample of older men and women (the InCHIANTI study).
      ). Finally, in a large case-control study, the odds ratio per LPA rs10455872 or rs3798220 risk alleles was 1.47 (95% CI: 1.33–1.62) for peripheral arterial disease (N = 5,215 cases) (
      • Helgadottir A.
      • Gretarsdottir S.
      • Thorleifsson G.
      • Holm H.
      • Patel R.S.
      • Gudnason T.
      • Jones G.T.
      • van Rij A.M.
      • Eapen D.J.
      • Baas A.F.
      • et al.
      Apolipoprotein(a) genetic sequence variants associated with systemic atherosclerosis and coronary atherosclerotic burden but not with venous thromboembolism.
      ).
      In contrast, high Lp(a) concentrations or LPA risk alleles have not been associated with early atherosclerosis measured as modest intima-media thickening in carotid or femoral arteries (
      • Helgadottir A.
      • Gretarsdottir S.
      • Thorleifsson G.
      • Holm H.
      • Patel R.S.
      • Gudnason T.
      • Jones G.T.
      • van Rij A.M.
      • Eapen D.J.
      • Baas A.F.
      • et al.
      Apolipoprotein(a) genetic sequence variants associated with systemic atherosclerosis and coronary atherosclerotic burden but not with venous thromboembolism.
      ,
      • Denti L.
      • Marchini L.
      • Pasolini G.
      • Baffoni M.T.
      • Ablondi F.
      • Valenti G.
      Lipoprotein Lp(a) and cerebrovascular disease in the elderly: correlations with the severity of extracranial carotid atherosclerosis assessed by ultrasonography.
      ,
      • Srámek A.
      • Reiber J.H.
      • Baak-Pablo R.
      • Sturk A.
      • Rosendaal F.R.
      Lipoprotein(a) and ultrasonographically determined early atherosclerotic changes in the carotid and femoral artery.
      ,
      • Grebe M.T.
      • Schoene E.
      • Schaefer C.A.
      • Boedeker R.H.
      • Kemkes-Matthes B.
      • Voss R.
      • Tillmanns H.H.
      Elevated lipoprotein(a) does not promote early atherosclerotic changes of the carotid arteries in young, healthy adults.
      ,
      • Kivimäki M.
      • Magnussen C.G.
      • Juonala M.
      • Kähönen M.
      • Kettunen J.
      • Loo B.M.
      • Lehtimäki T.
      • Viikari J.
      • Raitakari O.T.
      Conventional and Mendelian randomization analyses suggest no association between lipoprotein(a) and early atherosclerosis: the Young Finns Study.
      ,
      • Calmarza P.
      • Trejo J.M.
      • Lapresta C.
      • Lopez P.
      Relationship between lipoprotein(a) concentrations and intima-media thickness: a healthy population study.
      ,
      • Bos S.
      • Duvekot M.H.
      • Touw-Blommesteijn A.C.
      • Verhoeven A.J.
      • Mulder M.T.
      • Watts G.F.
      • Sijbrands E.J.
      • Roeters van Lennep J.E.
      Lipoprotein (a) levels are not associated with carotid plaques and carotid intima media thickness in statin-treated patients with familial hypercholesterolemia.
      ); however, there is controversy as to whether such intima-media thickness represents atherosclerosis per se. For example, in the Young Finns Study, including 939 men and 1,141 women, data from observational and Mendelian randomization analyses provided no support for early atherogenic effects of high Lp(a) concentrations (
      • Kivimäki M.
      • Magnussen C.G.
      • Juonala M.
      • Kähönen M.
      • Kettunen J.
      • Loo B.M.
      • Lehtimäki T.
      • Viikari J.
      • Raitakari O.T.
      Conventional and Mendelian randomization analyses suggest no association between lipoprotein(a) and early atherosclerosis: the Young Finns Study.
      ). Also, after pooling two studies and including 3,714 individuals, LPA rs10455872 or rs3798220 risk alleles were not associated with carotid intima-media thickness (
      • Helgadottir A.
      • Gretarsdottir S.
      • Thorleifsson G.
      • Holm H.
      • Patel R.S.
      • Gudnason T.
      • Jones G.T.
      • van Rij A.M.
      • Eapen D.J.
      • Baas A.F.
      • et al.
      Apolipoprotein(a) genetic sequence variants associated with systemic atherosclerosis and coronary atherosclerotic burden but not with venous thromboembolism.
      ).
      Taken together, a large number of studies unanimously show that high plasma Lp(a) concentrations are associated with high risk of coronary, carotid, and femoral atherosclerotic stenosis, with concordance between observational and causal genetic risk estimates. In contrast, there is no human evidence to support that high Lp(a) concentrations should cause early atherosclerosis in the form of increased intima-media thickness. That said, many researchers likely disagree with us on this topic, and many view aortic stenosis and carotid, femoral, or coronary stenosis as separate pathological entities.

      AORTIC VALVE STENOSIS

      In a 1995 study from Japan, it was observed in 347 men and 437 women that high age and high plasma Lp(a) concentrations were the best risk factors for aortic valve stenosis (sclerosis) (
      • Gotoh T.
      • Kuroda T.
      • Yamasawa M.
      • Nishinaga M.
      • Mitsuhashi T.
      • Seino Y.
      • Nagoh N.
      • Kayaba K.
      • Yamada S.
      • Matsuo H.
      Correlation between lipoprotein(a) and aortic valve sclerosis assessed by echocardiography (the JMS Cardiac Echo and Cohort Study).
      ). Likewise, in 1997, it was observed in 5,201 US individuals aged 65 and above, that age, male sex, and high plasma Lp(a) concentrations were the most important factors to discriminate between individuals with and without calcific aortic valve stenosis (disease) (
      • Stewart B.F.
      • Siscovick D.
      • Lind B.K.
      • Gardin J.M.
      • Gottdiener J.S.
      • Smith V.E.
      • Kitzman D.W.
      • Otto C.M.
      Clinical factors associated with calcific aortic valve disease. Cardiovascular Health Study.
      ). High Lp(a) concentration as an important risk factor for aortic valve stenosis was later confirmed (
      • Glader C.A.
      • Birgander L.S.
      • Soderberg S.
      • Ildgruben H.P.
      • Saikku P.
      • Waldenstrom A.
      • Dahlen G.H.
      Lipoprotein(a), Chlamydia pneumoniae, leptin and tissue plasminogen activator as risk markers for valvular aortic stenosis.
      ,
      • Bozbas H.
      • Yildirir A.
      • Atar I.
      • Pirat B.
      • Eroglu S.
      • Aydinalp A.
      • Ozin B.
      • Muderrisoglu H.
      Effects of serum levels of novel atherosclerotic risk factors on aortic valve calcification.
      ), and interestingly, in individuals with both high Lp(a) and high Chlamydia pneumoniae IgG antibodies the risk was particularly high (
      • Glader C.A.
      • Birgander L.S.
      • Soderberg S.
      • Ildgruben H.P.
      • Saikku P.
      • Waldenstrom A.
      • Dahlen G.H.
      Lipoprotein(a), Chlamydia pneumoniae, leptin and tissue plasminogen activator as risk markers for valvular aortic stenosis.
      ). Probably not too many noticed these early findings, and after all, this was observational evidence believed to be prone to confounding and reverse causation.
      Then in 2013, Thanassoulis et al. (
      • Thanassoulis G.
      • Campbell C.Y.
      • Owens D.S.
      • Smith J.G.
      • Smith A.V.
      • Peloso G.M.
      • Kerr K.F.
      • Pechlivanis S.
      • Budoff M.J.
      • Harris T.B.
      • et al.
      Genetic associations with valvular calcification and aortic stenosis.
      ) discovered that the LPA rs10455872 SNP, well-known as a high risk factor for myocardial infarction and coronary heart disease, was also found to be the best genetic causal risk factor for aortic valve calcification and stenosis: the per allele odds ratio for aortic valve calcification was 2.05 (95% CI: 1.63–2.57). This finding was replicated in additional White European, African-American, and Hispanic-American cohorts. In prospective analyses, this LPA SNP had a per allele hazard ratio for incident aortic valve stenosis of 1.68 (95% CI: 1.32–2.15) and 1.60 (95% CI: 1.12–2.28) in Swedish and Danish cohorts of the general population.
      In a subsequent study in 2014 based on the Copenhagen City Heart Study and the Copenhagen General Population Study combined, we showed a stepwise higher risk of aortic valve stenosis with stepwise higher extreme concentrations of Lp(a) (Fig. 9) (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Elevated lipoprotein(a) and risk of aortic valve stenosis in the general population.
      ). Multivariable adjusted hazard ratios for aortic valve stenosis were 1.2 (95% CI: 0.8–1.7) for 5–19 mg/dl, 1.6 (95% CI: 1.1–2.4) for 20–64 mg/dl, 2.0 (95% CI: 1.2–3.4) for 65–90 mg/dl, and 2.9 (95% CI: 1.8–4.9) for above 90 mg/dl, versus Lp(a) concentrations below 5 mg/dl. Also, combining LPA rs10455872, rs3798220, and KIV-2 number of repeats, instrumental variable analysis yielded a genetic causal risk ratio for aortic valve stenosis of 1.6 (95% CI: 1.2–2.1) for a 10-fold higher Lp(a) concentration, comparable to the observational hazard ratio of 1.4 (95% CI: 1.2–1.7) for a 10-fold higher plasma Lp(a) concentration. Interestingly, as observed in the same individuals from Copenhagen and for a comparable doubling in Lp(a) concentrations, the risk estimated for aortic valve stenosis appeared slightly higher than for myocardial infarction, with concordance between observational and causal genetic risk estimates (Fig. 7, compare middle and upper panels).
      Figure thumbnail gr9
      Fig. 9Observational associations between high plasma Lp(a) concentrations and risk of aortic valve stenosis in the Copenhagen City Heart Study and Copenhagen General Population Study combined. Hazard ratios were estimated by Cox proportional hazard regression models and were multivariable adjusted for age, sex, total cholesterol, HDL cholesterol, systolic blood pressure, smoking, and diabetes. Lp(a) in milligrams per deciliter is shown as median (interquartile range). Adapted from (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Elevated lipoprotein(a) and risk of aortic valve stenosis in the general population.
      ).
      Even more studies have now confirmed that high Lp(a) concentrations observationally and genetically represent strong causal risk factors for aortic valve calcification, stenosis, and stenosis progression (
      • Arsenault B.J.
      • Boekholdt S.M.
      • Dube M.P.
      • Rheaume E.
      • Wareham N.J.
      • Khaw K.T.
      • Sandhu M.S.
      • Tardif J.C.
      Lipoprotein(a) levels, genotype, and incident aortic valve stenosis: a prospective Mendelian randomization study and replication in a case-control cohort.
      ,
      • Capoulade R.
      • Chan K.L.
      • Yeang C.
      • Mathieu P.
      • Bosse Y.
      • Dumesnil J.G.
      • Tam J.W.
      • Teo K.K.
      • Mahmut A.
      • Yang X.
      • et al.
      Oxidized phospholipids, lipoprotein(a), and progression of calcific aortic valve stenosis.
      ,
      • Vongpromek R.
      • Bos S.
      • Ten Kate G.J.
      • Yahya R.
      • Verhoeven A.J.
      • de Feyter P.J.
      • Kronenberg F.
      • Roeters van Lennep J.E.
      • Sijbrands E.J.
      • Mulder M.T.
      Lipoprotein(a) levels are associated with aortic valve calcification in asymptomatic patients with familial hypercholesterolaemia.
      ,
      • Yang N.
      • Zhang G.
      • Li X.
      • Zhou L.
      Correlation analysis between serum lipoprotein (a) and the incidence of aortic valve sclerosis.
      ,
      • Hojo Y.
      • Kumakura H.
      • Kanai H.
      • Iwasaki T.
      • Ichikawa S.
      • Kurabayashi M.
      Lipoprotein(a) is a risk factor for aortic and mitral valvular stenosis in peripheral arterial disease.
      ,
      • Nsaibia M.J.
      • Mahmut A.
      • Boulanger M.C.
      • Arsenault B.J.
      • Bouchareb R.
      • Simard S.
      • Witztum J.L.
      • Clavel M.A.
      • Pibarot P.
      • Bosse Y.
      • et al.
      Autotaxin interacts with lipoprotein(a) and oxidized phospholipids in predicting the risk of calcific aortic valve stenosis in patients with coronary artery disease.
      ). This is true for Whites and Asians alike.
      Together, these studies document high Lp(a) concentrations as one of the strongest causal risk factors for aortic valve stenosis, with risk estimates slightly higher than for myocardial infarction. As both these diseases are among the main causes of heart failure, high Lp(a) concentrations may also be a strong causal risk factor for this condition.

      HEART FAILURE

      Given the role of high Lp(a) concentrations in myocardial infarction and aortic valve stenosis, it seemed natural to explore the impact of Lp(a) on the end product of these two diseases in the form of heart failure. We combined the Copenhagen City Heart Study and the Copenhagen General Population Study with 98,097 Danish individuals, of whom 4,122 were diagnosed with heart failure from 1976 through 2013. High Lp(a) concentrations were associated with multivariable adjusted hazard ratios for heart failure of 1.10 (95% CI: 0.97–1.25) for 8–19 mg/dl, 1.24 (95% CI: 1.08–1.42) for 20–67 mg/dl, 1.57 (95% CI: 1.32–1.87) for 68–153 mg/dl, and of 1.79 (95% CI: 1.18–2.73) for concentrations above153 mg/dl, versus Lp(a) concentrations below 8 mg/dl. Of all heart failure in the population, high Lp(a) had a population-attributable fraction of 9%. This means that if high Lp(a) was not present in the Danish population, then heart failure prevalence would be 9% lower than current levels.
      By combining all LPA risk genotypes, instrumental variable analysis yielded a genetic causal risk ratio for heart failure of 1.18 (95% CI: 1.04–1.34) per 10-fold higher Lp(a) concentrations, which was comparable to the corresponding observational hazard ratio of 1.22 (95% CI: 1.11–1.35). Finally, in mediation analysis, 63% (95% CI: 45–99%) of heart failure risk due to high Lp(a) was mediated via myocardial infarction and aortic valve stenosis combined. Although 63% is a very high number in a mediation analysis, we naturally cannot exclude that high Lp(a) could lead to heart failure via yet another mechanism, e.g., through occlusion of small blood vessels in the myocardium.

      VENOUS THROMBOEMBOLISM

      Because high Lp(a) is a causal factor for myocardial infarction, and as there is ample evidence that Lp(a) interferes with fibrinolysis and likely promotes thrombosis (
      • Boffa M.B.
      • Koschinsky M.L.
      Lipoprotein (a): truly a direct prothrombotic factor in cardiovascular disease?.
      ), it seems logical that high Lp(a) should also be a strong causal risk factor for venous thromboembolism, that is, deep venous thrombosis and pulmonary embolism. Interestingly however, this is probably not the case except at extremely high Lp(a) concentrations (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Genetic evidence that lipoprotein(a) associates with atherosclerotic stenosis rather than venous thrombosis.
      ). Importantly, Lp(a) likely does not have inherent pro-thrombotic properties, but it may be anti-fibrinolytic, i.e., a clot has to be forming for Lp(a) to potentially affect growth. This implies a first hit of some other pro-thrombotic risk factor that is worsened by Lp(a), which might explain why different results on venous thromboembolism have been reported.
      Kamstrup, Tybjærg-Hansen, and Nordestgaard (
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Genetic evidence that lipoprotein(a) associates with atherosclerotic stenosis rather than venous thrombosis.
      ) found that highest versus lowest tertile of Lp(a) did not associate with high risk of venous thrombosis; however, the extreme top 5% versus the lowest 22% of the concentration distribution of Lp(a) yielded an odds ratio of 1.7 (95% CI: 1.2–2.3) for risk of venous thrombosis. For the purpose of the present review, we updated this analysis and now included 53,908 individuals from the Copenhagen General Population Study and the Copenhagen City Heart Study with a total of 2,501 events of venous thromboembolism, and correspondingly found at extremely high Lp(a) concentrations above 100 mg/dl compared with concentrations below 5 mg/dl, a hazard ratio of 1.33 (95% CI: 1.06–1.69) for risk of venous thromboembolism (Fig. 10).
      Figure thumbnail gr10
      Fig. 10