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Prediction of cardiovascular risk by Lp(a) concentrations or genetic variants within the LPA gene region

Prediction of cardiovascular risk by Lp(a) concentrations or genetic variants within the LPA gene... In the middle of the 1990s the interest in Lp(a) vanished after a few badly performed studies almost erased Lp(a) from the map of biological targets. However, since roughly 10 years the interest has begun to grow again mainly for two reasons: first, genetic studies using easily accessible and high-throughput techniques for genotyping of single-nucleotide polymorphisms (SNPs) have allowed large studies in patients with cardiovascular disease and controls to be performed. This strengthened the earlier findings on a copy number variation in the LPA gene and its association with cardiovascular outcomes. Second, new therapies are on the horizon raising strong and justified hope that in a few years drugs will become available which tremendously lower Lp(a) concentrations. This review article should provide an introduction to the genetic determination of Lp(a) concentrations and considerations whether Lp(a) concentrations or genetic variants are important for the prediction of cardiovascular risk. Keywords Lipoprotein(a) · Apolipoprotein(a) · Cardiovascular disease · Copy number variation · Association study · Mendelian randomization Introduction fore the lipoprotein with the strongest genetic control. The discovery of the size polymorphism of apo(a) in serum [7] An astonishing characteristic of Lp(a) is the more than andK-IV CNV inthe LPA gene [4, 8–10] resulted in the 1000-fold range of concentrations between individuals from identification of the LPA gene as the major gene for Lp(a) less than 0.1 mg/dL to more than 300 mg/dL [1]. Lp(a) con- levels. centrations are not influenced much by age, sex, fasting Individuals expressing a low number of K-IV repeats re- state, inflammation [2, 3] or lifestyle factors such as diet sulting in so-called small apo(a) isoforms (up to 22 K-IV or physical activity. However, the concentrations are under repeats) have on average markedly higher Lp(a) concentra- strict genetic control by the LPA gene locus and here espe- tions than individuals carrying only large apo(a) isoforms cially by a size polymorphism of apo(a) caused by a variable (more than 22 K-IV repeats) [1]. number of so called kringle IV (K-IV) repeats in the LPA gene [1, 4]. Each of these up to more than 40 repeats has Genetic regulation of Lp(a) concentrations a size of 5.6 kB which results in a highly polymorphic and and risk for CHD informative copy number variation (CNV). The substantial differences in Lp(a) concentrations be- tween individuals are to a large extent genetically deter- The evidence is quite strong that high Lp(a) concentrations mined. Family studies revealed a heritability estimate of are associated with an increasing risk for coronary heart Lp(a) concentrations of about 90% [5, 6]. Lp(a) is there- disease (CHD) [1]. The Copenhagen City Heart Study observed for individuals from a general population with concentrations between 30 and 76 mg/dL (corresponding This article is part of the special issue “Lp(a) – Update 2018” th th to the 67 –90 percentile) a 1.6-fold increased risk for Florian Kronenberg incident myocardial infarction compared to individuals Florian.Kronenberg@i-med.ac.at with Lp(a) concentrations below 5 mg/dL (corresponding to the lower 22% of the population). This risk increased Division of Genetic Epidemiology, Department of Medical to 1.90 for persons with Lp(a) concentrations between 77 Genetics, Molecular and Clinical Pharmacology, Medical th th and 117 mg/dL (90 –95 percentile) and to 2.60 for indi- University of Innsbruck, Schöpfstr. 41, 6020 Innsbruck, th Austria viduals with Lp(a) concentrations above 117 mg/dL (>95 K 6 F. Kronenberg Determined at the me of concepon b c % of controls / patients with small apo(a) isoforms Molecular Weight of Apo(a) . 100 60 low (LMW) high (HMW) Controls CHD = small = large 80 Combined OR = 1.78 Small apo(a) p<0.001 isoforms 60 53 50 30 25-35% of the 30 population 20 13 9 10 7 7 0,6 11-16 17-19 20-22 23-25 26-28 29-31 32-34 35-37 >37 Null Tyrolean Welsh German Israeli Chinese Indian Number of KIV Repeats Populaons strong association strong association causal association High Lp(a) CVD Lp(a) concentraons Percenle mg/dL HR 95%CI th >95 >117 2.6 1.6 - 4.1 th th 90 -95 77-117 1.9 1.2 - 3.0 th th 67 -89 30-76 1.6 1.1 - 1.2 nd th 22 -66 5-29 1.2 0.9 - 1.6 nd <22 <5 reference Fig. 1 Mendelian randomization approach to demonstrate a causal association between Lp(a) concentrations and cardiovascular disease. Panel a shows the association between elevated Lp(a) concentrations and cardiovascular disease (CVD) as shown in the Copenhagen City Heart Study [11]. Panel b shows the association between the number of K-IV repeats in the LPA gene and Lp(a) concentrations: individuals with small apo(a) isoform have markedly higher median Lp(a) concentrations than individuals with large apo(a) isoforms [23]. Panel c shows the preponderance of small apo(a) isoforms in patients with CVD when compared to controls [27]. Since a low number of K-IV copies (11–22 copies) is associated with high Lp(a) levels and high Lp(a) levels are associated with CVD, it follows that a low number of K-IV copies has to be associated with CVD if the association of Lp(a) with CVD is causal. Figure is taken and adapted with permission from reference [29]. Lp(a) lipoprotein(a), apo(a) apolipoprotein(a), LMW low molecular weight, HMW high molecular weight, KIV kringle IV, CVD cardiovascular disease, CHD coronary heart disease, OR odds ratio, 95%CI 95% confidence interval percentile) ([11]; Fig. 1a). The concentration threshold When a biomarker is changed in diseased patients, the for an increased risk has been discussed controversially important discussion starts whether this biomarker is a risk and a European Atherosclerosis Society (EAS) consensus factor or a risk marker. If it is a risk factor, this parame- statement proposed 50 mg/dL [12]. Most importantly, such ter is causally related to disease and it might become an th a threshold corresponds to the 80 percentile of the concen- interesting drug target. If it is a biomarker, this parameter tration distribution in a Caucasian population and means might be interesting for diagnostic purposes because it is that 20% of the population have probably an increased changed secondarily to the disease (often also called re- risk for CVD due to elevated Lp(a) concentrations. From verse causation), it draws the attention to a disease but it a standpoint of public health relevance, this makes Lp(a) would not make sense to develop drugs which influence to a very important risk factor for CVD. that parameter (see recent discussion on this issue in ref- Besides numerous studies on CHD, during recent years erence [26]). This discussion took place for Lp(a) in the several studies found even a strong association between middle of the 1990s and Mendelian randomization studies high Lp(a) concentrations and aortic valve calcification, illustrated in Fig. 1 provided a strong support for causal- stroke [13], and stenosis [14–21], heart failure [22]as well ity. Genetic variants that are strongly associated with high as peripheral arterial disease [23–25]. This makes Lp(a) Lp(a) concentrations (Fig. 1b) show also an increased risk a risk factor for many CVD endpoints although the strength with CVD (Fig. 1c) which underscores the causal link be- of the association might differ between the various entities tween high Lp(a) concentrations and CVD. The first time of endpoints. applying such an approach used data on the small apo(a) Median Lp(a) mg/dL Lipoprotein(a) and cardiovascular risk 7 Table 1 Advantages and disadvantages of various methods to investigate the association of Lp(a) concentrations and genetic polymorphisms within the LPA gene region with clinical outcomes Method Advantages Disadvantages Lp(a) concentrations Some assays are still not fully standardized High-throughput Concentration of the biological active lipopro- tein is measured Some assays are not independent from apo(a) isoform No genetic counseling required Most suitable for risk prediction Western blot analysis of K-IV Laborious Only expressed isoforms are measured repeats No genetic counseling required since mea- sured in serum Very suitable for risk prediction (especially in certain patient groups) Pulsed-field gel electrophoresis Provides K-IV repeats at the DNA level for Ignores expression status of the isoforms (PFGE) or fiber-fluorescence in each apo(a) isoform Extremely laborious situ hybridization (FISH) Suitable for risk prediction Requires special DNA preparation Genetic counseling required Quantitative polymerase chain High-throughput Provides sum of K-IV repeats but does not distinguish reaction (qPCR) the two isoforms Ignores expression status of the two isoforms Less good for risk prediction Genetic counseling required Single nucleotide Easy to analyze in routine lab The two most widely used SNPs (rs10455872 and polymorphisms (SNPs) rs3798220) tag only half of the small apo(a) isoforms resulting in a large frequency of false negatives Ignores expression status Less good for risk prediction Genetic counseling required Lp(a) lipoprotein(a), K-IV kringle IV, apo(a) apolipoprotein(a) Whether genetic counseling is required depends of the laws of the respective countries isoforms determined by the number of K-IV repeats: small Measurement of Lp(a) concentrations isoforms with up to 22 K-IV repeats (which were called F, B, S1 and S2 in earlier times) were associated with a sig- From a clinical standpoint, the measurement of Lp(a) con- nificantly increased risk for CHD in 6 different populations centrations is the method of choice to estimate the risk as- [27]. A later meta-analysis including 7382 CHD cases and sociated with this atherogenic lipoprotein since we assume 8514 controls identified a 2.08-fold increased risk for car- that we are measuring the biologically active lipoprotein. riers of small apo(a) isoforms [28]. This strong association However, the measurement of Lp(a) is trickier compared to probably makes Lp(a) the most important genetic risk fac- other lipoproteins. A multitude of immunochemical Lp(a) tor for CVD if we keep the high frequency of small apo(a) assays have been developed over time and the use of dif- isoforms in the population in mind [29]. ferent calibrators and antibodies have resulted in a wide range of Lp(a) measurement values not readily comparable between different assays [30]. The major problem is the What methods are available for CVD risk repetitive K-IV structure which causes the size polymor- estimation? phism with dozens of isoforms. Most of the antibodies used in the various assays are not exactly characterized and are Table 1 provides an overview on the methods available and probably directed against the repetitive K-IV type 2 struc- their advantages and disadvantages to investigate the as- ture which ranges from 2 to more than 40 repeats. This may sociation between Lp(a) concentrations and their genetic result in a measurement bias where serum concentrations of determinants with clinical outcomes. small isoforms, which are usually associated with elevated levels, are underestimated, while serum concentrations of large isoforms, usually associated with low levels, are over- estimated. However, we have to distinguish between the rel- ative and the absolute bias of the measurement in relation K 8 F. Kronenberg to the apo(a) isoforms. Thorough comparisons between an requires a special DNA preparation to yield high-molecu- isoform-sensitive and an isoform-insensitive assay by Mar- lar DNA but allows determination of the number of K-IV covina and colleagues [31] revealed that the relative bias type 2 copies in separated alleles. Both alleles will also be can become quite high with an overestimation of 25–35% separated by fiber-fluorescence in situ hybridization (FISH) in carriers of large isoforms. However, this translates to an that allows the number of repeats to be counted under fluo- absolute bias in most of the samples of a few mg/dL. The rescence microscopy [32]. In contrast to these two methods, relative bias for most of the carriers of small isoforms is quantitative polymerase chain reaction (qPCR) is doable in around 10% which translates also only to a few mg/dL. high-throughput [11] but provides the total number (sum) However, in the rare cases with up to 16 K-IV repeats, the of the K-IV type 2 copies of the two alleles of the investi- relative and the absolute bias can become quite pronounced gated genome. This means that individuals with one short with an underestimation of the concentration and therefore and one large allele and individuals with two medium-sized the risk for outcomes. The proportion of these individuals alleles end up in the same risk category which might result in the general population is rather low: only 1.9% of the in an underestimation of the risk for the person with one population turned out to have such small isoforms when short and one large allele. This is in line with the largest we investigated more than 24,000 individuals with west- single-center case–control and prospective study up to now ern blot analyses (unpublished data). In summary, when describing that individuals in the lower quartile of the sum interpreting the data from Marcovina and colleagues [31], of copy number in their genome had an adjusted hazard we would expect that for most of the individuals this does ratio for myocardial infarction of 1.50 compared to those not really cause a major underestimation or overestimation in the highest quartile of copy number. This estimate is of the risk. Mainly in the grey zone around the proposed lower than that from studies performing the calculations threshold of Lp(a) concentrations, this might become an based on the expressed isoforms in western blot with a rel- issue. However, we have to keep in mind that this thresh- ative risk of 2.08 [28]. The K-IV type-2 CNV measured by old is also under discussion whether it should be at 30 or qPCR explained only 25% of the variability in Lp(a) levels 50 mg/dL. Moreover, it has to be added that these data are [11], which is also markedly lower than in other studies proven for the assays Marcovina and colleagues have inves- of European populations using methods such as apo(a) iso- tigated [31]. Whether the absolute and relative biases are forms by western blot or separated alleles by pulsed-field similar for other assays needs to be seen and some of the gel electrophoresis. These observations might be related to assays have been compared to the assay from Marcovina the circumstance that qPCR is not easy to standardize with and colleagues. a relatively high coefficient of variation which makes com- parison of results between laboratories difficult. Investigation of the K-IV repeat number by western Based on the peculiarity that roughly 30 to 50% of all blot and DNA analysis individuals express only one apo(a) isoform in serum al- though they have two isoforms at the DNA level, points to The apo(a) isoform size can be best investigated in serum some limitations of the DNA analysis. It raises the question or plasma by western blot using an SDS agarose gel elec- from a clinical standpoint why one should be interested in trophoresis. It has the advantage that only those isoforms an isoform that is not expressed. This raises also the ques- will be visible which are indeed expressed. This is one of tion whether it is better to simply consider Lp(a) concen- the peculiarities of Lp(a): although 95% of the subjects trations instead of the genetic determinants of the concen- are heterozygous on the DNA level, only 50–70% of the trations for risk prediction. However, earlier data suggested individuals show two isoforms in serum. In the remain- that Lp(a) from small apo(a) isoforms have a higher athero- ing 30–50% only a minor fraction is indeed homozygous genic potential [33]. This finding needs to be further evalu- (which cannot be visualized in the western blot) but the ma- ated. Furthermore, certain frequent disease conditions such jority of them express only one isoform in serum. The exact as chronic kidney disease result in pronounced changes of number of K-IV repeats can be measured with a precision Lp(a) concentrations. In these patients small apo(a) isoform of ±1–2 K-IV repeats. However, the method is laborious were demonstrated to be more predictive for CVD risk than and only available in a few laboratories. Until today, it is Lp(a) concentrations [34, 35] (for review see [1, 36]). still the best method to be used for Mendelian randomiza- tion studies to support causality between Lp(a) and various Investigation of single nucleotide polymorphisms outcomes (Fig. 1). (SNPs) Three methods to investigate apo(a) isoforms in terms of number of K-IV repeats on DNA level are available One of the reasons for a revival of the Lp(a) field ten years (Table 1). Pulsed-field gel electrophoresis (PFGE)/southern ago is based on the identification of SNPs that are strongly blotting of genomic DNA [9, 10] is extremely laborious, associated with CHD risk. Clarke and colleagues investi- K Lipoprotein(a) and cardiovascular risk 9 rs10455872 rs10455872 DIoGRAMplusC4D consortium. From all 49 genetic vari- or rs3798220 or rs3798220 ants that were shown to be independently associated with Non-Carriers Non-Carriers Apo(a) phenotypes Lp(a) concentrations, 40 were present in summary-level 97% 51% data retrieved from the CARDIoGRAMplusC4D consor- Do they get the wrong message? tium. Seven SNPs were even significantly associated with CAD on a genome-wide scale. This means that a SNP score Large Small with more SNPs than rs10455872 and rs3798220 might be 76% 24% better suitable for risk prediction [40]. SNPs in the kringle-IV type 2 region—a journey to 3% 49% a white spot on the genetic map Carriers Carriers Fig. 2 Apolipoprotein(a) [apo(a)] phenotypes and the carrier and non- The K-IV type 2 is a region that is difficult to resolve by carrier status of the two single-nucleotide polymorphisms rs10455872 conventional DNA analysis methods. Variants within the and rs3798220 derived from a large Caucasian general population sam- K-IV type 2 region cannot be detected in common se- ple of 5999 individuals quencing projects, leaving up to 70% of the LPA coding region currently unaddressed. We recently developed an gated27SNPsin the LPA gene region and observed two ultra-deep sequencing protocol and an easy-to-use variant SNPs (rs10455872 and rs3798220) to be strongly associated analysis pipeline to create a first map of genetic variation not only with Lp(a) concentrations but also with CHD [37]. in the K-IV type 2 region. We found dozens of loss-of- These SNPs are easy to analyze in a routine laboratory and function and splice site mutations, as well as >100 par- might have contributed to their popularity. However, these tially even common missense variants. This provides novel two SNPs have a minor allele frequency of 7 and 2%, re- candidates to explain the large ethnic and individual differ- spectively and about 15% of a typical Caucasian population ences in Lp(a) concentrations [41]. One of these variants carry at least one minor allele of the two SNPs. They were is a splice site variant (G4925A) in preferential associa- claimed to identify carriers of small apo(a) isoforms [37]. tion with the smaller apo(a) isoforms [42]. It has an ex- However, investigations in a large general Caucasian popu- ceptionally high carrier frequency of 22.1% in the general lation sample of 5999 subjects revealed that roughly half of population and explains 20.6% of the Lp(a) variance in all subjects expressing a small apo(a) isoform are not car- carriers of small apo(a) isoforms. It reduces Lp(a) concen- riers of the minor alleles of one or two of these SNPs [38]. trations dramatically by more than 30 mg/dL. Accordingly, This means that about half of the individuals who have the odds ratio for CVD was reduced from 1.39 for wild- a high risk for CHD based on an expressed small apo(a) type carriers of small isoforms to 1.19 in carriers of small isoform will not be detected when only these two SNPs are isoforms who were additionally positive for G4925A [42]. genotyped. These individuals might get the wrong message Functional studies pointed towards a reduction of splicing when only the SNP result and not also the Lp(a) concentra- efficiency highlighting splicing efficiency modulation by tion will be investigated and considered for risk counselling antisense oligos or transsplicing [43] as a potential novel (Fig. 2). Moreover, the situation might be different for var- Lp(a)-lowering approach [42]. ious ethnicities. For example, rs3798220 was not found in Africans. Allele frequencies in East and Southeast Asians Which approach is now the most suitable for ranged from 2.9 to 11.6%, and were very low (0.15%) in clinical risk assessment? CAD cases and controls from India. The variant was neither associated with small K-IV CNV alleles nor elevated Lp(a) concentrations in Asians [39]. The measurement of Lp(a) concentrations by a well-per- In a recent genome-wide association analysis including forming and standardized assay is usually first choice and 13,781 individuals we observed 2001 SNPs in the wider sufficient to get an estimate for the expected risk for clinical LPA gene region which were significantly associated with outcomes. The Lp(a) concentration might reflect best the bi- Lp(a) concentrations [40]. Many of the SNPs were strongly ologically active lipoprotein. Furthermore, since a protein correlated to each other and an in-depth analysis of the re- and not a genetic polymorphism is measured, no genetic gion identified 48 SNPs to be independently associated with counseling is required in most countries. Lp(a) concentrations. In addition to this region the SNP in There is at least some evidence (but we would wish to the APOE region that is responsible for the apoE2 allele have more) that two persons who have the same high Lp(a) (rs7412) was also associated with Lp(a) concentrations. In concentration but the one has a large isoform and the other a further step, we made a look-up in the data from the CAR- has a small isoform, that the latter has a higher risk which K 10 F. Kronenberg would mean that the smaller isoform (with fewer K-IV re- isoforms, which are usually associated with increased CVD peats) might be more atherogenic. Therefore, the measure- risk and overestimate Lp(a) concentrations of large isoforms ment of the isoform with western blot might provide a fur- which are usually associated with a lower CVD risk. Since ther argument for an increased CVD risk. From all methods small isoforms are overrepresented in CVD patients com- that investigate apo(a) genetic polymorphisms, it provides pared to a control group [27, 28], this phenomenon can the most comprehensive information and is second to the result in a dilution of the Lp(a) concentration differences Lp(a) concentrations very suitable for risk prediction. The between CVD patients and controls. Therefore, negative advantage can probably be explained by the fact that we studies on the association between Lp(a) concentrations and see only the isoforms which are indeed expressed in serum outcomes should exclude that an isoform-sensitivity of the or plasma and not those which are available on the DNA assay has contributed to these results. level but which are suppressed for whatever reasons. The measurement of the apo(a) isoforms adds also cer- Storage effects on Lp(a) concentrations tain information in acquired diseases which are associated with a secondary increase in Lp(a) concentrations. A typi- Some assays are strongly influenced by the effect of stor- cal example is chronic kidney disease: we observed espe- age conditions. This is especially the case for long-termed cially in patients treated by hemodialysis that they show stored samples from epidemiological studies. Extreme ex- an increase in Lp(a) concentrations and this relative in- amples are a nearly linear decrease in Lp(a) immunoreac- crease by the disease is especially observed in patients with tivity of 46% during 6 months of storage [46]or75% lower large apo(a) isoforms when compared to controls with the Lp(a) values after 600 days of storage compared to fresh same isoform categories [44]. Therefore, the risk for CVD samples [47] in two different studies. We observed with our events might be highest especially in those patients with assay in 310 samples on average only a small decrease of small isoforms (which we and others observed indeed [34, Lp(a) of 4.83% during a 25 months storage period but this 35, 45]) since their exposure to high Lp(a) concentrations was higher for individuals with small isoforms compared lasted already their entire life. In contrast, many patients to those with large isoforms [48]. with large isoforms might have experienced the increase in An effect of sample storage could also be a reason for Lp(a) only recently with the development of chronic kid- negative findings in epidemiological studies which used ney disease and the risk might no longer be sufficiently long-term stored samples und using an assay which is even predictable simply by the Lp(a) concentrations [36]. more strongly influenced by sample storage conditions The use of SNPs for risk estimation will have to compared to our assay [46, 47]. Some of the earlier studies find its place in the future. Using the famous two SNPs which used assays not appropriately performing in stored (rs10455872 and rs3798220) described 10 years ago might samples were also included in later meta-analyses and be less efficient since too many false negative results might might have contributed to an underestimation of the asso- be found as illustrated in Fig. 2 (individuals with small ciation between Lp(a) concentrations and cardiovascular apo(a) isoforms and high Lp(a) concentrations despite the disease [13] compared to another large single-center study non-mutated variants of these SNPs) [38]. A SNP score using one well-validated assay [11]. with a large and growing package of SNPs might be more informative [40]. Using SNPs or SNP scores for individu- Samples size in clinical and epidemiological studies als should always be done in combination with the Lp(a) concentrations. The same holds true for apo(a) isoforms It is a widely observed phenomenon in epidemiology that for which the Lp(a) concentration has to be known anyway the first group to report a significant test result (the winner) for the laboratory process which requires that roughly the will also report an effect size much larger than is likely to same amount of Lp(a) is put on the SDS agarose gel. be seen in subsequent replication studies [49]. This phe- nomenon of “winner’s curse” is often seen in Lp(a) re- search which is caused by an underpowered study with Individual risk assessment versus a pronounced right-skewed distribution of the Lp(a) con- epidemiological studies centrations where a large proportion of the population has low concentrations. Since the concentrations of Lp(a) are Apo(a) isoform-sensitivity of assays strongly determined by genetic variants with major effect sizes and less by other conditions, usually large sample The use of an apo(a) isoform-sensitive assay might result in sizes are required to prove a statistically significant differ- an underestimation of the association between Lp(a) con- ence which is not caused by chance findings. As discussed centrations and clinical outcomes. As mentioned above, earlier and demonstrated by simulations, small sample sizes such assays underestimate serum concentrations of small can cause any result between a patient and a control group K Lipoprotein(a) and cardiovascular risk 11 6. Austin MA, Sandholzer C, Selby JV, Newman B, Krauss RM, [44] or any correlation between Lp(a) concentrations and Utermann G (1992) Lipoprotein(a) in women twins: heritability another parameter [3] if the sample size is small enough. To and relationship to apolipoprotein(a) phenotypes. 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E, Kovanen PT, Kuivenhoven JA, Lesnik P, Masana L, Reiner Z, Taskinen MR, Tokgozoglu L, Tybjaerg-Hansen A (2010) Lipopro- Other methods to measure the K-IV repeat number are of tein(a) as a cardiovascular risk factor: current status. Eur Heart J academic interest. SNPs will have to find their role prob- 31:2844–2853 ably more as a SNP score and there is currently no major 13. Erqou S, Kaptoge S, Perry PL, Di AE, Thompson A, White added value to be used beside or instead the measurement IR, Marcovina SM, Collins R, Thompson SG, Danesh J (2009) Lipoprotein(a) concentration and the risk of coronary heart disease, of Lp(a) concentrations. stroke, and nonvascular mortality. JAMA 302:412–423 14. Thanassoulis G, Campbell CY, Owens DS, Smith JG, Smith AV, Compliance with ethical guidelines Peloso GM, Kerr KF, Pechlivanis S, Budoff MJ, Harris TB, Mal- hotra R, O’Brien KD, Kamstrup PR, Nordestgaard BG, Tybjaerg- Conflict of interest F. Kronenberg declares to have received speaker Hansen A, Allison MA, Aspelund T, Criqui MH, Heckbert SR, honoraria from Kaneka, Miltenyi Biotec and Amgen. He is member of Hwang SJ, Liu Y, Sjogren M, van der Pals J, Kalsch H, Muhleisen advisory boards from Kaneka and Amgen. TW, Nothen MM, Cupples LA, Caslake M, Di AE, Danesh J, Rot- ter JI, Sigurdsson S, Wong Q, Erbel R, Kathiresan S, Melander Ethical standards This article does not contain any studies with human O, Gudnason V, O’Donnell CJ, Post WS (2013) Genetic associa- participants or animals performed by any of the authors. tions with valvular calcification and aortic stenosis. N Engl J Med 368:503–512 Open Access This article is distributed under the terms of the 15. 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Kronenberg F, Kronenberg MF, Kiechl S, Trenkwalder E, San- Gimple LW (1995) Elevated serum lipoprotein(a) is a risk factor for ter P, Oberhollenzer F, Egger G, Utermann G, Willeit J (1999) clinical recurrence after coronary balloon angioplasty. Circulation Role of lipoprotein(a) and apolipoprotein(a) phenotype in athero- 91:1403–1409 genesis: prospective results from the Bruneck Study. Circulation 48. Kronenberg F, Trenkwalder E, Dieplinger H, Utermann G (1996) 100:1154–1160 Lipoprotein(a) in stored plasma samples and the ravages of time: 34. Kronenberg F, Neyer U, Lhotta K, Trenkwalder E, Auinger M, Prib- why epidemiological studies might fail. Arterioscler Thromb Vasc asnig A, Meisl T, König P, Dieplinger H (1999) The low molecu- Biol 16:1568–1572 lar weight apo(a) phenotype is an independent predictor for coro- 49. Ioannidis JP (2008) Why most discovered true associations are in- nary artery disease in hemodialysis patients: a prospective follow- flated. Epidemiology 19:640–648 up. J Am Soc Nephrol 10:1027–1036 35. Longenecker JC, Klag MJ, Marcovina SM, Liu YM, Jaar BG, Powe NR, Fink NE, Levey AS, Coresh J (2005) High lipoprotein(a) levels http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Clinical Research in Cardiology Supplements Springer Journals

Prediction of cardiovascular risk by Lp(a) concentrations or genetic variants within the LPA gene region

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Copyright © 2019 by The Author(s)
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Medicine & Public Health; Cardiology; Internal Medicine; Angiology; Cardiac Surgery; Diagnostic Radiology
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10.1007/s11789-019-00093-5
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Abstract

In the middle of the 1990s the interest in Lp(a) vanished after a few badly performed studies almost erased Lp(a) from the map of biological targets. However, since roughly 10 years the interest has begun to grow again mainly for two reasons: first, genetic studies using easily accessible and high-throughput techniques for genotyping of single-nucleotide polymorphisms (SNPs) have allowed large studies in patients with cardiovascular disease and controls to be performed. This strengthened the earlier findings on a copy number variation in the LPA gene and its association with cardiovascular outcomes. Second, new therapies are on the horizon raising strong and justified hope that in a few years drugs will become available which tremendously lower Lp(a) concentrations. This review article should provide an introduction to the genetic determination of Lp(a) concentrations and considerations whether Lp(a) concentrations or genetic variants are important for the prediction of cardiovascular risk. Keywords Lipoprotein(a) · Apolipoprotein(a) · Cardiovascular disease · Copy number variation · Association study · Mendelian randomization Introduction fore the lipoprotein with the strongest genetic control. The discovery of the size polymorphism of apo(a) in serum [7] An astonishing characteristic of Lp(a) is the more than andK-IV CNV inthe LPA gene [4, 8–10] resulted in the 1000-fold range of concentrations between individuals from identification of the LPA gene as the major gene for Lp(a) less than 0.1 mg/dL to more than 300 mg/dL [1]. Lp(a) con- levels. centrations are not influenced much by age, sex, fasting Individuals expressing a low number of K-IV repeats re- state, inflammation [2, 3] or lifestyle factors such as diet sulting in so-called small apo(a) isoforms (up to 22 K-IV or physical activity. However, the concentrations are under repeats) have on average markedly higher Lp(a) concentra- strict genetic control by the LPA gene locus and here espe- tions than individuals carrying only large apo(a) isoforms cially by a size polymorphism of apo(a) caused by a variable (more than 22 K-IV repeats) [1]. number of so called kringle IV (K-IV) repeats in the LPA gene [1, 4]. Each of these up to more than 40 repeats has Genetic regulation of Lp(a) concentrations a size of 5.6 kB which results in a highly polymorphic and and risk for CHD informative copy number variation (CNV). The substantial differences in Lp(a) concentrations be- tween individuals are to a large extent genetically deter- The evidence is quite strong that high Lp(a) concentrations mined. Family studies revealed a heritability estimate of are associated with an increasing risk for coronary heart Lp(a) concentrations of about 90% [5, 6]. Lp(a) is there- disease (CHD) [1]. The Copenhagen City Heart Study observed for individuals from a general population with concentrations between 30 and 76 mg/dL (corresponding This article is part of the special issue “Lp(a) – Update 2018” th th to the 67 –90 percentile) a 1.6-fold increased risk for Florian Kronenberg incident myocardial infarction compared to individuals Florian.Kronenberg@i-med.ac.at with Lp(a) concentrations below 5 mg/dL (corresponding to the lower 22% of the population). This risk increased Division of Genetic Epidemiology, Department of Medical to 1.90 for persons with Lp(a) concentrations between 77 Genetics, Molecular and Clinical Pharmacology, Medical th th and 117 mg/dL (90 –95 percentile) and to 2.60 for indi- University of Innsbruck, Schöpfstr. 41, 6020 Innsbruck, th Austria viduals with Lp(a) concentrations above 117 mg/dL (>95 K 6 F. Kronenberg Determined at the me of concepon b c % of controls / patients with small apo(a) isoforms Molecular Weight of Apo(a) . 100 60 low (LMW) high (HMW) Controls CHD = small = large 80 Combined OR = 1.78 Small apo(a) p<0.001 isoforms 60 53 50 30 25-35% of the 30 population 20 13 9 10 7 7 0,6 11-16 17-19 20-22 23-25 26-28 29-31 32-34 35-37 >37 Null Tyrolean Welsh German Israeli Chinese Indian Number of KIV Repeats Populaons strong association strong association causal association High Lp(a) CVD Lp(a) concentraons Percenle mg/dL HR 95%CI th >95 >117 2.6 1.6 - 4.1 th th 90 -95 77-117 1.9 1.2 - 3.0 th th 67 -89 30-76 1.6 1.1 - 1.2 nd th 22 -66 5-29 1.2 0.9 - 1.6 nd <22 <5 reference Fig. 1 Mendelian randomization approach to demonstrate a causal association between Lp(a) concentrations and cardiovascular disease. Panel a shows the association between elevated Lp(a) concentrations and cardiovascular disease (CVD) as shown in the Copenhagen City Heart Study [11]. Panel b shows the association between the number of K-IV repeats in the LPA gene and Lp(a) concentrations: individuals with small apo(a) isoform have markedly higher median Lp(a) concentrations than individuals with large apo(a) isoforms [23]. Panel c shows the preponderance of small apo(a) isoforms in patients with CVD when compared to controls [27]. Since a low number of K-IV copies (11–22 copies) is associated with high Lp(a) levels and high Lp(a) levels are associated with CVD, it follows that a low number of K-IV copies has to be associated with CVD if the association of Lp(a) with CVD is causal. Figure is taken and adapted with permission from reference [29]. Lp(a) lipoprotein(a), apo(a) apolipoprotein(a), LMW low molecular weight, HMW high molecular weight, KIV kringle IV, CVD cardiovascular disease, CHD coronary heart disease, OR odds ratio, 95%CI 95% confidence interval percentile) ([11]; Fig. 1a). The concentration threshold When a biomarker is changed in diseased patients, the for an increased risk has been discussed controversially important discussion starts whether this biomarker is a risk and a European Atherosclerosis Society (EAS) consensus factor or a risk marker. If it is a risk factor, this parame- statement proposed 50 mg/dL [12]. Most importantly, such ter is causally related to disease and it might become an th a threshold corresponds to the 80 percentile of the concen- interesting drug target. If it is a biomarker, this parameter tration distribution in a Caucasian population and means might be interesting for diagnostic purposes because it is that 20% of the population have probably an increased changed secondarily to the disease (often also called re- risk for CVD due to elevated Lp(a) concentrations. From verse causation), it draws the attention to a disease but it a standpoint of public health relevance, this makes Lp(a) would not make sense to develop drugs which influence to a very important risk factor for CVD. that parameter (see recent discussion on this issue in ref- Besides numerous studies on CHD, during recent years erence [26]). This discussion took place for Lp(a) in the several studies found even a strong association between middle of the 1990s and Mendelian randomization studies high Lp(a) concentrations and aortic valve calcification, illustrated in Fig. 1 provided a strong support for causal- stroke [13], and stenosis [14–21], heart failure [22]as well ity. Genetic variants that are strongly associated with high as peripheral arterial disease [23–25]. This makes Lp(a) Lp(a) concentrations (Fig. 1b) show also an increased risk a risk factor for many CVD endpoints although the strength with CVD (Fig. 1c) which underscores the causal link be- of the association might differ between the various entities tween high Lp(a) concentrations and CVD. The first time of endpoints. applying such an approach used data on the small apo(a) Median Lp(a) mg/dL Lipoprotein(a) and cardiovascular risk 7 Table 1 Advantages and disadvantages of various methods to investigate the association of Lp(a) concentrations and genetic polymorphisms within the LPA gene region with clinical outcomes Method Advantages Disadvantages Lp(a) concentrations Some assays are still not fully standardized High-throughput Concentration of the biological active lipopro- tein is measured Some assays are not independent from apo(a) isoform No genetic counseling required Most suitable for risk prediction Western blot analysis of K-IV Laborious Only expressed isoforms are measured repeats No genetic counseling required since mea- sured in serum Very suitable for risk prediction (especially in certain patient groups) Pulsed-field gel electrophoresis Provides K-IV repeats at the DNA level for Ignores expression status of the isoforms (PFGE) or fiber-fluorescence in each apo(a) isoform Extremely laborious situ hybridization (FISH) Suitable for risk prediction Requires special DNA preparation Genetic counseling required Quantitative polymerase chain High-throughput Provides sum of K-IV repeats but does not distinguish reaction (qPCR) the two isoforms Ignores expression status of the two isoforms Less good for risk prediction Genetic counseling required Single nucleotide Easy to analyze in routine lab The two most widely used SNPs (rs10455872 and polymorphisms (SNPs) rs3798220) tag only half of the small apo(a) isoforms resulting in a large frequency of false negatives Ignores expression status Less good for risk prediction Genetic counseling required Lp(a) lipoprotein(a), K-IV kringle IV, apo(a) apolipoprotein(a) Whether genetic counseling is required depends of the laws of the respective countries isoforms determined by the number of K-IV repeats: small Measurement of Lp(a) concentrations isoforms with up to 22 K-IV repeats (which were called F, B, S1 and S2 in earlier times) were associated with a sig- From a clinical standpoint, the measurement of Lp(a) con- nificantly increased risk for CHD in 6 different populations centrations is the method of choice to estimate the risk as- [27]. A later meta-analysis including 7382 CHD cases and sociated with this atherogenic lipoprotein since we assume 8514 controls identified a 2.08-fold increased risk for car- that we are measuring the biologically active lipoprotein. riers of small apo(a) isoforms [28]. This strong association However, the measurement of Lp(a) is trickier compared to probably makes Lp(a) the most important genetic risk fac- other lipoproteins. A multitude of immunochemical Lp(a) tor for CVD if we keep the high frequency of small apo(a) assays have been developed over time and the use of dif- isoforms in the population in mind [29]. ferent calibrators and antibodies have resulted in a wide range of Lp(a) measurement values not readily comparable between different assays [30]. The major problem is the What methods are available for CVD risk repetitive K-IV structure which causes the size polymor- estimation? phism with dozens of isoforms. Most of the antibodies used in the various assays are not exactly characterized and are Table 1 provides an overview on the methods available and probably directed against the repetitive K-IV type 2 struc- their advantages and disadvantages to investigate the as- ture which ranges from 2 to more than 40 repeats. This may sociation between Lp(a) concentrations and their genetic result in a measurement bias where serum concentrations of determinants with clinical outcomes. small isoforms, which are usually associated with elevated levels, are underestimated, while serum concentrations of large isoforms, usually associated with low levels, are over- estimated. However, we have to distinguish between the rel- ative and the absolute bias of the measurement in relation K 8 F. Kronenberg to the apo(a) isoforms. Thorough comparisons between an requires a special DNA preparation to yield high-molecu- isoform-sensitive and an isoform-insensitive assay by Mar- lar DNA but allows determination of the number of K-IV covina and colleagues [31] revealed that the relative bias type 2 copies in separated alleles. Both alleles will also be can become quite high with an overestimation of 25–35% separated by fiber-fluorescence in situ hybridization (FISH) in carriers of large isoforms. However, this translates to an that allows the number of repeats to be counted under fluo- absolute bias in most of the samples of a few mg/dL. The rescence microscopy [32]. In contrast to these two methods, relative bias for most of the carriers of small isoforms is quantitative polymerase chain reaction (qPCR) is doable in around 10% which translates also only to a few mg/dL. high-throughput [11] but provides the total number (sum) However, in the rare cases with up to 16 K-IV repeats, the of the K-IV type 2 copies of the two alleles of the investi- relative and the absolute bias can become quite pronounced gated genome. This means that individuals with one short with an underestimation of the concentration and therefore and one large allele and individuals with two medium-sized the risk for outcomes. The proportion of these individuals alleles end up in the same risk category which might result in the general population is rather low: only 1.9% of the in an underestimation of the risk for the person with one population turned out to have such small isoforms when short and one large allele. This is in line with the largest we investigated more than 24,000 individuals with west- single-center case–control and prospective study up to now ern blot analyses (unpublished data). In summary, when describing that individuals in the lower quartile of the sum interpreting the data from Marcovina and colleagues [31], of copy number in their genome had an adjusted hazard we would expect that for most of the individuals this does ratio for myocardial infarction of 1.50 compared to those not really cause a major underestimation or overestimation in the highest quartile of copy number. This estimate is of the risk. Mainly in the grey zone around the proposed lower than that from studies performing the calculations threshold of Lp(a) concentrations, this might become an based on the expressed isoforms in western blot with a rel- issue. However, we have to keep in mind that this thresh- ative risk of 2.08 [28]. The K-IV type-2 CNV measured by old is also under discussion whether it should be at 30 or qPCR explained only 25% of the variability in Lp(a) levels 50 mg/dL. Moreover, it has to be added that these data are [11], which is also markedly lower than in other studies proven for the assays Marcovina and colleagues have inves- of European populations using methods such as apo(a) iso- tigated [31]. Whether the absolute and relative biases are forms by western blot or separated alleles by pulsed-field similar for other assays needs to be seen and some of the gel electrophoresis. These observations might be related to assays have been compared to the assay from Marcovina the circumstance that qPCR is not easy to standardize with and colleagues. a relatively high coefficient of variation which makes com- parison of results between laboratories difficult. Investigation of the K-IV repeat number by western Based on the peculiarity that roughly 30 to 50% of all blot and DNA analysis individuals express only one apo(a) isoform in serum al- though they have two isoforms at the DNA level, points to The apo(a) isoform size can be best investigated in serum some limitations of the DNA analysis. It raises the question or plasma by western blot using an SDS agarose gel elec- from a clinical standpoint why one should be interested in trophoresis. It has the advantage that only those isoforms an isoform that is not expressed. This raises also the ques- will be visible which are indeed expressed. This is one of tion whether it is better to simply consider Lp(a) concen- the peculiarities of Lp(a): although 95% of the subjects trations instead of the genetic determinants of the concen- are heterozygous on the DNA level, only 50–70% of the trations for risk prediction. However, earlier data suggested individuals show two isoforms in serum. In the remain- that Lp(a) from small apo(a) isoforms have a higher athero- ing 30–50% only a minor fraction is indeed homozygous genic potential [33]. This finding needs to be further evalu- (which cannot be visualized in the western blot) but the ma- ated. Furthermore, certain frequent disease conditions such jority of them express only one isoform in serum. The exact as chronic kidney disease result in pronounced changes of number of K-IV repeats can be measured with a precision Lp(a) concentrations. In these patients small apo(a) isoform of ±1–2 K-IV repeats. However, the method is laborious were demonstrated to be more predictive for CVD risk than and only available in a few laboratories. Until today, it is Lp(a) concentrations [34, 35] (for review see [1, 36]). still the best method to be used for Mendelian randomiza- tion studies to support causality between Lp(a) and various Investigation of single nucleotide polymorphisms outcomes (Fig. 1). (SNPs) Three methods to investigate apo(a) isoforms in terms of number of K-IV repeats on DNA level are available One of the reasons for a revival of the Lp(a) field ten years (Table 1). Pulsed-field gel electrophoresis (PFGE)/southern ago is based on the identification of SNPs that are strongly blotting of genomic DNA [9, 10] is extremely laborious, associated with CHD risk. Clarke and colleagues investi- K Lipoprotein(a) and cardiovascular risk 9 rs10455872 rs10455872 DIoGRAMplusC4D consortium. From all 49 genetic vari- or rs3798220 or rs3798220 ants that were shown to be independently associated with Non-Carriers Non-Carriers Apo(a) phenotypes Lp(a) concentrations, 40 were present in summary-level 97% 51% data retrieved from the CARDIoGRAMplusC4D consor- Do they get the wrong message? tium. Seven SNPs were even significantly associated with CAD on a genome-wide scale. This means that a SNP score Large Small with more SNPs than rs10455872 and rs3798220 might be 76% 24% better suitable for risk prediction [40]. SNPs in the kringle-IV type 2 region—a journey to 3% 49% a white spot on the genetic map Carriers Carriers Fig. 2 Apolipoprotein(a) [apo(a)] phenotypes and the carrier and non- The K-IV type 2 is a region that is difficult to resolve by carrier status of the two single-nucleotide polymorphisms rs10455872 conventional DNA analysis methods. Variants within the and rs3798220 derived from a large Caucasian general population sam- K-IV type 2 region cannot be detected in common se- ple of 5999 individuals quencing projects, leaving up to 70% of the LPA coding region currently unaddressed. We recently developed an gated27SNPsin the LPA gene region and observed two ultra-deep sequencing protocol and an easy-to-use variant SNPs (rs10455872 and rs3798220) to be strongly associated analysis pipeline to create a first map of genetic variation not only with Lp(a) concentrations but also with CHD [37]. in the K-IV type 2 region. We found dozens of loss-of- These SNPs are easy to analyze in a routine laboratory and function and splice site mutations, as well as >100 par- might have contributed to their popularity. However, these tially even common missense variants. This provides novel two SNPs have a minor allele frequency of 7 and 2%, re- candidates to explain the large ethnic and individual differ- spectively and about 15% of a typical Caucasian population ences in Lp(a) concentrations [41]. One of these variants carry at least one minor allele of the two SNPs. They were is a splice site variant (G4925A) in preferential associa- claimed to identify carriers of small apo(a) isoforms [37]. tion with the smaller apo(a) isoforms [42]. It has an ex- However, investigations in a large general Caucasian popu- ceptionally high carrier frequency of 22.1% in the general lation sample of 5999 subjects revealed that roughly half of population and explains 20.6% of the Lp(a) variance in all subjects expressing a small apo(a) isoform are not car- carriers of small apo(a) isoforms. It reduces Lp(a) concen- riers of the minor alleles of one or two of these SNPs [38]. trations dramatically by more than 30 mg/dL. Accordingly, This means that about half of the individuals who have the odds ratio for CVD was reduced from 1.39 for wild- a high risk for CHD based on an expressed small apo(a) type carriers of small isoforms to 1.19 in carriers of small isoform will not be detected when only these two SNPs are isoforms who were additionally positive for G4925A [42]. genotyped. These individuals might get the wrong message Functional studies pointed towards a reduction of splicing when only the SNP result and not also the Lp(a) concentra- efficiency highlighting splicing efficiency modulation by tion will be investigated and considered for risk counselling antisense oligos or transsplicing [43] as a potential novel (Fig. 2). Moreover, the situation might be different for var- Lp(a)-lowering approach [42]. ious ethnicities. For example, rs3798220 was not found in Africans. Allele frequencies in East and Southeast Asians Which approach is now the most suitable for ranged from 2.9 to 11.6%, and were very low (0.15%) in clinical risk assessment? CAD cases and controls from India. The variant was neither associated with small K-IV CNV alleles nor elevated Lp(a) concentrations in Asians [39]. The measurement of Lp(a) concentrations by a well-per- In a recent genome-wide association analysis including forming and standardized assay is usually first choice and 13,781 individuals we observed 2001 SNPs in the wider sufficient to get an estimate for the expected risk for clinical LPA gene region which were significantly associated with outcomes. The Lp(a) concentration might reflect best the bi- Lp(a) concentrations [40]. Many of the SNPs were strongly ologically active lipoprotein. Furthermore, since a protein correlated to each other and an in-depth analysis of the re- and not a genetic polymorphism is measured, no genetic gion identified 48 SNPs to be independently associated with counseling is required in most countries. Lp(a) concentrations. In addition to this region the SNP in There is at least some evidence (but we would wish to the APOE region that is responsible for the apoE2 allele have more) that two persons who have the same high Lp(a) (rs7412) was also associated with Lp(a) concentrations. In concentration but the one has a large isoform and the other a further step, we made a look-up in the data from the CAR- has a small isoform, that the latter has a higher risk which K 10 F. Kronenberg would mean that the smaller isoform (with fewer K-IV re- isoforms, which are usually associated with increased CVD peats) might be more atherogenic. Therefore, the measure- risk and overestimate Lp(a) concentrations of large isoforms ment of the isoform with western blot might provide a fur- which are usually associated with a lower CVD risk. Since ther argument for an increased CVD risk. From all methods small isoforms are overrepresented in CVD patients com- that investigate apo(a) genetic polymorphisms, it provides pared to a control group [27, 28], this phenomenon can the most comprehensive information and is second to the result in a dilution of the Lp(a) concentration differences Lp(a) concentrations very suitable for risk prediction. The between CVD patients and controls. Therefore, negative advantage can probably be explained by the fact that we studies on the association between Lp(a) concentrations and see only the isoforms which are indeed expressed in serum outcomes should exclude that an isoform-sensitivity of the or plasma and not those which are available on the DNA assay has contributed to these results. level but which are suppressed for whatever reasons. The measurement of the apo(a) isoforms adds also cer- Storage effects on Lp(a) concentrations tain information in acquired diseases which are associated with a secondary increase in Lp(a) concentrations. A typi- Some assays are strongly influenced by the effect of stor- cal example is chronic kidney disease: we observed espe- age conditions. This is especially the case for long-termed cially in patients treated by hemodialysis that they show stored samples from epidemiological studies. Extreme ex- an increase in Lp(a) concentrations and this relative in- amples are a nearly linear decrease in Lp(a) immunoreac- crease by the disease is especially observed in patients with tivity of 46% during 6 months of storage [46]or75% lower large apo(a) isoforms when compared to controls with the Lp(a) values after 600 days of storage compared to fresh same isoform categories [44]. Therefore, the risk for CVD samples [47] in two different studies. We observed with our events might be highest especially in those patients with assay in 310 samples on average only a small decrease of small isoforms (which we and others observed indeed [34, Lp(a) of 4.83% during a 25 months storage period but this 35, 45]) since their exposure to high Lp(a) concentrations was higher for individuals with small isoforms compared lasted already their entire life. In contrast, many patients to those with large isoforms [48]. with large isoforms might have experienced the increase in An effect of sample storage could also be a reason for Lp(a) only recently with the development of chronic kid- negative findings in epidemiological studies which used ney disease and the risk might no longer be sufficiently long-term stored samples und using an assay which is even predictable simply by the Lp(a) concentrations [36]. more strongly influenced by sample storage conditions The use of SNPs for risk estimation will have to compared to our assay [46, 47]. Some of the earlier studies find its place in the future. Using the famous two SNPs which used assays not appropriately performing in stored (rs10455872 and rs3798220) described 10 years ago might samples were also included in later meta-analyses and be less efficient since too many false negative results might might have contributed to an underestimation of the asso- be found as illustrated in Fig. 2 (individuals with small ciation between Lp(a) concentrations and cardiovascular apo(a) isoforms and high Lp(a) concentrations despite the disease [13] compared to another large single-center study non-mutated variants of these SNPs) [38]. A SNP score using one well-validated assay [11]. with a large and growing package of SNPs might be more informative [40]. Using SNPs or SNP scores for individu- Samples size in clinical and epidemiological studies als should always be done in combination with the Lp(a) concentrations. The same holds true for apo(a) isoforms It is a widely observed phenomenon in epidemiology that for which the Lp(a) concentration has to be known anyway the first group to report a significant test result (the winner) for the laboratory process which requires that roughly the will also report an effect size much larger than is likely to same amount of Lp(a) is put on the SDS agarose gel. be seen in subsequent replication studies [49]. This phe- nomenon of “winner’s curse” is often seen in Lp(a) re- search which is caused by an underpowered study with Individual risk assessment versus a pronounced right-skewed distribution of the Lp(a) con- epidemiological studies centrations where a large proportion of the population has low concentrations. Since the concentrations of Lp(a) are Apo(a) isoform-sensitivity of assays strongly determined by genetic variants with major effect sizes and less by other conditions, usually large sample The use of an apo(a) isoform-sensitive assay might result in sizes are required to prove a statistically significant differ- an underestimation of the association between Lp(a) con- ence which is not caused by chance findings. As discussed centrations and clinical outcomes. As mentioned above, earlier and demonstrated by simulations, small sample sizes such assays underestimate serum concentrations of small can cause any result between a patient and a control group K Lipoprotein(a) and cardiovascular risk 11 6. Austin MA, Sandholzer C, Selby JV, Newman B, Krauss RM, [44] or any correlation between Lp(a) concentrations and Utermann G (1992) Lipoprotein(a) in women twins: heritability another parameter [3] if the sample size is small enough. To and relationship to apolipoprotein(a) phenotypes. 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E, Kovanen PT, Kuivenhoven JA, Lesnik P, Masana L, Reiner Z, Taskinen MR, Tokgozoglu L, Tybjaerg-Hansen A (2010) Lipopro- Other methods to measure the K-IV repeat number are of tein(a) as a cardiovascular risk factor: current status. Eur Heart J academic interest. SNPs will have to find their role prob- 31:2844–2853 ably more as a SNP score and there is currently no major 13. Erqou S, Kaptoge S, Perry PL, Di AE, Thompson A, White added value to be used beside or instead the measurement IR, Marcovina SM, Collins R, Thompson SG, Danesh J (2009) Lipoprotein(a) concentration and the risk of coronary heart disease, of Lp(a) concentrations. stroke, and nonvascular mortality. JAMA 302:412–423 14. Thanassoulis G, Campbell CY, Owens DS, Smith JG, Smith AV, Compliance with ethical guidelines Peloso GM, Kerr KF, Pechlivanis S, Budoff MJ, Harris TB, Mal- hotra R, O’Brien KD, Kamstrup PR, Nordestgaard BG, Tybjaerg- Conflict of interest F. Kronenberg declares to have received speaker Hansen A, Allison MA, Aspelund T, Criqui MH, Heckbert SR, honoraria from Kaneka, Miltenyi Biotec and Amgen. He is member of Hwang SJ, Liu Y, Sjogren M, van der Pals J, Kalsch H, Muhleisen advisory boards from Kaneka and Amgen. TW, Nothen MM, Cupples LA, Caslake M, Di AE, Danesh J, Rot- ter JI, Sigurdsson S, Wong Q, Erbel R, Kathiresan S, Melander Ethical standards This article does not contain any studies with human O, Gudnason V, O’Donnell CJ, Post WS (2013) Genetic associa- participants or animals performed by any of the authors. tions with valvular calcification and aortic stenosis. N Engl J Med 368:503–512 Open Access This article is distributed under the terms of the 15. 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