Imputation Server PGS: an automated approach to calculate polygenic risk scores on imputation servers.

Polygenic scores (PGS) enable the prediction of genetic predisposition for a wide range of traits and diseases by calculating the weighted sum of allele dosages for genetic variants associated with the trait or disease in question. Present approaches for calculating PGS from genotypes are often inefficient and labor-intensive, limiting transferability into clinical applications. Here, we present 'Imputation Server PGS', an extension of the Michigan Imputation Server designed to automate a standardized calculation of polygenic scores based on imputed genotypes. This extends the widely used Michigan Imputation Server with new functionality, bringing the simplicity and efficiency of modern imputation to the PGS field. The service currently supports over 4489 published polygenic scores from publicly available repositories and provides extensive quality control, including ancestry estimation to report population stratification. An interactive report empowers users to screen and compare thousands of scores in a fast and intuitive way. Imputation Server PGS provides a user-friendly web service, facilitating the application of polygenic scores to a wide range of genetic studies and is freely available at https://imputationserver.sph.umich.edu.

Toll-Like Receptor 3 Mediates Aortic Stenosis Through a Conserved Mechanism of Calcification.

BACKGROUND: Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling. METHODS: Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan (Bgn), Tlr3, and IFN-α/ß receptor alpha chain (Ifnar1)-deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans. RESULTS: Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn-/-, Tlr3-/-, and Ifnar1-/- mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1-BGN-TLR3-interferon-α/ß receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans. CONCLUSIONS: This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD.

Development of an LC-MRM-MS-Based Candidate Reference Measurement Procedure for Standardization of Serum Apolipoprotein (a) Tests.

BACKGROUND: Medical results generated by European CE Marking for In Vitro Diagnostic or in-house tests should be traceable to higher order reference measurement systems (RMS), such as International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)-endorsed reference measurement procedures (RMPs) and reference materials. Currently, serum apolipoprotein (a) [apo(a)] is recognized as a novel risk factor for cardiovascular risk assessment and patient management. The former RMS for serum apo(a) is no longer available; consequently, an International System of Units (SI)-traceable, ideally multiplexed, and sustainable RMS for apo(a) is needed. METHODS: A mass spectrometry (MS)-based candidate RMP (cRMP) for apo(a) was developed using quantitative bottom-up proteomics targeting 3 proteotypic peptides. The method was provisionally validated according to ISO 15193 using a single human serum based calibrator traceable to the former WHO-IFCC RMS. RESULTS: The quantitation of serum apo(a) was by design independent of its size polymorphism, was linear from 3.8 to 456 nmol/L, and had a lower limit of quantitation for apo(a) of 3.8 nmol/L using peptide LFLEPTQADIALLK. Interpeptide agreement showed Pearson Rs of 0.987 and 0.984 for peptides GISSTVTGR and TPENYPNAGLTR, and method comparison indicated good correspondence (slopes 0.977, 1.033, and 1.085 for LFLEPTQADIALLK, GISSTVTGR, and TPENYPNAGLTR). Average within-laboratory imprecision of the cRMP was 8.9%, 11.9%, and 12.8% for the 3 peptides. CONCLUSIONS: A robust, antibody-independent, MS-based cRMP was developed as higher order RMP and an essential part of the apo(a) traceability chain and future RMS. The cRMP fulfils predefined analytical performance specifications, making it a promising RMP candidate in an SI-traceable MS-based RMS for apo(a).

Commutability Assessment of Candidate Reference Materials for Lipoprotein(a) by Comparison of a MS-based Candidate Reference Measurement Procedure with Immunoassays.

BACKGROUND: Elevated concentrations of lipoprotein(a) [Lp(a)] are directly related to an increased risk of cardiovascular diseases, making it a relevant biomarker for clinical risk assessment. However, the lack of global standardization of current Lp(a) measurement procedures (MPs) leads to inconsistent patient care. The International Federation for Clinical Chemistry and Laboratory Medicine working group on quantitating apolipoproteins by mass spectrometry (MS) aims to develop a next-generation SI (International system of units)-traceable reference measurement system consisting of a MS-based, peptide-calibrated reference measurement procedure (RMP) and secondary serum-based reference materials (RMs) certified for their apolipoprotein(a) [apo(a)] content. To reach measurement standardization through this new measurement system, 2 essential requirements need to be fulfilled: a sufficient correlation among the MPs and appropriate commutability of future serum-based RMs. METHODS: The correlation among the candidate RMP (cRMP) and immunoassay-based MPs was assessed by measuring a panel of 39 clinical samples (CS). In addition, the commutability of 14 different candidate RMs was investigated. RESULTS: Results of the immunoassay-based MPs and the cRMPs demonstrated good linear correlations for the CS but some significant sample-specific differences were also observed. The results of the commutability study show that RMs based on unspiked human serum pools can be commutable with CS, whereas human pools spiked with recombinant apo(a) show different behavior compared to CS. CONCLUSIONS: The results of this study show that unspiked human serum pools are the preferred candidate secondary RMs in the future SI-traceable Lp(a) Reference Measurement System.

The TMEM189 gene encodes plasmanylethanolamine desaturase which introduces the characteristic vinyl ether double bond into plasmalogens.

A significant fraction of the glycerophospholipids in the human body is composed of plasmalogens, particularly in the brain, cardiac, and immune cell membranes. A decline in these lipids has been observed in such diseases as Alzheimer's and chronic obstructive pulmonary disease. Plasmalogens contain a characteristic 1-O-alk-1'-enyl ether (vinyl ether) double bond that confers special biophysical, biochemical, and chemical properties to these lipids. However, the genetics of their biosynthesis is not fully understood, since no gene has been identified that encodes plasmanylethanolamine desaturase (E.C. 1.14.99.19), the enzyme introducing the crucial alk-1'-enyl ether double bond. The present work identifies this gene as transmembrane protein 189 (TMEM189). Inactivation of the TMEM189 gene in human HAP1 cells led to a total loss of plasmanylethanolamine desaturase activity, strongly decreased plasmalogen levels, and accumulation of plasmanylethanolamine substrates and resulted in an inability of these cells to form labeled plasmalogens from labeled alkylglycerols. Transient expression of TMEM189 protein, but not of other selected desaturases, recovered this deficit. TMEM189 proteins contain a conserved protein motif (pfam10520) with eight conserved histidines that is shared by an alternative type of plant desaturase but not by other mammalian proteins. Each of these histidines is essential for plasmanylethanolamine desaturase activity. Mice homozygous for an inactivated Tmem189 gene lacked plasmanylethanolamine desaturase activity and had dramatically lowered plasmalogen levels in their tissues. These results assign the TMEM189 gene to plasmanylethanolamine desaturase and suggest that the previously characterized phenotype of Tmem189-deficient mice may be caused by a lack of plasmalogens.

The kringle IV type 2 domain variant 4925G>A causes the elusive association signal of the LPA pentanucleotide repeat.

Lipoprotein(a) [Lp(a)] concentrations are regulated by the LPA gene mainly via the large kringle IV-type 2 (KIV-2) copy number variation and multiple causal variants. Early studies suggested an effect of long pentanucleotide repeat (PNR) alleles (10 and 11 repeats, PNR10 and PNR11) in the LPA promoter on gene transcription and found an association with lower Lp(a). Subsequent in vitro studies showed no effects on mRNA transcription, but the association with strongly decreased Lp(a) remained consistent. We investigated the isolated and combined effect of PNR10, PNR11, and the frequent splice site variant KIV-2 4925G>A on Lp(a) concentrations in the Cooperative Health Research in the Region of Augsburg F4 study by multiple quantile regression in single-SNP and joint models. Data on Lp(a), apolipoprotein(a) Western blot isoforms, and variant genotypes were available for 2,858 individuals. We found a considerable linkage disequilibrium between KIV-2 4925G>A and the alleles PNR10 and PNR11. In single-variant analysis adjusted for age, sex, and the shorter apo(a) isoform, we determined that both PNR alleles were associated with a highly significant Lp(a) decrease (PNR10: ß = -14.43 mg/dl, 95% CI: -15.84, -13.02, P = 3.33e-84; PNR11: ß = -17.21 mg/dl, 95% CI: -20.19, -14.23, P = 4.01e-29). However, a joint model, adjusting the PNR alleles additionally for 4925G>A, abolished the effect on Lp(a) (PNR10: ß = +0.44 mg/dl, 95% CI: -1.73, 2.60, P = 0.69; PNR11: ß = -1.52 mg/dl, 95% CI: -6.05, 3.00, P = 0.51). Collectively, we conclude that the previously reported Lp(a) decrease observed in pentanucleotide alleles PNR10 or PNR11 carriers results from a linkage disequilibrium with the frequent splicing mutation KIV-2 4925G>A.

Lipoprotein(a) and SARS-CoV-2 infections: Susceptibility to infections, ischemic heart disease and thromboembolic events.

BACKGROUND: Comorbidities including ischemic heart disease (IHD) worsen outcomes after SARS-CoV-2 infections. High lipoprotein(a) [Lp(a)] concentrations are a strong risk factor for IHD and possibly for thromboembolic events. We therefore evaluated whether SARS-CoV-2 infections modify the risk of high Lp(a) concentrations for IHD or thromboembolic events during the first 8.5 months follow-up of the pandemic. METHOD: Cohort study using data from the UK Biobank during the SARS-CoV-2 pandemic. Baseline Lp(a) was compared between SARS-CoV-2 positive patients and the population controls. RESULTS: SARS-CoV-2 positive patients had Lp(a) concentrations similar to the population controls. The risk for IHD increased with higher Lp(a) concentrations in both, the population controls (n = 435,104) and SARS-CoV-2 positive patients (n = 6937). The causality of the findings was supported by a genetic risk score for Lp(a). A SARS-CoV-2 infection modified the association with a steeper increase in risk for infected patients (interaction p-value = 0.03). Although SARS-CoV-2 positive patients had a five-times higher frequency of thromboembolic events compared to the population controls (1.53% vs. 0.31%), the risk was not influenced by Lp(a). CONCLUSIONS: SARS-CoV-2 infections enforce the association between high Lp(a) and IHD but the risk for thromboembolic events is not influenced by Lp(a).

Lysis reagents, cell numbers, and calculation method influence high-throughput measurement of HDL-mediated cholesterol efflux capacity.

HDL-mediated cholesterol efflux capacity (CEC) may protect against cardiovascular disease. However, CEC assays are not standardized, hampering their application in large cohorts and comparison between studies. To improve standardization, we systematically investigated technical differences between existing protocols that influence assay performance that have not been previously addressed. CEC was measured in 96-well plates using J774A.1 macrophages labeled with BODIPY-cholesterol and incubated for 4 h with 2% apolipoprotein B-depleted human serum. The time zero method, which calculates CEC using control wells, and the per-well method, which calculates CEC based on the actual content of BODIPY-cholesterol in each well, were compared in 506 samples. We showed that the per-well method had a considerably lower sample rejection rate (4.74% vs. 13.44%) and intra-assay (4.48% vs. 5.28%) and interassay coefficients of variation (two controls: 7.85%, 9.86% vs. 13.58%, 15.29%) compared with the time zero method. Correction for plate-to-plate differences using four controls on each plate also improved assay performance of both methods. In addition, we observed that the lysis reagent used had a significant effect. Compared with cholic acid, lysis with sodium hydroxide results in higher (P = 0.0082) and Triton X-100 in lower (P = 0.0028) CEC values. Furthermore, large cell seeding errors (30% variation) greatly biased CEC for both referencing methods (P < 0.0001) as measured by a resazurin assay. In conclusion, lysis reagents, cell numbers, and assay setup greatly impact the quality and reliability of CEC quantification and should be considered when this method is newly established in a laboratory.

Meta-analysis uncovers genome-wide significant variants for rapid kidney function decline.

Rapid decline of glomerular filtration rate estimated from creatinine (eGFRcrea) is associated with severe clinical endpoints. In contrast to cross-sectionally assessed eGFRcrea, the genetic basis for rapid eGFRcrea decline is largely unknown. To help define this, we meta-analyzed 42 genome-wide association studies from the Chronic Kidney Diseases Genetics Consortium and United Kingdom Biobank to identify genetic loci for rapid eGFRcrea decline. Two definitions of eGFRcrea decline were used: 3 mL/min/1.73m2/year or more ("Rapid3"; encompassing 34,874 cases, 107,090 controls) and eGFRcrea decline 25% or more and eGFRcrea under 60 mL/min/1.73m2 at follow-up among those with eGFRcrea 60 mL/min/1.73m2 or more at baseline ("CKDi25"; encompassing 19,901 cases, 175,244 controls). Seven independent variants were identified across six loci for Rapid3 and/or CKDi25: consisting of five variants at four loci with genome-wide significance (near UMOD-PDILT (2), PRKAG2, WDR72, OR2S2) and two variants among 265 known eGFRcrea variants (near GATM, LARP4B). All these loci were novel for Rapid3 and/or CKDi25 and our bioinformatic follow-up prioritized variants and genes underneath these loci. The OR2S2 locus is novel for any eGFRcrea trait including interesting candidates. For the five genome-wide significant lead variants, we found supporting effects for annual change in blood urea nitrogen or cystatin-based eGFR, but not for GATM or LARP4B. Individuals at high compared to those at low genetic risk (8-14 vs. 0-5 adverse alleles) had a 1.20-fold increased risk of acute kidney injury (95% confidence interval 1.08-1.33). Thus, our identified loci for rapid kidney function decline may help prioritize therapeutic targets and identify mechanisms and individuals at risk for sustained deterioration of kidney function.

Towards an SI-Traceable Reference Measurement System for Seven Serum Apolipoproteins Using Bottom-Up Quantitative Proteomics: Conceptual Approach Enabled by Cross-Disciplinary/Cross-Sector Collaboration.

Current dyslipidemia management in patients with atherosclerotic cardiovascular disease (ASCVD) is based on traditional serum lipids. Yet, there is some indication from basic research that serum apolipoproteins A-I, (a), B, C-I, C-II, C-III, and E may give better pathophysiological insight into the root causes of dyslipidemia. To facilitate the future adoption of clinical serum apolipoprotein (apo) profiling for precision medicine, strategies for accurate testing should be developed in advance. Recent discoveries in basic science and translational medicine set the stage for the IFCC Working Group on Apolipoproteins by Mass Spectrometry. Main drivers were the convergence of unmet clinical needs in cardiovascular disease (CVD) patients with enabling technology and metrology. First, the residual cardiovascular risk after accounting for established risk factors demonstrates that the current lipid panel is too limited to capture the full complexity of lipid metabolism in patients. Second, there is a need for accurate test results in highly polymorphic and atherogenic apolipoproteins such as apo(a). Third, sufficient robustness of mass spectrometry technology allows reproducible protein quantification at the molecular level. Fourth, several calibration hierarchies in the revised ISO 17511:2020 guideline facilitate metrological traceability of test results, the highest achievable standard being traceability to SI. This article outlines the conceptual approach aimed at achieving a novel, multiplexed Reference Measurement System (RMS) for seven apolipoproteins based on isotope dilution mass spectrometry and peptide-based calibration. This RMS should enable standardization of existing and emerging apolipoprotein assays to SI, within allowable limits of measurement uncertainty, through a sustainable network of Reference Laboratories.

The haemochromatosis gene Hfe and Kupffer cells control LDL cholesterol homeostasis and impact on atherosclerosis development.

AIMS: Imbalances of iron metabolism have been linked to the development of atherosclerosis. However, subjects with hereditary haemochromatosis have a lower prevalence of cardiovascular disease. The aim of our study was to understand the underlying mechanisms by combining data from genome-wide association study analyses in humans, CRISPR/Cas9 genome editing, and loss-of-function studies in mice. METHODS AND RESULTS: Our analysis of the Global Lipids Genetics Consortium (GLGC) dataset revealed that single nucleotide polymorphisms (SNPs) in the haemochromatosis gene HFE associate with reduced low-density lipoprotein cholesterol (LDL-C) in human plasma. The LDL-C lowering effect could be phenocopied in dyslipidaemic ApoE-/- mice lacking Hfe, which translated into reduced atherosclerosis burden. Mechanistically, we identified HFE as a negative regulator of LDL receptor expression in hepatocytes. Moreover, we uncovered liver-resident Kupffer cells (KCs) as central players in cholesterol homeostasis as they were found to acquire and transfer LDL-derived cholesterol to hepatocytes in an Abca1-dependent fashion, which is controlled by iron availability. CONCLUSION: Our results disentangle novel regulatory interactions between iron metabolism, KC biology and cholesterol homeostasis which are promising targets for treating dyslipidaemia but also provide a mechanistic explanation for reduced cardiovascular morbidity in subjects with haemochromatosis.

A comprehensive map of single-base polymorphisms in the hypervariable LPA kringle IV type 2 copy number variation region.

Lipoprotein (a) [Lp(a)] concentrations are among the strongest genetic risk factors for cardiovascular disease and present pronounced interethnic and interindividual differences. Approximately 90% of Lp(a) variance is controlled by the LPA gene, which contains a 5.6-kb-large copy number variation [kringle IV type 2 (KIV-2) repeat] that generates >40 protein isoforms. Variants within the KIV-2 region are not called in common sequencing projects, leaving up to 70% of the LPA coding region currently unaddressed. To completely assess the variability in LPA, we developed a sequencing strategy for this region and report here the first map of genetic variation in the KIV-2 region, a comprehensively evaluated ultradeep sequencing protocol, and an easy-to-use variant analysis pipeline. We sequenced 123 Central-European individuals and reanalyzed public data of 2,504 individuals from 26 populations. We found 14 different loss-of-function and splice-site mutations, as well as >100, partially even common, missense variants. Some coding variants were frequent in one population but absent in others. This provides novel candidates to explain the large ethnic and individual differences in Lp(a) concentrations. Importantly, our approach and pipeline are also applicable to other similar copy number variable regions, allowing access to regions that are not captured by common genome sequencing.

Association of mitochondrial iron deficiency and dysfunction with idiopathic restless legs syndrome.

BACKGROUND: Restless legs syndrome is a sensorimotor neurological disorder of the limbs that impairs quality of life and disturbs sleep. However, there has been progress in understanding the disease involving the dopaminergic system as well as iron metabolism. The exact pathophysiological mechanisms of restless legs syndrome remain elusive. We tried to elucidate the underlying mechanisms in iron metabolism in restless legs syndrome subjects on a systemic, cellular, and mitochondrial level. METHODS: We conducted a study prospectively recruiting 168 restless legs syndrome patients and 119 age-matched healthy controls focusing on iron metabolism using human monocytes as surrogates. RESULTS: Evaluation of systemic iron metabolism parameters in the circulation showed no significant difference between patients and controls. We observed a significant reduction in mRNA levels of heme oxygenase 1 and mitochondrial iron genes like mitoferrin 1 and 2 in monocytes isolated from restless legs syndrome patients, indicating mitochondrial iron deficiency. Interestingly, we also observed reduced expression of iron regulatory protein 2 along with impaired activity of mitochondrial aconitase and reduced mitochondrial superoxide formation in restless legs syndrome subjects. Along this line, patients had reduced mitochondrial respiratory capacity that improved in restless legs syndrome subjects under treatment with dopaminergic drugs compared with untreated patients. CONCLUSIONS: Our data suggest that restless legs syndrome is linked to mitochondrial iron deficiency and associated impairment of mitochondrial function. This is partly corrected by treatment with dopaminergic drugs compared with untreated patients, which may be linked to an effect of dopamine on cellular iron homeostasis. © 2018 International Parkinson and Movement Disorder Society.

Genetic Factors Explain a Major Fraction of the 50% Lower Lipoprotein(a) Concentrations in Finns.

OBJECTIVE: Lp(a) (lipoprotein(a)) concentrations are widely genetically determined by the LPA isoforms and show 5-fold interpopulation differences. Two- to 3-fold differences have been reported even within Europe. Finns represent a distinctive population isolate within Europe and have been repeatedly reported to present lower Lp(a) concentrations than Central Europeans. The significance of this finding was unclear for a long time because of the difficult comparability of Lp(a) assays. Recently, a large standardized study in >50 000 individuals from 7 European populations confirmed this observation but could not provide insights into the causes. APPROACH AND RESULTS: We investigated Lp(a) concentrations, LPA isoforms, and genotypes of established genetic variants affecting Lp(a) concentrations (LPA variants, APOE isoforms, and PCSK9 R46L) in the Finnish YFS (Cardiovascular Risk in Young Finns Study) population (n=2281) and 3 Non-Finnish Central European populations (n=10 003). We observed ≈50% lower Lp(a) concentrations in Finns. The isoform distribution was shifted toward longer isoforms, and the percentage of low-molecular-weight isoform carriers was reduced. Most interestingly, however, Lp(a) was reduced in each single-isoform group. In contrast to the known inverse relationship between LPA isoforms and Lp(a) concentrations, especially very short isoforms presented unexpectedly low Lp(a) concentrations in Finns. The investigated genetic variants, as well as age, sex, and renal function, explained 71.8% of the observed population differences. CONCLUSIONS: The population differences in Lp(a) concentrations between Finnish and Central European populations originate not only from a different LPA isoform distribution but suggest the existence of novel functional variation in the small-isoform range.

Phenome-wide association analysis of LDL-cholesterol lowering genetic variants in PCSK9.

BACKGROUND: We characterised the phenotypic consequence of genetic variation at the PCSK9 locus and compared findings with recent trials of pharmacological inhibitors of PCSK9. METHODS: Published and individual participant level data (300,000+ participants) were combined to construct a weighted PCSK9 gene-centric score (GS). Seventeen randomized placebo controlled PCSK9 inhibitor trials were included, providing data on 79,578 participants. Results were scaled to a one mmol/L lower LDL-C concentration. RESULTS: The PCSK9 GS (comprising 4 SNPs) associations with plasma lipid and apolipoprotein levels were consistent in direction with treatment effects. The GS odds ratio (OR) for myocardial infarction (MI) was 0.53 (95% CI 0.42; 0.68), compared to a PCSK9 inhibitor effect of 0.90 (95% CI 0.86; 0.93). For ischemic stroke ORs were 0.84 (95% CI 0.57; 1.22) for the GS, compared to 0.85 (95% CI 0.78; 0.93) in the drug trials. ORs with type 2 diabetes mellitus (T2DM) were 1.29 (95% CI 1.11; 1.50) for the GS, as compared to 1.00 (95% CI 0.96; 1.04) for incident T2DM in PCSK9 inhibitor trials. No genetic associations were observed for cancer, heart failure, atrial fibrillation, chronic obstructive pulmonary disease, or Alzheimer's disease - outcomes for which large-scale trial data were unavailable. CONCLUSIONS: Genetic variation at the PCSK9 locus recapitulates the effects of therapeutic inhibition of PCSK9 on major blood lipid fractions and MI. While indicating an increased risk of T2DM, no other possible safety concerns were shown; although precision was moderate.

A genome-wide association meta-analysis on apolipoprotein A-IV concentrations.

Apolipoprotein A-IV (apoA-IV) is a major component of HDL and chylomicron particles and is involved in reverse cholesterol transport. It is an early marker of impaired renal function. We aimed to identify genetic loci associated with apoA-IV concentrations and to investigate relationships with known susceptibility loci for kidney function and lipids. A genome-wide association meta-analysis on apoA-IV concentrations was conducted in five population-based cohorts (n = 13,813) followed by two additional replication studies (n = 2,267) including approximately 10 M SNPs. Three independent SNPs from two genomic regions were significantly associated with apoA-IV concentrations: rs1729407 near APOA4 (P = 6.77 × 10 - 44), rs5104 in APOA4 (P = 1.79 × 10-24) and rs4241819 in KLKB1 (P = 5.6 × 10-14). Additionally, a look-up of the replicated SNPs in downloadable GWAS meta-analysis results was performed on kidney function (defined by eGFR), HDL-cholesterol and triglycerides. From these three SNPs mentioned above, only rs1729407 showed an association with HDL-cholesterol (P = 7.1 × 10 - 07). Moreover, weighted SNP-scores were built involving known susceptibility loci for the aforementioned traits (53, 70 and 38 SNPs, respectively) and were associated with apoA-IV concentrations. This analysis revealed a significant and an inverse association for kidney function with apoA-IV concentrations (P = 5.5 × 10-05). Furthermore, an increase of triglyceride-increasing alleles was found to decrease apoA-IV concentrations (P = 0.0078). In summary, we identified two independent SNPs located in or next the APOA4 gene and one SNP in KLKB1 The association of KLKB1 with apoA-IV suggests an involvement of apoA-IV in renal metabolism and/or an interaction within HDL particles. Analyses of SNP-scores indicate potential causal effects of kidney function and by lesser extent triglycerides on apoA-IV concentrations.

A novel but frequent variant in LPA KIV-2 is associated with a pronounced Lp(a) and cardiovascular risk reduction.

AIMS: Lp(a) concentrations represent a major cardiovascular risk factor and are almost entirely controlled by one single locus (LPA). However, many genetic factors in LPA governing the enormous variance of Lp(a) levels are still unknown. Since up to 70% of the LPA coding sequence are located in a difficult to access hypervariable copy number variation named KIV-2, we hypothesized that it may contain novel functional variants with pronounced effects on Lp(a) concentrations. We performed a large scale mutation analysis in the KIV-2 using an extreme phenotype approach. METHODS AND RESULTS: We compiled an discovery set of 123 samples showing discordance between LPA isoform phenotype and Lp(a) concentrations and controls. Using ultra-deep sequencing, we identified a splice site variant (G4925A) in preferential association with the smaller LPA isoforms. Follow-up in a European general population (n = 2892) revealed an exceptionally high carrier frequency of 22.1% in the general population. The variant explains 20.6% of the Lp(a) variance in carriers of low molecular weight (LMW) apo(a) isoforms (P = 5.75e-38) and reduces Lp(a) concentrations by 31.3 mg/dL. Accordingly the odds ratio for cardiovascular disease was reduced from 1.39 [95% confidence interval (CI): 1.17-1.66, P = 1.89e-04] for wildtype LMW individuals to 1.19 [95%CI: 0.92; 1.56, P = 0.19] in LMW individuals who were additionally positive for G4925A. Functional studies point towards a reduction of splicing efficiency by this novel variant. CONCLUSION: A highly frequent but until now undetected variant in the LPA KIV-2 region is strongly associated with reduced Lp(a) concentrations and reduced cardiovascular risk in LMW individuals.

A genome-wide association meta-analysis on lipoprotein (a) concentrations adjusted for apolipoprotein (a) isoforms.

High lipoprotein (a) [Lp(a)] concentrations are an independent risk factor for cardiovascular outcomes. Concentrations are strongly influenced by apo(a) kringle IV repeat isoforms. We aimed to identify genetic loci associated with Lp(a) concentrations using data from five genome-wide association studies (n = 13,781). We identified 48 independent SNPs in the LPA and 1 SNP in the APOE gene region to be significantly associated with Lp(a) concentrations. We also adjusted for apo(a) isoforms to identify loci affecting Lp(a) levels independently from them, which resulted in 31 SNPs (30 in the LPA, 1 in the APOE gene region). Seven SNPs showed a genome-wide significant association with coronary artery disease (CAD) risk. A rare SNP (rs186696265; MAF ∼1%) showed the highest effect on Lp(a) and was also associated with increased risk of CAD (odds ratio = 1.73, P = 3.35 × 10-30). Median Lp(a) values increased from 2.1 to 91.1 mg/dl with increasing number of Lp(a)-increasing alleles. We found the APOE2-determining allele of rs7412 to be significantly associated with Lp(a) concentrations (P = 3.47 × 10-10). Each APOE2 allele decreased Lp(a) by 3.34 mg/dl corresponding to ∼15% of the population's mean values. Performing a gene-based test of association, including suspected Lp(a) receptors and regulators, resulted in one significant association of the TLR2 gene with Lp(a) (P = 3.4 × 10-4). In summary, we identified a large number of independent SNPs in the LPA gene region, as well as the APOE2 allele, to be significantly associated with Lp(a) concentrations.

Is High-Density Lipoprotein Cholesterol Causally Related to Kidney Function? Evidence From Genetic Epidemiological Studies.

OBJECTIVE: A recent observational study with almost 2 million men reported an association between low high-density lipoprotein (HDL) cholesterol and worse kidney function. The causality of this association would be strongly supported if genetic variants associated with HDL cholesterol were also associated with kidney function. APPROACH AND RESULTS: We used 68 genetic variants (single-nucleotide polymorphisms [SNPs]) associated with HDL cholesterol in genome-wide association studies including >188 000 subjects and tested their association with estimated glomerular filtration rate (eGFR) using summary statistics from another genome-wide association studies meta-analysis of kidney function including ≤133 413 subjects. Fourteen of the 68 SNPs (21%) had a P value <0.05 compared with the 5% expected by chance (Binomial test P=5.8×10-6). After Bonferroni correction, 6 SNPs were still significantly associated with eGFR. The genetic variants with the strongest associations with HDL cholesterol concentrations were not the same as those with the strongest association with kidney function and vice versa. An evaluation of pleiotropy indicated that the effects of the HDL-associated SNPs on eGFR were not mediated by HDL cholesterol. In addition, we performed a Mendelian randomization analysis. This analysis revealed a positive but nonsignificant causal effect of HDL cholesterol-increasing variants on eGFR. CONCLUSIONS: In summary, our findings indicate that HDL cholesterol does not causally influence eGFR and propose pleiotropic effects on eGFR for some HDL cholesterol-associated SNPs. This may cause the observed association by mechanisms other than the mere HDL cholesterol concentration.

Linkage and Association Analysis Identifies TRAF1 Influencing Common Carotid Intima-Media Thickness.

BACKGROUND AND PURPOSE: Carotid intima-media thickness is a marker for subclinical atherosclerosis that predicts subsequent clinical cardiovascular events. The aim of this study was to identify chromosomal loci with linkage or association to common carotid intima-media thickness. METHODS: Nuclear families were recruited using the single parental proband sib-pair design. Genotype data were available for 546 individuals from 132 nuclear families of the Bonn IMT Family Study using the Affymetrix GeneChip Human Mapping 250K Sty chip. Multipoint logarithm of the odds (LOD) scores were determined with the quantitative trait locus statistic implemented in multipoint engine for rapid likelihood. Linkage analysis and family-based association tests were conducted. Data from 2471 German participants from the HNR (Heinz Nixdorf Recall) Study were used for subsequent replication. RESULTS: Two new genomic regions with suggestive linkage (LOD>2) were identified on chromosome 4 (LOD=2.26) and on chromosome 17 (LOD=2.01). Previously reported linkage findings were replicated on chromosomes 13 and 14. Fifteen single nucleotide polymorhisms, located on chromosomes 4, 6, and 9, revealed P<10-4 in the family-based association analyses. One of these signals was replicated in HNR (rs2416804, 1-sided P=1.60×10-3, located in the gene TRAF1). CONCLUSIONS: This study presents the first genome-wide linkage and association study of common carotid intima-media thickness in the German population. Alleles of rs2416804 in TRAF1 were identified as being linked and associated with carotid intima-media thickness. Further studies are needed to evaluate the contribution of this locus to the development of atherosclerosis.