Publications by authors named "Stefan Coassin"

60 Publications

Frequent LPA KIV-2 Variants Lower Lipoprotein(a) Concentrations and Protect Against Coronary Artery Disease.

J Am Coll Cardiol 2021 Aug;78(5):437-449

Institute of Genetic Epidemiology, Department of Genetics and Pharmacology, Medical University of Innsbruck, Innsbruck, Austria. Electronic address:

Background: Lipoprotein(a) (Lp(a)) concentrations are a major independent risk factor for coronary artery disease (CAD) and are mainly determined by variation in LPA. Up to 70% of the LPA coding sequence is located in the hypervariable kringle IV type 2 (KIV-2) region. It is hardly accessible by conventional technologies, but may contain functional variants.

Objectives: This study sought to investigate the new, very frequent splicing variant KIV-2 4733G>A on Lp(a) and CAD.

Methods: We genotyped 4733G>A in the GCKD (German Chronic Kidney Disease) study (n = 4,673) by allele-specific polymerase chain reaction, performed minigene assays, identified proxy single nucleotide polymorphisms and used them to characterize its effect on CAD by survival analysis in UK Biobank (n = 440,234). Frequencies in ethnic groups were assessed in the 1000 Genomes Project.

Results: The 4733G>A variant (38.2% carrier frequency) was found in most isoform sizes. It reduces allelic expression without abolishing protein production, lowers Lp(a) by 13.6 mg/dL (95% CI: 12.5-14.7; P < 0.0001) and is the strongest variance-explaining factor after the smaller isoform. Splicing of minigenes was modified. Compound heterozygosity (4.6% of the population) for 4733G>A and 4925G>A, another KIV-2 splicing mutation, reduces Lp(a) by 31.8 mg/dL and most importantly narrows the interquartile range by 9-fold (from 42.1 to 4.6 mg/dL) when compared to the wild type. In UK Biobank 4733G>A alone and compound heterozygosity with 4925G>A reduced HR for CAD by 9% (95% CI: 7%-11%) and 12% (95% CI: 7%-16%) (both P < 0.001). Frequencies in ethnicities differ notably.

Conclusions: Functional variants in the previously inaccessible LPA KIV-2 region cooperate in determining Lp(a) variance and CAD risk. Even a moderate but lifelong genetic Lp(a) reduction translates to a noticeable CAD risk reduction.
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http://dx.doi.org/10.1016/j.jacc.2021.05.037DOI Listing
August 2021

The size of apolipoprotein (a) is an independent determinant of the reduction in lipoprotein (a) induced by PCSK9 inhibitors.

Cardiovasc Res 2021 Jul 26. Epub 2021 Jul 26.

Université de La Réunion, INSERM UMR 1188 DéTROI, Sainte-Clotilde, France.

Aims: Lipoprotein (a) [Lp(a)] is a lipoprotein species causatively associated with atherosclerosis. Unlike statins, PCSK9 inhibitors (PCSK9i) reduce Lp(a), but this reduction is highly variable. Levels of Lp(a) are chiefly governed by the size of its signature protein, apolipoprotein (a) [apo(a)]. Whether this parameter determines some of the reduction in Lp(a) induced by PCSK9i remains unknown. We aimed to investigate if the Lp(a) lowering efficacy of PCSK9i is modulated by the size of apo(a), which is genetically determined by the variable number of KIV domains present on that protein.

Methods And Results: The levels of Lp(a) and the size of apo(a) were assessed in plasma samples from 268 patients before and after treatment with PCSK9i. Patients were recruited at the Outpatient Lipid Clinic of the Charité Hospital (Berlin) between 2015 and 2020. They were hypercholesterolemic at very high CVD risk with LDL-cholesterol levels above therapeutic targets despite maximally tolerated lipid-lowering therapy. Patients received either Alirocumab (75 or 150 mg) or Evolocumab (140 mg) every 2 weeks. Apo(a), apoB100, and apoE concentrations as well as apoE major isoforms were determined by liquid chromatography high-resolution mass spectrometry. Apo(a) isoforms sizes were determined by Western Blot. PCSK9i sharply reduced LDL-cholesterol (-57%), apoB100 (-47%) and Lp(a) (-36%). There was a positive correlation between the size of apo(a) and the relative reduction in Lp(a) induced by PCSK9i (r = 0.363, p = 0.0001). The strength of this association remained unaltered after adjustment for baseline Lp(a) levels and all other potential confounding factors. In patients with two detectable apo(a) isoforms, there was also a positive correlation between the size of apo(a) and the reduction in Lp(a), separately for the smaller (r = 0.350, p = 0.0001) and larger (r = 0.324, p = 0.0003) isoforms. The relative contribution of the larger isoform to the total concentration of apo(a) was reduced from 29% to 15% (p < 0.0001).

Conclusions: The size of apo(a) is an independent determinant of the response to PCSK9i. Each additional kringle domain is associated with a 3% additional reduction in Lp(a). This explains in part the variable efficacy of PCSK9i and allows to identify patients who will benefit most from these therapies in terms of Lp(a) lowering.

Translational Perspective: Unlike statins, PCSK9 inhibitors reduce the circulating levels of the highly atherogenic Lipoprotein (a). The underlying mechanism remains a matter of considerable debate. The size of apo(a), the signature protein of Lp(a), is extremely variable (300 to more than 800 kDa) and depends on its number of kringle domains. We now show that each increase in apo(a) size by one kringle domain is associated with a 3% additional reduction in Lp(a) following PCSK9i treatment and that apo(a) size polymorphism is an independent predictor of the reduction in Lp(a) induced by these drugs. In an era of personalized medicine, this allows to identify patients who will benefit most from PCSK9i in terms of Lp(a) lowering.
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http://dx.doi.org/10.1093/cvr/cvab247DOI Listing
July 2021

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

J Intern Med 2021 Jun 7. Epub 2021 Jun 7.

Institute of Genetic Epidemiology, Department of Genetics and Pharmacology, Medical University of Innsbruck, Innsbruck, Austria.

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. UK Biobank received ethical approval from the North West Multi-Centre Research Ethics Committee (REC reference: 11/NW/0382). All participants gave written informed consent before enrolment in the study, which was conducted in accordance with the principles of the Declaration of Helsinki.

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 = 6,937). 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 5-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). This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1111/joim.13338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8242884PMC
June 2021

Cis-epistasis at the LPA locus and risk of cardiovascular diseases.

Cardiovasc Res 2021 Apr 20. Epub 2021 Apr 20.

Estonian Genome Center, Institute of Genomics, University of Tartu, 51010, Tartu, Estonia.

Aims: Coronary artery disease (CAD) has a strong genetic predisposition. However, despite substantial discoveries made by genome-wide association studies (GWAS), a large proportion of heritability awaits identification. Non-additive genetic-effects might be responsible for part of the unaccounted genetic variance. Here we attempted a proof-of-concept study to identify non-additive genetic effects, namely epistatic interactions, associated with CAD.

Methods And Results: We tested for epistatic interactions in ten CAD case-control studies and UK Biobank with focus on 8,068 SNPs at 56 loci with known associations with CAD risk. We identified a SNP pair located in cis at the LPA locus, rs1800769 and rs9458001, to be jointly associated with risk for CAD (odds ratio [OR]=1.37, p = 1.07 × 10-11), peripheral arterial disease (OR = 1.22, p = 2.32 × 10-4), aortic stenosis (OR = 1.47, p = 6.95 × 10-7), hepatic lipoprotein(a) (Lp(a)) transcript levels (beta = 0.39, p = 1.41 × 10-8), and Lp(a) serum levels (beta = 0.58, p = 8.7 × 10-32), while individual SNPs displayed no association. Further exploration of the LPA locus revealed a strong dependency of these associations on a rare variant, rs140570886, that was previously associated with Lp(a) levels. We confirmed increased CAD risk for heterozygous (relative OR = 1.46, p = 9.97 × 10-32) and individuals homozygous for the minor allele (relative OR = 1.77, p = 0.09) of rs140570886. Using forward model selection, we also show that epistatic interactions between rs140570886, rs9458001, and rs1800769 modulate the effects of the rs140570886 risk allele.

Conclusions: These results demonstrate the feasibility of a large-scale knowledge-based epistasis scan and provide rare evidence of an epistatic interaction in a complex human disease. We were directed to a variant (rs140570886) influencing risk through additive genetic as well as epistatic effects. In summary, this study provides deeper insights into the genetic architecture of a locus important for cardiovascular diseases.

Translational Perspective: Genetic variants identified by GWAS studies explain about a quarter of the heritability of coronary artery disease by additive genetic effects. Our study demonstrates that non-additive effects contribute to the genetic architecture of the disease as well and identifies complex interaction patterns at the LPA locus, which affect LPA expression, Lp(a) plasma levels and risk of atherosclerosis. This proof-of-concept study encourages systematic searches for epistatic interactions in further studies to shed new light on the aetiology of the disease.
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http://dx.doi.org/10.1093/cvr/cvab136DOI Listing
April 2021

When the genome bluffs: a tandem duplication event during generation of a novel Agmo knockout mouse model fools routine genotyping.

Cell Biosci 2021 Mar 16;11(1):54. Epub 2021 Mar 16.

Institute of Biological Chemistry, Biocenter, Medical University of Innsbruck, Innsbruck, Austria.

Background: Genome editing in mice using either classical approaches like homologous recombination or CRISPR/Cas9 has been reported to harbor off target effects (insertion/deletion, frame shifts or gene segment duplications) that lead to mutations not only in close proximity to the target site but also outside. Only the genomes of few engineered mouse strains have been sequenced. Since the role of the ether-lipid cleaving enzyme alkylglycerol monooxygenase (AGMO) in physiology and pathophysiology remains enigmatic, we created a knockout mouse model for AGMO using EUCOMM stem cells but unforeseen genotyping issues that did not agree with Mendelian distribution and enzyme activity data prompted an in-depth genomic validation of the mouse model.

Results: We report a gene segment tandem duplication event that occurred during the generation of an Agmo knockout-first allele by homologous recombination. Only low homology was seen between the breakpoints. While a single copy of the recombinant 18 kb cassette was integrated correctly around exon 2 of the Agmo gene, whole genome nanopore sequencing revealed a 94 kb duplication in the Agmo locus that contains Agmo wild-type exons 1-3. The duplication fooled genotyping by routine PCR, but could be resolved using qPCR-based genotyping, targeted locus amplification sequencing and nanopore sequencing. Despite this event, this Agmo knockout mouse model lacks AGMO enzyme activity and can therefore be used to study its physiological role.

Conclusions: A duplication event occurred at the exact locus of the homologous recombination and was not detected by conventional quality control filters such as FISH or long-range PCR over the recombination sites. Nanopore sequencing provides a cost convenient method to detect such underrated off-target effects, suggesting its use for additional quality assessment of gene editing in mice and also other model organisms.
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http://dx.doi.org/10.1186/s13578-021-00566-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962373PMC
March 2021

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.

Clin Chem 2021 03;67(3):478-489

Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden, The Netherlands.

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.
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http://dx.doi.org/10.1093/clinchem/hvaa239DOI Listing
March 2021

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

Kidney Int 2021 04 31;99(4):926-939. Epub 2020 Oct 31.

Division of Nephrology, University of Washington, Seattle, Washington, USA; Kidney Research Institute, University of Washington, Seattle, Washington, USA.

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/1.73m/year or more ("Rapid3"; encompassing 34,874 cases, 107,090 controls) and eGFRcrea decline 25% or more and eGFRcrea under 60 mL/min/1.73m at follow-up among those with eGFRcrea 60 mL/min/1.73m 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.
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http://dx.doi.org/10.1016/j.kint.2020.09.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010357PMC
April 2021

Investigation of a nonsense mutation located in the complex KIV-2 copy number variation region of apolipoprotein(a) in 10,910 individuals.

Genome Med 2020 08 21;12(1):74. Epub 2020 Aug 21.

Institute of Genetic Epidemiology, Department of Genetics and Pharmacology, Medical University of Innsbruck, Schöpfstrasse 41, A-6020, Innsbruck, Austria.

Background: The concentrations of the highly atherogenic lipoprotein(a) [Lp(a)] are mainly genetically determined by the LPA gene locus. However, up to 70% of the coding sequence is located in the complex so-called kringle IV type 2 (KIV-2) copy number variation, a region hardly accessible by common genotyping and sequencing technologies. Despite its size, little is known about genetic variants in this complex region. The R21X variant is a functional variant located in this region, but it has never been analyzed in large cohorts.

Methods: We typed R21X in 10,910 individuals from three European populations using a newly developed high-throughput allele-specific qPCR assay. R21X allelic location was determined by separating the LPA alleles using pulsed-field gel electrophoresis (PFGE) and typing them separately. Using GWAS data, we identified a proxy SNP located outside of the KIV-2. Linkage disequilibrium was determined both statistically and by long-range haplotyping using PFGE. Worldwide frequencies were determined by reanalyzing the sequencing data of the 1000 Genomes Project with a dedicated pipeline.

Results: R21X carriers (frequency 0.016-0.021) showed significantly lower mean Lp(a) concentrations (- 11.7 mg/dL [- 15.5; - 7.82], p = 3.39e-32). The variant is located mostly on medium-sized LPA alleles. In the 1000 Genome data, R21X mostly occurs in Europeans and South Asians, is absent in Africans, and shows varying frequencies in South American populations (0 to 0.022). Of note, the best proxy SNP was another LPA null mutation (rs41272114, D' = 0.958, R = 0.281). D' was very high in all 1000G populations (0.986-0.996), although rs41272114 frequency varies considerably (0-0.182). Co-localization of both null mutations on the same allele was confirmed by PFGE-based long-range haplotyping.

Conclusions: We performed the largest epidemiological study on an LPA KIV-2 variant so far, showing that it is possible to assess LPA KIV-2 mutations on a large scale. Surprisingly, in all analyzed populations, R21X was located on the same haplotype as the splice mutation rs41272114, creating "double-null" LPA alleles. Despite being a nonsense variant, the R21X status does not provide additional information beyond the rs41272114 genotype. This has important implications for studies using LPA loss-of-function mutations as genetic instruments and emphasizes the complexity of LPA genetics.
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http://dx.doi.org/10.1186/s13073-020-00771-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442989PMC
August 2020

Mechanistic insights into lipoprotein(a): from infamous to 'inflammous'.

Eur Heart J 2020 06;41(24):2272-2274

Institute of Genetic Epidemiology, Department of Genetics and Pharmacology, Medical University of Innsbruck, Innsbruck, Austria.

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http://dx.doi.org/10.1093/eurheartj/ehaa420DOI Listing
June 2020

A genome-wide analysis of DNA methylation identifies a novel association signal for Lp(a) concentrations in the LPA promoter.

PLoS One 2020 28;15(4):e0232073. Epub 2020 Apr 28.

Department of Genetics and Pharmacology, Institute of Genetic Epidemiology, Medical University of Innsbruck, Innsbruck, Austria.

Lipoprotein(a) [Lp(a)] is a major cardiovascular risk factor, which is largely genetically determined by one major gene locus, the LPA gene. Many aspects of the transcriptional regulation of LPA are poorly understood and the role of epigenetics has not been addressed yet. Therefore, we conducted an epigenome-wide analysis of DNA methylation on Lp(a) levels in two population-based studies (total n = 2208). We identified a CpG site in the LPA promoter which was significantly associated with Lp(a) concentrations. Surprisingly, the identified CpG site was found to overlap the SNP rs76735376. We genotyped this SNP de-novo in three studies (total n = 7512). The minor allele of rs76735376 (1.1% minor allele frequency) was associated with increased Lp(a) values (p = 1.01e-59) and explained 3.5% of the variation of Lp(a). Statistical mediation analysis showed that the effect on Lp(a) is rather originating from the base change itself and is not mediated by DNA methylation levels. This finding is supported by eQTL data from 208 liver tissue samples from the GTEx project, which shows a significant association of the rs76735376 minor allele with increased LPA expression. To evaluate, whether the association signal at rs76735376 may actually be derived from a stronger eQTL signal in LD with this SNP, eQTL association results of all correlated SNPs (r2≥0.1) were integrated with genetic association results. This analysis pinpointed to rs10455872 as the potential trigger of the effect of rs76735376. Furthermore, both SNPs coincide with short apo(a) isoforms. Adjusting for both, rs10455872 and the apo(a) isoforms diminished the effect size of rs76735376 to 5.38 mg/dL (p = 0.0463). This indicates that the effect of rs76735376 can be explained by both an independent effect of the SNP and a strong correlation with rs10455872 and apo(a) isoforms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0232073PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7188291PMC
July 2020

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

Eur Heart J 2020 10;41(40):3949-3959

Department of Cardiac Surgery, Medical University of Innsbruck, Anichstraße 35, 6020 Innsbruck, Austria.

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.
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http://dx.doi.org/10.1093/eurheartj/ehaa140DOI Listing
October 2020

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

Proc Natl Acad Sci U S A 2020 04 24;117(14):7792-7798. Epub 2020 Mar 24.

Institute of Biological Chemistry, Biocenter, Medical University of Innsbruck, A-6020 Innsbruck, Austria;

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--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 (). Inactivation of the 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 gene lacked plasmanylethanolamine desaturase activity and had dramatically lowered plasmalogen levels in their tissues. These results assign the gene to plasmanylethanolamine desaturase and suggest that the previously characterized phenotype of -deficient mice may be caused by a lack of plasmalogens.
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http://dx.doi.org/10.1073/pnas.1917461117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7149458PMC
April 2020

Expression in Vascular Endothelial Cells Is Modulated by a Coronary Artery Disease-Associated Genetic Variant and Influences Monocyte Transendothelial Migration.

J Am Heart Assoc 2020 02 11;9(4):e014333. Epub 2020 Feb 11.

Shantou University Medical College Shantou China.

Background Genome-wide association studies have shown an association between the single-nucleotide polymorphism on chromosome 15q26.1 and coronary artery disease susceptibility. The underlying biological mechanism is, however, not fully understood. is located in the FES Upstream Region () gene, which is expressed in vascular endothelial cells (ECs). We investigated whether has an influence on expression in ECs and whether FURIN affects EC behavior. Methods and Results Quantitative reverse transcription-polymerase chain reaction analysis showed that cultured vascular ECs from individuals carrying the coronary artery disease risk allele of had higher expression than cells from noncarriers. In support, luciferase reporter analyses in ECs indicated that the risk allele had higher transcriptional activity than the nonrisk allele. Electrophoretic mobility shift assays using EC nuclear protein extracts detected a DNA-protein complex with allele-specific differential binding of a nuclear protein. Knockdown of FURIN in ECs reduced endothelin-1 secretion, nuclear factor-κB activity, vascular cell adhesion molecule-1, and MCP1 (monocyte chemotactic protein-1) expression and monocyte-endothelial adhesion and transmigration. A population-based study showed an association of the risk allele with higher circulating MCP1 levels and greater carotid intima-media thickness. Conclusions The coronary artery disease risk variant at the 15q26.1 locus modulates expression in vascular ECs. FURIN levels in ECs affect monocyte-endothelial adhesion and migration.
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http://dx.doi.org/10.1161/JAHA.119.014333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070217PMC
February 2020

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

BMC Cardiovasc Disord 2019 10 29;19(1):240. Epub 2019 Oct 29.

Department Primary Care & Population Health, University College London, London, UK.

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.
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http://dx.doi.org/10.1186/s12872-019-1187-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6820948PMC
October 2019

Temporary adhesion of the proseriate flatworm Minona ileanae.

Philos Trans R Soc Lond B Biol Sci 2019 10 9;374(1784):20190194. Epub 2019 Sep 9.

Institute of Zoology and Center of Molecular Biosciences Innsbruck, University of Innsbruck, 6020 Innsbruck, Austria.

Flatworms can very rapidly attach to and detach from many substrates. In the presented work, we analysed the adhesive system of the marine proseriate flatworm Minona ileanae. We used light-, scanning- and transmission electron microscopy to analyse the morphology of the adhesive organs, which are located at the ventral side of the tail-plate. We performed transcriptome sequencing and differential RNA-seq for the identification of tail-specific transcripts. Using in situ hybridization expression screening, we identified nine transcripts that were expressed in the cells of the adhesive organs. Knock-down of five of these transcripts by RNA interference led to a reduction of the animal's attachment capacity. Adhesive proteins in footprints were confirmed using mass spectrometry and antibody staining. Additionally, lectin labelling of footprints revealed the presence of several sugar moieties. Furthermore, we determined a genome size of about 560 Mb for M. ileanae. We demonstrated the potential of Oxford Nanopore sequencing of genomic DNA as a cost-effective tool for identifying the number of repeats within an adhesive protein and for combining transcripts that were fragments of larger genes. A better understanding of the molecules involved in flatworm bioadhesion can pave the way towards developing innovative glues with reversible adhesive properties. This article is part of the theme issue 'Transdisciplinary approaches to the study of adhesion and adhesives in biological systems'.
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http://dx.doi.org/10.1098/rstb.2019.0194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6745481PMC
October 2019

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

J Lipid Res 2019 01 9;60(1):186-199. Epub 2018 Nov 9.

Division of Genetic Epidemiology Department of Medical Genetics, Molecular and Clinical Pharmacology, Medical University of Innsbruck, Innsbruck, Austria

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 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 coding region currently unaddressed. To completely assess the variability in , 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.
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http://dx.doi.org/10.1194/jlr.M090381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314250PMC
January 2019

Plasmid-normalized quantification of relative mitochondrial DNA copy number.

Sci Rep 2018 10 18;8(1):15347. Epub 2018 Oct 18.

Division of Genetic Epidemiology, Department of Medical Genetics, Molecular and Clinical Pharmacology, Medical University of Innsbruck, Innsbruck, Austria.

Alterations of mitochondrial DNA (mtDNA) copy number have been associated with a wide variety of phenotypes and diseases. Unfortunately, the literature provides scarce methodical information about duplex targeting of nuclear and mtDNA that meets the quality criteria for qPCR. Therefore, we established a method for mtDNA copy number quantification using a quantitative PCR assay that allows for simultaneous targeting of a single copy nuclear gene (beta-2-microglobulin) and the t-RNA gene on the mtDNA. We include a plasmid containing both targets in order to normalize against differences in emission intensities of the fluorescent dyes Yakima Yellow and FAM. Applying the plasmid calibrator on an internal control reduced the intra-assay variability from 21% (uncorrected) to 7% (plasmid-corrected). Moreover, we noted that DNA samples isolated with different methods revealed different numbers of mtDNA copies, thus highlighting an important influence of the pre-analytical procedures. In summary, we developed a precise assay for mitochondrial copy number detection relative to nuclear DNA. Our method is applicable to comparative mitochondrial DNA copy number studies since the use of the dual insert plasmid allows correcting for the unequal emission intensities of the different fluorescent labels of the two targets.
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http://dx.doi.org/10.1038/s41598-018-33684-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6194030PMC
October 2018

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

Mov Disord 2019 01 11;34(1):114-123. Epub 2018 Oct 11.

Department of Internal Medicine II, Medical University of Innsbruck, Innsbruck, Austria.

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.
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http://dx.doi.org/10.1002/mds.27482DOI Listing
January 2019

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

Arterioscler Thromb Vasc Biol 2018 05 22;38(5):1230-1241. Epub 2018 Mar 22.

From the Division of Genetic Epidemiology, Department of Medical Genetics, Molecular and Clinical Pharmacology, Medical University of Innsbruck, Austria (G.E., C.L., F.K., S.C.)

Objective: Lp(a) (lipoprotein(a)) concentrations are widely genetically determined by the 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, isoforms, and genotypes of established genetic variants affecting Lp(a) concentrations ( variants, isoforms, and 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 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 isoform distribution but suggest the existence of novel functional variation in the small-isoform range.
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http://dx.doi.org/10.1161/ATVBAHA.118.310865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943067PMC
May 2018

Evaluating the Causal Relation of ApoA-IV with Disease-Related Traits - A Bidirectional Two-sample Mendelian Randomization Study.

Sci Rep 2017 08 18;7(1):8734. Epub 2017 Aug 18.

Division of Genetic Epidemiology, Department of Medical Genetics, Molecular and Clinical Pharmacology, Medical University of Innsbruck, Innsbruck, 6020, Austria.

Apolipoprotein A-IV (apoA-IV) has been observed to be associated with lipids, kidney function, adiposity- and diabetes-related parameters. To assess the causal relationship of apoA-IV with these phenotypes, we conducted bidirectional Mendelian randomization (MR) analyses using publicly available summary-level datasets from GWAS consortia on apoA-IV concentrations (n = 13,813), kidney function (estimated glomerular filtration rate (eGFR), n = 133,413), lipid traits (HDL cholesterol, LDL cholesterol, triglycerides, n = 188,577), adiposity-related traits (body-mass-index (n = 322,206), waist-hip-ratio (n = 210,088)) and fasting glucose (n = 133,010). Main analyses consisted in inverse-variance weighted and multivariable MR, whereas MR-Egger regression and weighted median estimation were used as sensitivity analyses. We found that eGFR is likely to be causal on apoA-IV concentrations (53 SNPs; causal effect estimate per 1-SD increase in eGFR = -0.39; 95% CI = [-0.54, -0.24]; p-value = 2.4e-07). Triglyceride concentrations were also causally associated with apoA-IV concentrations (40 SNPs; causal effect estimate per 1-SD increase in triglycerides = -0.06; 95% CI = [-0.08, -0.04]; p-value = 4.8e-07), independently of HDL-C and LDL-C concentrations (causal effect estimate from multivariable MR = -0.06; 95% CI = [-0.10, -0.02]; p-value = 0.0014). Evaluating the inverse direction of causality revealed a possible causal association of apoA-IV on HDL-cholesterol (2 SNPs; causal effect estimate per one percent increase in apoA-IV = -0.40; 95% CI = [-0.60, -0.21]; p-value = 5.5e-05).
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http://dx.doi.org/10.1038/s41598-017-07213-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5562707PMC
August 2017

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

J Lipid Res 2017 09 16;58(9):1834-1844. Epub 2017 May 16.

Division of Genetic Epidemiology, Department of Medical Genetics, Molecular and Clinical Pharmacology, Medical University of Innsbruck, 6020 Innsbruck, Austria

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 and 1 SNP in the 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 , 1 in the 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, = 3.35 × 10). Median Lp(a) values increased from 2.1 to 91.1 mg/dl with increasing number of Lp(a)-increasing alleles. We found the -determining allele of rs7412 to be significantly associated with Lp(a) concentrations ( = 3.47 × 10). Each 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 gene with Lp(a) ( = 3.4 × 10). In summary, we identified a large number of independent SNPs in the gene region, as well as the allele, to be significantly associated with Lp(a) concentrations.
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http://dx.doi.org/10.1194/jlr.M076232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5580897PMC
September 2017

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

Eur Heart J 2017 Jun;38(23):1823-1831

Division of Genetic Epidemiology, Department of Medical Genetics, Molecular and Clinical Pharmacology, Medical University of Innsbruck, Schoepfstrasse 41, 6020 Innsbruck, Austria.

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.
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http://dx.doi.org/10.1093/eurheartj/ehx174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837733PMC
June 2017

PCSK9 genetic variants and risk of type 2 diabetes: a mendelian randomisation study.

Lancet Diabetes Endocrinol 2017 02 29;5(2):97-105. Epub 2016 Nov 29.

Centre for Population Health Sciences, Usher Institute of Population Health Sciences and Informatics, University of Edinburgh, Edinburgh, UK.

Background: Statin treatment and variants in the gene encoding HMG-CoA reductase are associated with reductions in both the concentration of LDL cholesterol and the risk of coronary heart disease, but also with modest hyperglycaemia, increased bodyweight, and modestly increased risk of type 2 diabetes, which in no way offsets their substantial benefits. We sought to investigate the associations of LDL cholesterol-lowering PCSK9 variants with type 2 diabetes and related biomarkers to gauge the likely effects of PCSK9 inhibitors on diabetes risk.

Methods: In this mendelian randomisation study, we used data from cohort studies, randomised controlled trials, case control studies, and genetic consortia to estimate associations of PCSK9 genetic variants with LDL cholesterol, fasting blood glucose, HbA, fasting insulin, bodyweight, waist-to-hip ratio, BMI, and risk of type 2 diabetes, using a standardised analysis plan, meta-analyses, and weighted gene-centric scores.

Findings: Data were available for more than 550 000 individuals and 51 623 cases of type 2 diabetes. Combined analyses of four independent PCSK9 variants (rs11583680, rs11591147, rs2479409, and rs11206510) scaled to 1 mmol/L lower LDL cholesterol showed associations with increased fasting glucose (0·09 mmol/L, 95% CI 0·02 to 0·15), bodyweight (1·03 kg, 0·24 to 1·82), waist-to-hip ratio (0·006, 0·003 to 0·010), and an odds ratio for type diabetes of 1·29 (1·11 to 1·50). Based on the collected data, we did not identify associations with HbA (0·03%, -0·01 to 0·08), fasting insulin (0·00%, -0·06 to 0·07), and BMI (0·11 kg/m, -0·09 to 0·30).

Interpretation: PCSK9 variants associated with lower LDL cholesterol were also associated with circulating higher fasting glucose concentration, bodyweight, and waist-to-hip ratio, and an increased risk of type 2 diabetes. In trials of PCSK9 inhibitor drugs, investigators should carefully assess these safety outcomes and quantify the risks and benefits of PCSK9 inhibitor treatment, as was previously done for statins.

Funding: British Heart Foundation, and University College London Hospitals NHS Foundation Trust (UCLH) National Institute for Health Research (NIHR) Biomedical Research Centre.
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http://dx.doi.org/10.1016/S2213-8587(16)30396-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5266795PMC
February 2017

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

Stroke 2016 12 8;47(12):2904-2909. Epub 2016 Nov 8.

From the Institute of Medical Biometry and Statistics (IMBS), University of Lübeck, University Medical Center Schleswig-Holstein, Campus Lübeck, Germany (N.H., M.V., A.Z.); Clinical Epidemiology, Integrated Research and Treatment Center, Center for Sepsis Control and Care (CSCC), Jena University Hospital, Germany (M.H.G., A.S.); Institute for Medical Informatics, Biometry and Epidemiology (IMIBE), University of Duisburg-Essen, Germany (M.H.G., R.E., K.-H.J., S.M.); Division of Genetic Epidemiology, Department of Medical Genetics, Molecular and Clinical Pharmacology, Medical University of Innsbruck, Austria (S.C., F.K.); Institute of Human Genetics (S.H.) and Department of Epileptology (S.M.-H.), University of Bonn, Germany; Department of Genomics, Life & Brain Research Center, Bonn, Germany (S.H.); Institute of Occupational, Social and Environmental Medicine, Center for Health and Society, Faculty of Medicine, University of Düsseldorf, Germany (F.H., B.H.); Department of Neurology, University Hospital Bonn, Germany (S.M.-H., T.K.); Cologne Center of Genomics, University of Cologne, Germany (G.N., P.N.); Department of Medical Biometry and Epidemiology, University Medical Center Hamburg-Eppendorf, Germany (M.V.); Center for Clinical Trials, University of Lübeck, Germany (A.Z.); and School of Mathematics, Statistics and Computer Science, University of KwaZulu-Natal, Pietermaritzburg, South Africa (A.Z.).

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 in the family-based association analyses. One of these signals was replicated in HNR (rs2416804, 1-sided P=1.60×10, 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.
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http://dx.doi.org/10.1161/STROKEAHA.116.013943DOI Listing
December 2016

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

Arterioscler Thromb Vasc Biol 2016 11 29;36(11):2252-2258. Epub 2016 Sep 29.

From the Division of Genetic Epidemiology, Department of Medical Genetics, Molecular and Clinical Pharmacology, Medical University of Innsbruck, Austria (S.C., S.F., C.L., F.K.); and Division of Genetic Epidemiology, Institute for Medical Biometry and Statistics, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany (A.K.).

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). 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.
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http://dx.doi.org/10.1161/ATVBAHA.116.308393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5084637PMC
November 2016

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

Hum Mol Genet 2016 08 12;25(16):3635-3646. Epub 2016 Jul 12.

Department of Medicine, Internal Medicine, Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland.

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   ), rs5104 in APOA4 (P = 1.79 × 10) and rs4241819 in KLKB1 (P = 5.6 × 10). 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   ). 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). 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.
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http://dx.doi.org/10.1093/hmg/ddw211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5179953PMC
August 2016

Influence of DNA extraction methods on relative telomere length measurements and its impact on epidemiological studies.

Sci Rep 2016 05 3;6:25398. Epub 2016 May 3.

Division of Genetic Epidemiology, Department of Medical Genetics, Molecular and Clinical Pharmacology, Medical University of Innsbruck, Innsbruck, Austria.

Measurement of telomere length is widely used in epidemiologic studies. Insufficient standardization of the measurements processes has, however, complicated the comparison of results between studies. We aimed to investigate whether DNA extraction methods have an influence on measured values of relative telomere length (RTL) and whether this has consequences for epidemiological studies. We performed four experiments with RTL measurement in quadruplicate by qPCR using DNA extracted with different methods: 1) a standardized validation experiment including three extraction methods (magnetic-particle-method EZ1, salting-out-method INV, phenol-chloroform-isoamyl-alcohol PCI) each in the same 20 samples demonstrated pronounced differences in RTL with lowest values with EZ1 followed by INV and PCI-isolated DNA; 2) a comparison of 307 samples from an epidemiological study showing EZ1-measurements 40% lower than INV-measurements; 3) a matching-approach of two similar non-diseased control groups including 143 pairs of subjects revealed significantly shorter RTL in EZ1 than INV-extracted DNA (0.844 ± 0.157 vs. 1.357 ± 0.242); 4) an association analysis of RTL with prevalent cardiovascular disease detected a stronger association with INV than with EZ1-extracted DNA. In summary, DNA extraction methods have a pronounced influence on the measured RTL-values. This might result in spurious or lost associations in epidemiological studies under certain circumstances.
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http://dx.doi.org/10.1038/srep25398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4853716PMC
May 2016

Update of the effect estimates for common variants associated with carotid intima media thickness within four independent samples: The Bonn IMT Family Study, the Heinz Nixdorf Recall Study, the SAPHIR Study and the Bruneck Study.

Atherosclerosis 2016 06 4;249:83-7. Epub 2016 Apr 4.

Division of Genetic Epidemiology, Department of Medical Genetics, Molecular and Clinical Pharmacology, Medical University of Innsbruck, Innsbruck, Austria. Electronic address:

Carotid intima media thickness (cIMT) is a marker for subclinical atherosclerosis. The most recent genome-wide association meta-analysis (GWAMA) from the CHARGE consortium identified four genomic regions showing either significant (ZHX2, APOC1, PINX1) or suggestive evidence (SLC17A4) for an association. Here we assess these four cIMT loci in a pooled analysis of four independent studies including 5446 individuals by providing updated unbiased effect estimates of the cIMT association signals. The pooled estimates of our four independent samples pointed in the same direction and were similar to those of the GWAMA. When updating the independent second stage replication results from the earlier CHARGE GWAMA by our estimates, effect size estimates were closer to those of the original CHARGE discovery. A fine-mapping approach within a ±50 kb region around each lead SNP from CHARGE revealed 27 variants with larger estimated effect sizes than the lead SNPs but only three of them showed a r(2) > 0.40 with these respective lead SNPs from CHARGE. Some variants are located within potential functional loci.
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http://dx.doi.org/10.1016/j.atherosclerosis.2016.03.042DOI Listing
June 2016

Genetic associations at 53 loci highlight cell types and biological pathways relevant for kidney function.

Nat Commun 2016 Jan 21;7:10023. Epub 2016 Jan 21.

Unit of Genetic Epidemiology and Bioinformatics, Department of Epidemiology, University Medical Center Groningen, PO Box 30001, Groningen 9700 RB, The Netherlands.

Reduced glomerular filtration rate defines chronic kidney disease and is associated with cardiovascular and all-cause mortality. We conducted a meta-analysis of genome-wide association studies for estimated glomerular filtration rate (eGFR), combining data across 133,413 individuals with replication in up to 42,166 individuals. We identify 24 new and confirm 29 previously identified loci. Of these 53 loci, 19 associate with eGFR among individuals with diabetes. Using bioinformatics, we show that identified genes at eGFR loci are enriched for expression in kidney tissues and in pathways relevant for kidney development and transmembrane transporter activity, kidney structure, and regulation of glucose metabolism. Chromatin state mapping and DNase I hypersensitivity analyses across adult tissues demonstrate preferential mapping of associated variants to regulatory regions in kidney but not extra-renal tissues. These findings suggest that genetic determinants of eGFR are mediated largely through direct effects within the kidney and highlight important cell types and biological pathways.
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http://dx.doi.org/10.1038/ncomms10023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4735748PMC
January 2016
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