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Cambodia harbours a variety of human aboriginal populations that have scarcely been studied in terms of genetic diversity of entire mitochondrial genomes. Here we present the matrilineal gene pool of 299 Cambodian refugees from three different ethnic groups (Cham, Khmer, and Khmer Loeu) deriving from 16 Cambodian districts. After establishing a DNA-saving high-throughput strategy for mitochondrial whole-genome Sanger sequencing, a HaploGrep based workflow was used for quality control, haplogroup classification and phylogenetic reconstruction. The application of diverse phylogenetic algorithms revealed an exciting picture of the genetic diversity of Cambodia, especially in relation to populations from Southeast Asia and from the whole world. A total of 224 unique haplotypes were identified, which were mostly classified under haplogroups B5a1, F1a1, or categorized as newly defined basal haplogroups or basal sub-branches of R, N and M clades. The presence of autochthonous maternal lineages could be confirmed as reported in previous studies. The exceptional homogeneity observed between and within the three investigated Cambodian ethnic groups indicates genetic isolation of the whole population. Between ethnicities, genetic barriers were not detected. The mtDNA data presented here increases the phylogenetic resolution in Cambodia significantly, thereby highlighting the need for an update of the current human mtDNA phylogeny.

Obesity has complex links to respiratory health. Mendelian randomization (MR) enables assessment of causality of body mass index (BMI) effects on airflow obstruction and mid-expiratory flow. In the adult SAPALDIA cohort, recruiting 9,651 population-representative samples aged 18-60 years at baseline (female 51%), BMI and the ratio of forced expiratory volume in 1 second (FEV 1 ) to forced vital capacity (FVC) as well as forced mid-expiratory flow (FEF25-75%) were measured three times over 20 follow-up years. The causal effects of BMI in childhood and adulthood on FEV1/FVC and FEF25-75% were assessed in predictive (BMI averaged over 1st and 2nd, lung function (LF) averaged over 2nd and 3rd follow-up; N = 2,850) and long-term cross-sectional models (BMI and LF averaged over all follow-ups; N = 2,728) by Mendelian Randomization analyses with the use of weighted BMI allele score as an instrument variable and two-stage least squares (2SLS) method. Three different BMI allele scores were applied to specifically capture the part of BMI in adulthood that likely reflects tracking of genetically determined BMI in childhood. The main causal effects were derived from models containing BMI (instrumented by BMI genetic score), age, sex, height, and packyears smoked as covariates. BMI interactions were instrumented by the product of the instrument (BMI genetic score) and the relevant concomitant variable. Causal effects of BMI on FEV1/FVC and FEF25-75% were observed in both the predictive and long-term cross-sectional models. The causal BMI- LF effects were negative and attenuated with increasing age, and stronger if instrumented by gene scores associated with childhood BMI. This non-standard MR approach interrogating causal effects of multiplicative interaction suggests that the genetically rooted part of BMI patterns in childhood may be of particular relevance for the level of small airway function and airflow obstruction later in life. The methodological relevance of the results is first to point to the importance of a life course perspective in studies on the etiological role of BMI in respiratory health, and second to point out novel methodological aspects to be considered in future MR studies on the causal effects of obesity related phenotypes., Competing Interests: DS obtained funding for grants from the following companies: Astra-Zeneca, PanGas, Wenmann, Curetis, Boston Scientific, Cucassia, and also from Swiss Lung Leagues. DS was paid for lectures including service on speaker's bureaus by the following companies: Astra-Zeneca, Novartis, GSK, Roche, Zambon, Pfizer, Schwabe Pharma and Vifor. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Probst-Hensch, Jeong, Stolz, Pons, Soccal, Bettschart, Jarvis, Holloway, Kronenberg, Imboden, Schindler and Lovison.)

Background: After the 2002/2003 severe acute respiratory syndrome outbreak, 30% of survivors exhibited persisting structural pulmonary abnormalities. The long-term pulmonary sequelae of coronavirus disease 2019 (COVID-19) are yet unknown, and comprehensive clinical follow-up data are lacking., Methods: In this prospective, multicentre, observational study, we systematically evaluated the cardiopulmonary damage in subjects recovering from COVID-19 at 60 and 100 days after confirmed diagnosis. We conducted a detailed questionnaire, clinical examination, laboratory testing, lung function analysis, echocardiography and thoracic low-dose computed tomography (CT)., Results: Data from 145 COVID-19 patients were evaluated, and 41% of all subjects exhibited persistent symptoms 100 days after COVID-19 onset, with dyspnoea being most frequent (36%). Accordingly, patients still displayed an impaired lung function, with a reduced diffusing capacity in 21% of the cohort being the most prominent finding. Cardiac impairment, including a reduced left ventricular function or signs of pulmonary hypertension, was only present in a minority of subjects. CT scans unveiled persisting lung pathologies in 63% of patients, mainly consisting of bilateral ground-glass opacities and/or reticulation in the lower lung lobes, without radiological signs of pulmonary fibrosis. Sequential follow-up evaluations at 60 and 100 days after COVID-19 onset demonstrated a vast improvement of symptoms and CT abnormalities over time., Conclusion: A relevant percentage of post-COVID-19 patients presented with persisting symptoms and lung function impairment along with radiological pulmonary abnormalities >100 days after the diagnosis of COVID-19. However, our results indicate a significant improvement in symptoms and cardiopulmonary status over time., Competing Interests: Conflict of interest: T. Sonnweber has nothing to disclose. Conflict of interest: S. Sahanic has nothing to disclose. Conflict of interest: A. Pizzini has nothing to disclose. Conflict of interest: A. Luger has nothing to disclose. Conflict of interest: C. Schwabl has nothing to disclose. Conflict of interest: B. Sonnweber has nothing to disclose. Conflict of interest: K. Kurz has nothing to disclose. Conflict of interest: S. Koppelstätter has nothing to disclose. Conflict of interest: D. Haschka has nothing to disclose. Conflict of interest: V. Petzer has nothing to disclose. Conflict of interest: A. Boehm has nothing to disclose. Conflict of interest: M. Aichner has nothing to disclose. Conflict of interest: P. Tymoszuk has nothing to disclose. Conflict of interest: D. Lener has nothing to disclose. Conflict of interest: M. Theurl has nothing to disclose. Conflict of interest: A. Lorsbach-Köhler has nothing to disclose. Conflict of interest: A. Tancevski has nothing to disclose. Conflict of interest: A. Schapfl has nothing to disclose. Conflict of interest: M. Schaber has nothing to disclose. Conflict of interest: R. Hilbe has nothing to disclose. Conflict of interest: M. Nairz has nothing to disclose. Conflict of interest: B. Puchner has nothing to disclose. Conflict of interest: D. Hüttenberger has nothing to disclose. Conflict of interest: C. Tschurtschenthaler has nothing to disclose. Conflict of interest: M. Aßhoff has nothing to disclose. Conflict of interest: A. Peer has nothing to disclose. Conflict of interest: F. Hartig has nothing to disclose. Conflict of interest: R. Bellmann has nothing to disclose. Conflict of interest: M. Joannidis has nothing to disclose. Conflict of interest: C. Gollmann-Tepeköylü has nothing to disclose. Conflict of interest: J. Holfeld has nothing to disclose. Conflict of interest: G. Feuchtner has nothing to disclose. Conflict of interest: A. Egger has nothing to disclose. Conflict of interest: G. Hoermann has nothing to disclose. Conflict of interest: A. Schroll has nothing to disclose. Conflict of interest: G. Fritsche has nothing to disclose. Conflict of interest: S. Wildner has nothing to disclose. Conflict of interest: R. Bellmann-Weiler has nothing to disclose. Conflict of interest: R. Kirchmair has nothing to disclose. Conflict of interest: R. Helbok has nothing to disclose. Conflict of interest: H. Prosch has nothing to disclose. Conflict of interest: D. Rieder has nothing to disclose. Conflict of interest: Z. Trajanoski has nothing to disclose. Conflict of interest: F. Kronenberg has nothing to disclose. Conflict of interest: E. Wöll has nothing to disclose. Conflict of interest: G. Weiss has nothing to disclose. Conflict of interest: G. Widmann has nothing to disclose. Conflict of interest: J. Löffler-Ragg has nothing to disclose. Conflict of interest: I. Tancevski reports an Investigator Initiated Study (IIS) grant from Boehringer Ingelheim (IIS 1199-0424)., (Copyright ©ERS 2021.)

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 2 /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 2 at follow-up among those with eGFRcrea 60 mL/min/1.73m 2 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., (Copyright © 2020 International Society of Nephrology. All rights reserved.)

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., (© American Association for Clinical Chemistry 2020.)

Metabolite levels in urine may provide insights into genetic mechanisms shaping their related pathways. We therefore investigate the cumulative contribution of rare, exonic genetic variants on urine levels of 1487 metabolites and 53,714 metabolite ratios among 4864 GCKD study participants. Here we report the detection of 128 significant associations involving 30 unique genes, 16 of which are known to underlie inborn errors of metabolism. The 30 genes are strongly enriched for shared expression in liver and kidney (odds ratio = 65, p-FDR = 3e-7), with hepatocytes and proximal tubule cells as driving cell types. Use of UK Biobank whole-exome sequencing data links genes to diseases connected to the identified metabolites. In silico constraint-based modeling of gene knockouts in a virtual whole-body, organ-resolved metabolic human correctly predicts the observed direction of metabolite changes, highlighting the potential of linking population genetics to modeling. Our study implicates candidate variants and genes for inborn errors of metabolism.

Within-species contamination is a major issue in sequencing studies, especially for mitochondrial studies. Contamination can be detected by analyzing the nuclear genome or by inspecting polymorphic sites in the mitochondrial genome (mtDNA). Existing methods using the nuclear genome are computationally expensive, and no appropriate tool for detecting sample contamination in large-scale mtDNA data sets is available. Here we present haplocheck, a tool that requires only the mtDNA to detect contamination in both targeted mitochondrial and whole-genome sequencing studies. Our in silico simulations and amplicon mixture experiments indicate that haplocheck detects mtDNA contamination accurately and is independent of the phylogenetic distance within a sample mixture. By applying haplocheck to The 1000 Genomes Project Consortium data, we further evaluate the application of haplocheck as a fast proxy tool for nDNA-based contamination detection using the mtDNA and identify the mitochondrial copy number within a mixture as a critical component for the overall accuracy. The haplocheck tool is available both as a command-line tool and as a cloud web service producing interactive reports that facilitates the navigation through the phylogeny of contaminated samples., (© 2021 Weissensteiner et al.; Published by Cold Spring Harbor Laboratory Press.)

Objective: Increased risk of atherosclerotic cardiovascular disease in subjects with type 2 diabetes is linked to elevated levels of triglyceride-rich lipoproteins and their remnants. The metabolic effects of PCSK9 (proprotein convertase subtilisin/kexin 9) inhibitors on this dyslipidemia were investigated using stable-isotope-labeled tracers. Approach and Results: Triglyceride transport and the metabolism of apos (apolipoproteins) B48, B100, C-III, and E after a fat-rich meal were investigated before and on evolocumab treatment in 13 subjects with type 2 diabetes. Kinetic parameters were determined for the following: apoB48 in chylomicrons; triglyceride in VLDL 1 (very low-density lipoprotein) and VLDL 2 ; and apoB100 in VLDL 1 , VLDL 2 , IDL (intermediate-density lipoprotein), and LDL (low-density lipoprotein). Evolocumab did not alter the kinetics of apoB48 in chylomicrons or apoB100 or triglyceride in VLDL 1 . In contrast, the fractional catabolic rates of VLDL 2 -apoB100 and VLDL 2 -triglyceride were both increased by about 45%, which led to a 28% fall in the VLDL 2 plasma level. LDL-apoB100 was markedly reduced by evolocumab, which was linked to metabolic heterogeneity in this fraction. Evolocumab increased clearance of the more rapidly metabolized LDL by 61% and decreased production of the more slowly cleared LDL by 75%. ApoC-III kinetics were not altered by evolocumab, but the apoE fractional catabolic rates increased by 45% and the apoE plasma level fell by 33%. The apoE fractional catabolic rates was associated with the decrease in VLDL 2 - and IDL-apoB100 concentrations., Conclusions: Evolocumab had only minor effects on lipoproteins that are involved in triglyceride transport (chylomicrons and VLDL 1 ) but, in contrast, had a profound impact on lipoproteins that carry cholesterol (VLDL 2 , IDL, LDL). Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02948777.

Massive parallel sequencing technologies are promising a highly sensitive detection of low-level mutations, especially in mitochondrial DNA (mtDNA) studies. However, processes from DNA extraction and library construction to bioinformatic analysis include several varying tasks. Further, there is no validated recommendation for the comprehensive procedure. In this study, we examined potential pitfalls on the sequencing results based on two-person mtDNA mixtures. Therefore, we compared three DNA polymerases, six different variant callers in five mixtures between 50% and 0.5% variant allele frequencies generated with two different amplification protocols. In total, 48 samples were sequenced on Illumina MiSeq. Low-level variant calling at the 1% variant level and below was performed by comparing trimming and PCR duplicate removal as well as six different variant callers. The results indicate that sensitivity, specificity, and precision highly depend on the investigated polymerase but also vary based on the analysis tools. Our data highlight the advantage of prior standardization and validation of the individual laboratory setup with a DNA mixture model. Finally, we provide an artificial heteroplasmy benchmark dataset that can help improve somatic variant callers or pipelines, which may be of great interest for research related to cancer and aging.

Background and Aims: Peripheral artery disease (PAD) affects more than 200 million people worldwide. Increased low-density lipoprotein cholesterol (LDL-C)levels are a risk factor for PAD and the concentrations are influenced by proprotein convertase subtilisin/kexin type 9 (PCSK9). PCSK9 regulates the recycling of the LDL receptors to the cell membrane surface. Only a limited number of mostly small studies investigated the association between serum PCSK9 concentrations and PAD of different definition, which revealed contrasting results., Methods: Serum PCSK9, lipoprotein(a) [Lp(a)] and other lipoprotein concentrations were measured in male participants of the CAVASIC study, a case-control study of 248 patients with intermittent claudication and 251 age and diabetes-matched controls., Results: PAD patients had significantly higher PCSK9 concentrations when compared to controls (250 ± 77 vs. 222 ± 68 ng/mL, p < 0.001). Logistic regression analysis with adjustment for age revealed that an increase in PCSK9 concentrations of 100 ng/mL was associated with a 1.78-fold higher risk for PAD (95%CI 1.38-2.33, p = 1.43 × 10 -5 ). The association attenuated, but was still significant when adjusting additionally for age, Lp(a)-corrected LDL cholesterol, HDL cholesterol, high-sensitivity-CRP, statin treatment, hypertension, diabetes mellitus and smoking (OR = 1.49, 95%CI 1.03-2.18, p = 0.035). The strongest association was observed when both PCSK9 concentrations were above the median and Lp(a) concentrations were above 30 mg/dL (OR = 3.35, 95%CI 1.49-7.71, p = 0.0038)., Conclusions: Our findings suggest an association of higher PCSK9 concentrations with PAD, which was independent of other lipid parameters and classical cardiovascular risk factors., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The harm caused by the long interdialytic interval in three-times-per-week haemodialysis regimens (3×WHD) may relate to fluid accumulation and associated high ultrafiltration rate (UFR). Four-times-per-week haemodialysis (4×WHD) may offer a solution, but its impact on mortality, hospitalization and vascular access complications is unknown., Methods: From the AROii cohort of incident in-centre haemodialysis patients, 3×WHD patients with a UFR >10 mL/kg/h were identified. The hazard for the outcomes of mortality, hospitalization and vascular access complications in those who switched to 4×WHD compared with staying on 3×WHD was estimated using a marginal structural Cox proportional hazards model. Adjustment included baseline patient and treatment characteristics with inverse probability weighting used to adjust for time-varying UFR and cardiovascular comorbidities., Results: From 10 637 European 3×WHD patients, 3842 (36%) exceeded a UFR >10 mL/kg/h. Of these, 288 (7.5%) started 4×WHD and at baseline were more comorbid. Event rates while receiving 4×WHD compared with 3×WHD were 12.6 compared with 10.8 per 100 patient years for mortality, 0.96 compared with 0.65 per year for hospitalization and 14.7 compared with 8.0 per 100 patient years for vascular access complications. Compared with 3×WHD, the unadjusted hazard ratio (HR) for mortality on 4×WHD was 1.05 [95% confidence interval (CI) 0.78-1.42]. Following adjustment for baseline demographics, time-varying treatment probability and censoring risks, this HR was 0.73 (95% CI 0.50-1.05; P = 0.095). Despite these adjustments on 4×WHD, the HR for hospitalization remained elevated and vascular access complications were similar to 3×WHD., Conclusions: This observational study was not able to demonstrate a mortality benefit in patients switched to 4×WHD. To demonstrate the true benefits of 4×WHD requires a large, well-designed clinical trial. Our data may help in the design of such a study., (© The Author(s) 2020. Published by Oxford University Press on behalf of ERA-EDTA.)

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., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

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 2  = 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.

Telomere length is known to be inversely associated with aging and has been proposed as a marker for aging-related diseases. Telomere attrition can be accelerated by oxidative stress and inflammation, both commonly present in patients with chronic kidney disease. Here, we investigated whether relative telomere length is associated with mortality in a large cohort of patients with chronic kidney disease stage G3 and A1-3 or G1-2 with overt proteinuria (A3) at enrollment. Relative telomere length was quantified in peripheral blood by a quantitative PCR method in 4,955 patients from the GCKD study, an ongoing prospective observational cohort. Complete four-year follow-up was available from 4,926 patients in whom we recorded 354 deaths. Relative telomere length was a strong and independent predictor of all-cause mortality. Each decrease of 0.1 relative telomere length unit was highly associated with a 14% increased risk of death (hazard ratio1.14 [95% confidence interval 1.06-1.22]) in a model adjusted for age, sex, baseline eGFR, urine albumin/creatinine ratio, diabetes mellitus, prevalent cardiovascular disease, LDL-cholesterol, HDL-cholesterol, smoking, body mass index, systolic and diastolic blood pressure, C-reactive protein and serum albumin. This translated to a 75% higher risk for those in the lowest compared to the highest quartile of relative telomere length. The association was mainly driven by 117 cardiovascular deaths (1.20 [1.05-1.35]) as well as 67 deaths due to infections (1.27 [1.07-1.50]). Thus, our findings support an association of shorter telomere length with all-cause mortality, cardiovascular mortality and death due to infections in patients with moderate chronic kidney disease., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

While a shift in energy metabolism is essential to cancers, the knowledge about the involvement of the mitochondrial genome in tumorigenesis and progression in oral squamous cell carcinoma (OSCC) is still very limited. In this study, we evaluated 37 OSCC tumors and the corresponding benign mucosa tissue pairs by deep sequencing of the complete mitochondrial DNA (mtDNA). After extensive quality control, we identified 287 variants, 137 in tumor and 150 in benign samples exceeding the 1% threshold. Variant heteroplasmy levels were significantly increased in cancer compared to benign tissues ( p = 0.0002). Furthermore, pairwise high heteroplasmy frequency difference variants (∆HF% > 20) with potential functional impact were increased in the cancer tissues ( p = 0.024). Fourteen mutations were identified in the protein-coding region, out of which thirteen were detected in cancer and only one in benign tissue. After eight years of follow-up, the risk of mortality was higher for patients who harbored at least one ∆HF% > 20 variant in mtDNA protein-coding regions relative to those with no mutations (HR = 4.6, (95%CI = 1.3-17); p = 0.019 in primary tumor carriers). Haplogroup affiliation showed an impact on survival time, which however needs confirmation in a larger study. In conclusion, we observed a significantly higher accumulation of somatic mutations in the cancer tissues associated with a worse prognosis.

Background: Elevated plasma concentrations of symmetric and asymmetric dimethylarginine (SDMA and ADMA, respectively) and a lower plasma concentration of the structurally related homoarginine are commonly observed in patients with chronic kidney disease (CKD) and independently predict total mortality as well as progression of renal disease. We aimed to identify drugs that may alter this adverse metabolite pattern in a favourable fashion., Methods: Plasma ADMA, SDMA, homoarginine and l-arginine were determined by liquid chromatography-tandem mass spectrometry in 4756 CKD patients ages 18-74 years with an estimated glomerular filtration rate (eGFR) of 30-60 mL/min/1.73 m2 or an eGFR >60 mL/min/1.73 m2 and overt proteinuria who were enrolled in the German Chronic Kidney Disease (GCKD) study. Associations between laboratory, clinical and medication data were assessed., Results: Intake of several commonly used drugs was independently associated with plasma concentrations of homoarginine and/or related metabolites. Among these, the peroxisome proliferator-activated receptor alpha (PPAR-α) agonist fenofibrate was associated with the most profound differences in ADMA, SDMA and homoarginine plasma concentrations: 66 patients taking fenofibrate had a multivariable adjusted odds ratio (OR) of 5.83 [95% confidence interval (CI) 2.82-12.03, P < 0.001] to have a plasma homoarginine concentration above the median. The median homoarginine plasma concentration in patients taking fenofibrate was 2.30 µmol/L versus 1.55 in patients not taking the drug (P < 0.001). In addition, fibrates were significantly associated with lower plasma SDMA and higher l-arginine concentrations. In contrast, glucocorticoids were associated with lower plasma homoarginine, with adjusted ORs of 0.52 (95% CI 0.40-0.67, P < 0.001) and 0.53 (95% CI 0.31-0.90, P = 0.018) for prednisolone and methylprednisolone, respectively., Conclusions: In a large cohort of CKD patients, intake of fenofibrate and glucocorticoids were independently associated with higher and lower plasma homoarginine concentrations, respectively. Effects on plasma homoarginine and methylarginines warrant further investigation as potential mechanisms mediating beneficial or adverse drug effects., (© The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Background: The extension of the interdialytic interval due to due to dialysis session non-attendance varies according to which session of the week the patient misses. The impact of this on subsequent hospitalization and mortality is unknown., Methods: The ARO cohort study prospectively collected data from hemodialysis patients across 15 European countries on demography, comorbidity, laboratory, hospitalisation, mortality and individual hemodialysis sessions from 2007 to 2014. Event rates for death and hospitalisation according to dialysis day of the week were calculated for patients who attended the three previous scheduled hemodialysis sessions, who then on the next scheduled dialysis day either attended or did not attend. The hazard ratio for these events following non-attendance for the first compared to the second dialysis session of the week was estimated using Cox proportional hazards model adjusted for patient demographics., Results: 3.8 million hemodialysis sessions in 9397 patients were analysed. The non-attendance rates for Monday/Wednesday/Friday sessions were 0.8, 0.9% & 1.4% respectively, and for Tuesday/Thursday/Saturday sessions were 0.6, 1.0% & 1.2% respectively. Compared to those who attended, for the 48-72 h between non-attendance and the next scheduled haemodialysis session, mortality significantly increased from 4.86 to 51.9/100 pt-yrs and hospitalisation increased from 0.58 to 2.1/yr. As time from the two-day break increased, the risk associated with non-attendance lessened: compared to missing the second hemodialysis session, missing the first session had a hazard ratio for mortality of 2.04 (95% CI 1.27-3.29), and for hospitalisation 1.78 (95% CI 1.29-2.47). In patients who attended their scheduled dialysis session and the three preceding, after the two-day break there were absolute increases in mortality (8.3 vs. 4.9/100 pt-yrs) and hospitalisation (1.0 vs. 0.6/yr for the rest of the week) comparable to previous studies., Conclusions: In addition to hospitalisation and mortality increases seen after the two-day break, additional harm may be manifested in the greater increases in mortality and hospitalisation observed after non-attendance for the first hemodialysis session after the two-day break compared to missing other sessions.

Aims/hypothesis: Microvascular disease in type 2 diabetes is a significant cause of end-stage renal disease, blindness and peripheral neuropathy. The strict control of known risk factors, e.g. lifestyle, hyperglycaemia, hypertension and dyslipidaemia, reduces the incidence of microvascular complications, but a residual risk remains. Lipoprotein (a) [Lp(a)] is a strong risk factor for macrovascular disease in the general population. We hypothesised that plasma Lp(a) levels and the LPA gene SNPs rs10455872 and rs3798220 are associated with the incident development of microvascular complications in type 2 diabetes., Methods: Analyses were performed of data from the DiaGene study, a prospective study for complications of type 2 diabetes, collected in the city of Eindhoven, the Netherlands (n = 1886 individuals with type 2 diabetes, mean follow-up time = 6.97 years). To assess the relationship between plasma Lp(a) levels and the LPA SNPs with each newly developed microvascular complication (retinopathy n = 223, nephropathy n = 246, neuropathy n = 236), Cox proportional hazards models were applied and adjusted for risk factors for microvascular complications (age, sex, mean arterial pressure, non-HDL-cholesterol, HDL-cholesterol, BMI, duration of type 2 diabetes, HbA 1c and smoking)., Results: No significant associations of Lp(a) plasma levels and the LPA SNPs rs10455872 and rs3798220 with prevalent or incident microvascular complications in type 2 diabetes were found. In line with previous observations the LPA SNPs rs10455872 and rs3798220 did influence the plasma Lp(a) levels., Conclusions/interpretation: Our data show no association between Lp(a) plasma levels and the LPA SNPs with known effect on Lp(a) plasma levels with the development of microvascular complications in type 2 diabetes. This indicates that Lp(a) does not play a major role in the development of microvascular complications. However, larger studies are needed to exclude minimal effects of Lp(a) on the development of microvascular complications.

Background: Few epigenome-wide association studies (EWAS) on air pollutants exist, and none have been done on transportation noise exposures, which also contribute to environmental burden of disease., Objective: We performed mutually independent EWAS on transportation noise and air pollution exposures., Methods: We used data from two time points of the Swiss Cohort Study on Air Pollution and Lung and Heart Diseases in Adults (SAPALDIA) from 1,389 participants contributing 2,542 observations. We applied multiexposure linear mixed-effects regressions with participant-level random intercept to identify significant Cytosine-phosphate-Guanine (CpG) sites and differentially methylated regions (DMRs) in relation to 1-y average aircraft, railway, and road traffic day-evening-night noise (Lden); nitrogen dioxide ( NO 2 ); and particulate matter (PM) with aerodynamic diameter < 2.5 μ m ( PM 2.5 ). We performed candidate (CpG-based; cross-systemic phenotypes, combined into "allostatic load") and agnostic (DMR-based) pathway enrichment tests, and replicated previously reported air pollution EWAS signals., Results: We found no statistically significant CpGs at false discovery rate < 0.05 . However, 14, 48, 183, 8, and 71 DMRs independently associated with aircraft, railway, and road traffic Lden; NO 2 ; and PM 2.5 , respectively, with minimally overlapping signals. Transportation Lden and air pollutants tendentially associated with decreased and increased methylation, respectively. We observed significant enrichment of candidate DNA methylation related to C-reactive protein and body mass index (aircraft, road traffic Lden, and PM 2.5 ), renal function and "allostatic load" (all exposures). Agnostic functional networks related to cellular immunity, gene expression, cell growth/proliferation, cardiovascular, auditory, embryonic, and neurological systems development were enriched. We replicated increased methylation in cg08500171 ( NO 2 ) and decreased methylation in cg17629796 ( PM 2.5 )., Conclusions: Mutually independent DNA methylation was associated with source-specific transportation noise and air pollution exposures, with distinct and shared enrichments for pathways related to inflammation, cellular development, and immune responses. These findings contribute in clarifying the pathways linking these exposures and age-related diseases but need further confirmation in the context of mediation analyses. https://doi.org/10.1289/EHP6174.

Background: Dysfunctional mitochondria have an influence on inflammation and increased oxidative stress due to an excessive production of reactive oxygen species. The mitochondrial DNA copy number (mtDNA-CN) is a potential biomarker for mitochondrial dysfunction and has been associated with various diseases. However, results were partially contrasting which might have been caused by methodological difficulties to quantify mtDNA-CN., Objective: We aimed to investigate whether mtDNA-CN is associated with peripheral arterial disease (PAD) as well as all-cause mortality and cardiovascular events during seven years of follow-up., Methods: A total of 236 male patients with PAD from the Cardiovascular Disease in Intermittent Claudication (CAVASIC) study were compared with 249 age- and diabetes-matched controls. MtDNA-CN was measured with a well-standardized plasmid-normalized quantitative PCR-based assay determining the ratio between mtDNA-CN and nuclear DNA., Results: Individuals in the lowest quartile of mtDNA-CN had a twofold increased risk for PAD which, however, was no longer significant after adjusting for leukocytes and platelets. About 67 of the 236 patients had already experienced a cardiovascular event at baseline and those in the lowest mtDNA-CN quartile had a 2.34-fold increased risk for these events (95% CI 1.08-5.13). During follow-up, 37 PAD patients died and 66 patients experienced a cardiovascular event. Patients in the lowest mtDNA-CN quartile had hazard ratios of 2.66 (95% CI 1.27-5.58) for all-cause-mortality and 1.82 (95% CI 1.02-3.27) for cardiovascular events compared with the combined quartile 2-4 (adjusted for age, smoking, CRP, diabetes, prevalent cardiovascular disease, leukocytes and platelets)., Conclusion: This investigation supports the hypothesis of mitochondrial dysfunction in peripheral arterial disease and shows an association of low mtDNA-CNs with all-cause-mortality and prevalent and incident cardiovascular disease in PAD patients with intermittent claudication., (© 2020 The Authors. Journal of Internal Medicine published by John Wiley & Sons Ltd on behalf of Association for Publication of The Journal of Internal Medicine.)

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., Competing Interests: The authors have declared that no competing interests exist.

Scope: Previous work identified three metabolically homogeneous subgroups of individuals ("metabotypes") using k-means cluster analysis based on fasting serum levels of triacylglycerol, total cholesterol, HDL cholesterol, and glucose. The aim is to reproduce these findings and describe metabotype groups by dietary habits and by incident disease occurrence., Methods and Results: 1744 participants from the KORA F4 study and 2221 participants from the KORA FF4 study are assigned to the three metabotype clusters previously identified by minimizing the Euclidean distances. In both KORA studies, the assignment of participants results in three metabolically distinct clusters, with cluster 3 representing the group of participants with the most unfavorable metabolic characteristics. Individuals of cluster 3 are further characterized by the highest incident disease occurrence during follow-up; they also reveal the most unfavorable diet with significantly lowest intakes of vegetables, dairy products, and fibers, and highest intakes of total, red, and processed meat., Conclusion: The three metabotypes originally identified in an Irish population are successfully reproduced. In addition to this validation approach, the observed differences in disease incidence across metabotypes represent an important new finding that strongly supports the metabotyping approach as a tool for risk stratification., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The joint consensus panel of the European Atherosclerosis Society (EAS) and the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) recently addressed present and future challenges in the laboratory diagnostics of atherogenic lipoproteins. Total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDLC), LDL cholesterol (LDLC), and calculated non-HDLC (=total - HDLC) constitute the primary lipid panel for estimating risk of atherosclerotic cardiovascular disease (ASCVD) and can be measured in the nonfasting state. LDLC is the primary target of lipid-lowering therapies. For on-treatment follow-up, LDLC shall be measured or calculated by the same method to attenuate errors in treatment decisions due to marked between-method variations. Lipoprotein(a) [Lp(a)]-cholesterol is part of measured or calculated LDLC and should be estimated at least once in all patients at risk of ASCVD, especially in those whose LDLC declines poorly upon statin treatment. Residual risk of ASCVD even under optimal LDL-lowering treatment should be also assessed by non-HDLC or apolipoprotein B (apoB), especially in patients with mild-to-moderate hypertriglyceridemia (2-10 mmol/L). Non-HDLC includes the assessment of remnant lipoprotein cholesterol and shall be reported in all standard lipid panels. Additional apoB measurement can detect elevated LDL particle (LDLP) numbers often unidentified on the basis of LDLC alone. Reference intervals of lipids, lipoproteins, and apolipoproteins are reported for European men and women aged 20-100 years. However, laboratories shall flag abnormal lipid values with reference to therapeutic decision thresholds.

Rewiring of energy metabolism and adaptation of mitochondria are considered to impact on prostate cancer development and progression. Here, we report on mitochondrial respiration, DNA mutations and gene expression in paired benign/malignant human prostate tissue samples. Results reveal reduced respiratory capacities with NADH-pathway substrates glutamate and malate in malignant tissue and a significant metabolic shift towards higher succinate oxidation, particularly in high-grade tumors. The load of potentially deleterious mitochondrial-DNA mutations is higher in tumors and associated with unfavorable risk factors. High levels of potentially deleterious mutations in mitochondrial Complex I-encoding genes are associated with a 70% reduction in NADH-pathway capacity and compensation by increased succinate-pathway capacity. Structural analyses of these mutations reveal amino acid alterations leading to potentially deleterious effects on Complex I, supporting a causal relationship. A metagene signature extracted from the transcriptome of tumor samples exhibiting a severe mitochondrial phenotype enables identification of tumors with shorter survival times.

Background: There is little information in haemodialysis (HD) patients on whether temporal changes in serum calcium, phosphate or intact parathyroid hormone (iPTH) are associated with mortality., Methods: We analysed associations of phosphate, total calcium and iPTH with all-cause and cardiovascular mortality in 8817 incident HD patients from the European second Analyzing Data, Recognizing Excellence and Optimizing Outcomes (AROii) cohort enrolled in 2007-09, which were prospectively followed for a median of 3 years, using time-dependent Cox proportional hazards models. We evaluated changes in risk over time depending on changes in phosphate, calcium or iPTH., Results: The association of phosphate and iPTH with all-cause mortality was U-shaped, with the lowest risk ranges between 1.20 and 1.89 mmol/L for phosphate and between 239 and 710 ng/L for iPTH. For total calcium, the associations were J-shaped, with an increased risk for all-cause mortality at levels >2.36 mmol/L. Lowest risk ranges for cardiovascular mortality did not change markedly for all three parameters. If iPTH was below the lowest risk range at baseline (iPTH <239 ng/L), a subsequent increase in levels was associated with improved survival. For phosphate, an increase or decrease out of the lowest risk range was associated with increased mortality risk. For calcium, this was only the case when the values increased above the lowest risk range., Conclusion: In the AROii cohort, the ranges of bone mineral biomarkers associated with the lowest mortality ranges were largely consistent with the current Kidney Disease: Improving Global Outcomes chronic kidney disease-mineral and bone disorder guideline recommendations. Allowing a suppressed iPTH to increase was associated with a lower mortality, whereas shifts of phosphate or calcium outside the lowest risk range increased mortality., (© The Author(s) 2019. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

The kidneys integrate information from continuous systemic processes related to the absorption, distribution, metabolism and excretion (ADME) of metabolites. To identify underlying molecular mechanisms, we performed genome-wide association studies of the urinary concentrations of 1,172 metabolites among 1,627 patients with reduced kidney function. The 240 unique metabolite-locus associations (metabolite quantitative trait loci, mQTLs) that were identified and replicated highlight novel candidate substrates for transport proteins. The identified genes are enriched in ADME-relevant tissues and cell types, and they reveal novel candidates for biotransformation and detoxification reactions. Fine mapping of mQTLs and integration with single-cell gene expression permitted the prioritization of causal genes, functional variants and target cell types. The combination of mQTLs with genetic and health information from 450,000 UK Biobank participants illuminated metabolic mediators, and hence, novel urinary biomarkers of disease risk. This comprehensive resource of genetic targets and their substrates is informative for ADME processes in humans and is relevant to basic science, clinical medicine and pharmaceutical research.

The joint consensus panel of the European Atherosclerosis Society (EAS) and the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) recently addressed present and future challenges in the laboratory diagnostics of atherogenic lipoproteins. Total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and calculated non-HDL cholesterol (=total - HDL cholesterol) constitute the primary lipid panel for estimating risk of atherosclerotic cardiovascular disease (ASCVD) and can be measured in the nonfasting state. LDL cholesterol is the primary target of lipid-lowering therapies. For on-treatment follow-up, LDL cholesterol shall be measured or calculated by the same method to attenuate errors in treatment decisions due to marked between-method variations. Lipoprotein(a)-cholesterol is part of measured or calculated LDL cholesterol and should be estimated at least once in all patients at risk of ASCVD, especially in those whose LDL cholesterol decline poorly upon statin treatment. Residual risk of ASCVD even under optimal LDL-lowering treatment should be also assessed by non-HDL cholesterol or apolipoprotein B, especially in patients with mild-to-moderate hypertriglyceridemia (2-10 mmol/L). Non-HDL cholesterol includes the assessment of remnant lipoprotein cholesterol and shall be reported in all standard lipid panels. Additional apolipoprotein B measurement can detect elevated LDL particle numbers often unidentified on the basis of LDL cholesterol alone. Reference intervals of lipids, lipoproteins, and apolipoproteins are reported for European men and women aged 20-100 years. However, laboratories shall flag abnormal lipid values with reference to therapeutic decision thresholds., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Genome sequencing and gene-based therapies appear poised to advance the management of rare lipoprotein disorders and associated dyslipidaemias. However, in practice, underdiagnosis and undertreatment of these disorders are common, in large part due to interindividual variability in the genetic causes and phenotypic presentation of these conditions. To address these challenges, the European Atherosclerosis Society formed a task force to provide practical clinical guidance focusing on patients with extreme concentrations (either low or high) of plasma low-density lipoprotein cholesterol, triglycerides, or high-density lipoprotein cholesterol. The task force also recognises the scarcity of quality information regarding the prevalence and outcomes of these conditions. Collaborative registries are needed to improve health policy for the care of patients with rare dyslipidaemias., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Despite a plethora of studies on the effect of urate-lowering therapy (ULT) in patients with chronic kidney disease (CKD), current guidelines on the treatment of hyperuricaemia and gout vary, especially concerning the need for dose adjustment of allopurinol, whose main metabolite is accumulating with declining renal function. Data on allopurinol dosing and its relationship to renal function, co-medication and sex and the resulting urate level in large cohorts are missing., Methods: We studied a subgroup of 2378 patients of the German Chronic Kidney Disease (GCKD) study to determine prescription patterns of ULT among CKD patients under nephrological care and the relationship of ULT dose to urate levels. Prescription and dosing of ULT were manually abstracted from the patient's paper charts at the baseline visit, in which all currently used medications and their dosing were recorded., Results: In this cohort, 39.6% were women, the mean estimated glomerular filtration rate (eGFR) was 51.3 ± 19.3 mL/min/1.73 m 2 and the mean age was 59.0 ± 12.4 years. Of the 2378 examined patients, 666 (28.0%) received ULT. The dose of ULT was available for 572 patients. The main ULT agent was allopurinol (94.4%), followed by febuxostat (2.9%) and benzbromarone (2.6%). Of the 540 patients who used allopurinol with a reported daily dose, 480 had an eGFR <60 mL/min/1.73 m 2 and 320 had an eGFR <45 mL/min/1.73 m 2 , 31.5% of the latter ( n  = 101) received a dose >150 mg/day, the recommended maximal dose for this level of eGFR. The prescribed dose was not related to eGFR: the median eGFR for patients taking 100, 150 and 300 mg/day was 40 [interquartile range (IQR) 32-49], 43 (34-52) and 42 (35-54) mL/min/1.73 m 2 , respectively. Patients with lower doses of allopurinol had higher serum urate levels than patients with higher (than recommended) allopurinol doses. Sex, alcohol intake, eGFR, use of diuretics and treatment with allopurinol were independent determinants of serum urate levels in multivariate regression analysis., Conclusions: The most frequently used drug to lower serum urate levels in this CKD cohort was allopurinol. Even in patients regularly seen by nephrologists, the dose of allopurinol is often not adjusted to the current eGFR. Patients with higher ULT doses achieved better control of their serum urate levels. Lowering of serum urate in CKD patients requires balancing potential adverse effects of allopurinol with suboptimal control of serum urate levels., (© The Author(s) 2019. Published by Oxford University Press on behalf of ERA-EDTA.)

Purpose and Methods: A meta-analysis using data from seven German population-based cohorts was performed by the German Epidemiological consortium of Peripheral Arterial Disease (GEPArD) to investigate whether one question about claudication is more efficient for PAD screening than established questionnaires. Claudication was defined on the basis of the answer to one question asking for pain in the leg during normal walking. This simple question was compared with established questionnaires, including the Edinburgh questionnaire. The associations of claudication with continuous ABI values and decreased ABI were analyzed by linear and logistic regression analysis, respectively. The results of the studies were pooled in a random effect meta-analysis, which included data from 27,945 individuals (14,052 women, age range 20-84 years)., Results: Meta-analysis revealed a significant negative association between claudication and ABI, which was stronger in men (β = -0.07; 95%CI -0.10, -0.04) than in women (β = -0.02; 95%CI -0.02, -0.01). Likewise, the presence of claudication symptoms was related to an increased odds of a decreased ABI in both men (Odds ratio = 5.40; 95%CI 4.20, 6.96) and women (Odds ratio = 1.99; 95%CI 1.58, 2.51)., Conclusions: Asking only one question about claudication was able to identify many individuals with a high likelihood of a reduced ABI with markedly higher sensitivity and only slightly reduced specificity compared to more complex questionnaires. At least in men, this question should be established as first screening step., Competing Interests: The authors have declared that no competing interests exist.

Background and Aims: We aimed to evaluate the effect of statin treatment initiation on lipoprotein(a) [Lp(a)] levels in patients with dyslipidemia, and the interactions with the apolipoprotein(a) [apo(a)] phenotype, LPA single nucleotide polymorphisms (SNPs) and change in LDL cholesterol., Methods: The study population consisted of patients with dyslipidemia, predominantly familial hypercholesterolemia, who first initiated statin treatment (initiation group; n = 39) or were already on stable statin treatment for at least 4 months (control group; n = 42). Plasma Lp(a) levels were determined with a particle-enhanced immunoturbidimetric assay before and at least 2 months after start of statin treatment in individuals of the initiation group, and at two time points with an interval of at least 2 months in the control group. High and low molecular weight (HMW and LMW, respectively) apo(a) phenotype was determined by immunoblotting, and the common LPA SNPs rs10455872, rs3798220 and rs41272110 by Taqman assay., Results: Plasma Lp(a) levels did not increase significantly in the initiation group (median 20.5 (IQR 10.9-80.7) to 23.3 (10.8-71.8) mg/dL; p = 0.09) nor in the control group (30.9 (IQR 9.2-147.0) to 31.7 (IQR 10.9-164.0) mg/dL; p = 0.61). In patients with the LMW apo(a) phenotype, Lp(a) levels increased significantly from 66.4 (IQR 23.5-148.3) to 97.4 (IQR 24.9-160.4) mg/dL (p = 0.026) in the initiation group, but not in the control group and not in patients characterized by the HMW apo(a) phenotype. Interactions with common LPA SNPs and change in LDL cholesterol were not significant., Conclusions: Statins affect Lp(a) levels differently in patients with dyslipidemia depending on the apo(a) phenotype. Statins increase Lp(a) levels exclusively in patients with the LMW apo(a) phenotype., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Elevated serum urate levels cause gout and correlate with cardiometabolic diseases via poorly understood mechanisms. We performed a trans-ancestry genome-wide association study of serum urate in 457,690 individuals, identifying 183 loci (147 previously unknown) that improve the prediction of gout in an independent cohort of 334,880 individuals. Serum urate showed significant genetic correlations with many cardiometabolic traits, with genetic causality analyses supporting a substantial role for pleiotropy. Enrichment analysis, fine-mapping of urate-associated loci and colocalization with gene expression in 47 tissues implicated the kidney and liver as the main target organs and prioritized potentially causal genes and variants, including the transcriptional master regulators in the liver and kidney, HNF1A and HNF4A. Experimental validation showed that HNF4A transactivated the promoter of ABCG2, encoding a major urate transporter, in kidney cells, and that HNF4A p.Thr139Ile is a functional variant. Transcriptional coregulation within and across organs may be a general mechanism underlying the observed pleiotropy between urate and cardiometabolic traits.

Increased levels of the urinary albumin-to-creatinine ratio (UACR) are associated with higher risk of kidney disease progression and cardiovascular events, but underlying mechanisms are incompletely understood. Here, we conduct trans-ethnic (n = 564,257) and European-ancestry specific meta-analyses of genome-wide association studies of UACR, including ancestry- and diabetes-specific analyses, and identify 68 UACR-associated loci. Genetic correlation analyses and risk score associations in an independent electronic medical records database (n = 192,868) reveal connections with proteinuria, hyperlipidemia, gout, and hypertension. Fine-mapping and trans-Omics analyses with gene expression in 47 tissues and plasma protein levels implicate genes potentially operating through differential expression in kidney (including TGFB1, MUC1, PRKCI, and OAF), and allow coupling of UACR associations to altered plasma OAF concentrations. Knockdown of OAF and PRKCI orthologs in Drosophila nephrocytes reduces albumin endocytosis. Silencing fly PRKCI further impairs slit diaphragm formation. These results generate a priority list of genes and pathways for translational research to reduce albuminuria.

Damage of mitochondrial DNA (mtDNA) with reduction in copy number has been proposed as a biomarker for mitochondrial dysfunction and oxidative stress. Chronic kidney disease (CKD) is associated with increased mortality and risk of cardiovascular disease, but the underlying mechanisms remain incompletely understood. Here we investigated the prognostic role of mtDNA copy number for cause-specific mortality in 4812 patients from the German Chronic Kidney Disease study, an ongoing prospective observational national cohort study of patients with CKD stage G3 and A1-3 or G1-2 with overt proteinuria (A3) at enrollment. MtDNA was quantified in whole blood using a plasmid-normalized PCR-based assay. At baseline, 1235 patients had prevalent cardiovascular disease. These patients had a significantly lower mtDNA copy number than patients without cardiovascular disease (fully-adjusted model: odds ratio 1.03, 95% confidence interval [CI] 1.01-1.05 per 10 mtDNA copies decrease). After four years of follow-up, we observed a significant inverse association between mtDNA copy number and all-cause mortality, adjusted for kidney function and cardiovascular disease risk factors (hazard ratio 1.37, 95% CI 1.09-1.73 for quartile 1 compared to quartiles 2-4). When grouped by causes of death, estimates pointed in the same direction for all causes but in a fully-adjusted model decreased copy numbers were significantly lower only in infection-related death (hazard ratio 1.82, 95% CI 1.08-3.08). A similar association was observed for hospitalizations due to infections in 644 patients (hazard ratio 1.19, 95% CI 1.00-1.42 in the fully-adjusted model). Thus, our data support a role of mitochondrial dysfunction in increased cardiovascular disease and mortality risks as well as susceptibility to infections in patients with CKD., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Previous reports link differential DNA methylation (DNAme) to environmental exposures that are associated with lung function. Direct evidence on lung function DNAme is, however, limited. We undertook an agnostic epigenome-wide association study (EWAS) on pre-bronchodilation lung function and its change in adults.In a discovery-replication EWAS design, DNAme in blood and spirometry were measured twice, 6-15 years apart, in the same participants of three adult population-based discovery cohorts (n=2043). Associated DNAme markers (p<5×10 -7 ) were tested in seven replication cohorts (adult: n=3327; childhood: n=420). Technical bias-adjusted residuals of a regression of the normalised absolute β-values on control probe-derived principle components were regressed on level and change of forced expiratory volume in 1 s (FEV 1 ), forced vital capacity (FVC) and their ratio (FEV 1 /FVC) in the covariate-adjusted discovery EWAS. Inverse-variance-weighted meta-analyses were performed on results from discovery and replication samples in all participants and never-smokers.EWAS signals were enriched for smoking-related DNAme. We replicated 57 lung function DNAme markers in adult, but not childhood samples, all previously associated with smoking. Markers not previously associated with smoking failed replication. cg05575921 ( AHRR (aryl hydrocarbon receptor repressor)) showed the statistically most significant association with cross-sectional lung function (FEV 1 /FVC: p discovery =3.96×10 -21 and p combined =7.22×10 -50 ). A score combining 10 DNAme markers previously reported to mediate the effect of smoking on lung function was associated with lung function (FEV 1 /FVC: p=2.65×10 -20 ).Our results reveal that lung function-associated methylation signals in adults are predominantly smoking related, and possibly of clinical utility in identifying poor lung function and accelerated decline. Larger studies with more repeat time-points are needed to identify lung function DNAme in never-smokers and in children., Competing Interests: Conflict of interest: M. Imboden has nothing to disclose. Conflict of interest: M. Wielscher has nothing to disclose. Conflict of interest: F.I. Rezwan has nothing to disclose. Conflict of interest: A.F.S. Amaral has nothing to disclose. Conflict of interest: E. Schaffner has nothing to disclose. Conflict of interest: A. Jeong has nothing to disclose. Conflict of interest: A. Beckmeyer-Borowko has nothing to disclose. Conflict of interest: S.E. Harris reports grants from Medical Research Council, Biotechnology and Biological Sciences Research Council, Age UK and The Wellcome Trust, during the conduct of the study. Conflict of interest: J.M. Starr has nothing to disclose. Conflict of interest: I.J. Deary reports grants from Age UK and Medical Research Council, during the conduct of the study. Conflict of interest: C. Flexeder has nothing to disclose. Conflict of interest: M. Waldenberger has nothing to disclose. Conflict of interest: A. Peters has nothing to disclose. Conflict of interest: H. Schulz reports grants from German Federal Ministry of Education and Research (BMBF), during the conduct of the study. Conflict of interest: S. Chen has nothing to disclose. Conflict of interest: S.K. Sunny has nothing to disclose. Conflict of interest: W.J.J. Karmaus has nothing to disclose. Conflict of interest: Y. Jiang has nothing to disclose. Conflict of interest: G. Erhart has nothing to disclose. Conflict of interest: F. Kronenberg has nothing to disclose. Conflict of interest: R. Arathimos has nothing to disclose. Conflict of interest: G.C. Sharp has nothing to disclose. Conflict of interest: A.J. Henderson reports grants from Medical Research Council and Wellcome Trust, during the conduct of the study. Conflict of interest: Y. Fu has nothing to disclose. Conflict of interest: P. Piirilä has nothing to disclose. Conflict of interest: K.H. Pietiläinen has nothing to disclose. Conflict of interest: M. Ollikainen has nothing to disclose. Conflict of interest: A. Johansson has nothing to disclose. Conflict of interest: U. Gyllensten has nothing to disclose. Conflict of interest: M de Vries has nothing to disclose. Conflict of interest: D.A. van der Plaat has nothing to disclose. Conflict of interest: K. de Jong has nothing to disclose. Conflict of interest: H.M. Boezen has nothing to disclose. Conflict of interest: I.P. Hall reports grants from GSK and Boehringer Ingelheim, outside the submitted work. Conflict of interest: M.D. Tobin reports grants from Pfizer and GSK, outside the submitted work. Conflict of interest: M-R. Jarvelin has nothing to disclose. Conflict of interest: J.W. Holloway reports grants from European Union and National Institutes of Health, during the conduct of the study. Conflict of interest: D. Jarvis reports grants from European Union, Medical Research Council and Asthma UK, during the conduct of the study. Conflict of interest: N.M. Probst-Hensch has nothing to disclose., (Copyright ©ERS 2019.)

Smoking is associated with shorter leucocyte telomere length (LTL), a biomarker of increased morbidity and reduced longevity. This association is widely interpreted as evidence that smoking causes accelerated LTL attrition in adulthood, but the evidence for this is inconsistent. We analysed the association between smoking and LTL dynamics in 18 longitudinal cohorts. The dataset included data from 12 579 adults (4678 current smokers and 7901 non-smokers) over a mean follow-up interval of 8.6 years. Meta-analysis confirmed a cross-sectional difference in LTL between smokers and non-smokers, with mean LTL 84.61 bp shorter in smokers (95% CI: 22.62 to 146.61). However, LTL attrition was only 0.51 bp yr -1 faster in smokers than in non-smokers (95% CI: -2.09 to 1.08), a difference that equates to only 1.32% of the estimated age-related loss of 38.33 bp yr -1 . Assuming a linear effect of smoking, 167 years of smoking would be required to generate the observed cross-sectional difference in LTL. Therefore, the difference in LTL between smokers and non-smokers is extremely unlikely to be explained by a linear, causal effect of smoking. Selective adoption, whereby individuals with short telomeres are more likely to start smoking, needs to be considered as a more plausible explanation for the observed pattern of telomere dynamics., Competing Interests: The authors declare no competing interests.

Importance: Genetic and epidemiologic data suggest that lipoprotein(a) (Lp[a]) is one of the strongest genetically determined risk factors for coronary heart disease (CHD). Specific therapies to lower Lp(a) are on the horizon, but the required reduction of Lp(a) to translate into clinically relevant lowering of CHD outcomes is a matter of debate., Objective: To estimate the required Lp(a)-lowering effect size that may be associated with a reduction of CHD outcomes compared with the effect size of low-density lipoprotein cholesterol (LDL-C)-lowering therapies., Design, Setting, and Participants: Genetic epidemiologic study using a mendelian randomization analysis to estimate the required Lp(a)-lowering effect size for a clinically meaningful effect on outcomes. We used the effect estimates for Lp(a) from a genome-wide association study (GWAS) and meta-analysis on Lp(a) published in 2017 of 5 different primarily population-based studies of European ancestry. All Lp(a) measurements were performed in 1 laboratory. Genetic estimates for 27 single-nucleotide polymorphisms on Lp(a) concentrations were used. Odds ratios for these 27 single-nucleotide polymorphisms associated with CHD risk were retrieved from a subsample of the CHD Exome+ consortium., Exposures: Genetic LPA score, plasma Lp(a) concentrations, and observations of statin therapies on CHD outcomes., Main Outcomes and Measures: Coronary heart disease., Results: The study included 13 781 individuals from the Lp(a)-GWAS-Consortium from 5 primarily population-based studies and 20 793 CHD cases and 27 540 controls from a subsample of the CHD Exome+ consortium. Four of the studies were similar in age distribution (means between 51 and 59 years), and 1 cohort was younger; mean age, 32 years. The frequency of women was similar between 51% and 55%. We estimated that the required reduction in Lp(a) effect size would be 65.7 mg/dL (95% CI, 46.3-88.3) to reach the same potential effect on clinical outcomes that can be reached by lowering LDL-C by 38.67 mg/dL (to convert to millimoles per liter, multiply by 0.0259)., Conclusions and Relevance: This mendelian randomization analysis estimated a required Lp(a)-lowering effect size of 65.7 mg/dL to reach the same effect as a 38.67-mg/dL lowering of LDL-C. However, this estimate is determined by the observed effect estimates of single-nucleotide polymorphisms on Lp(a) concentrations and is therefore influenced by the standardization of the Lp(a) assay used. As a consequence, calculations of the required Lp(a)-lowering potential of a drug to be clinically effective might have been overestimated in the past.

Chronic kidney disease (CKD) is responsible for a public health burden with multi-systemic complications. Through trans-ancestry meta-analysis of genome-wide association studies of estimated glomerular filtration rate (eGFR) and independent replication (n = 1,046,070), we identified 264 associated loci (166 new). Of these, 147 were likely to be relevant for kidney function on the basis of associations with the alternative kidney function marker blood urea nitrogen (n = 416,178). Pathway and enrichment analyses, including mouse models with renal phenotypes, support the kidney as the main target organ. A genetic risk score for lower eGFR was associated with clinically diagnosed CKD in 452,264 independent individuals. Colocalization analyses of associations with eGFR among 783,978 European-ancestry individuals and gene expression across 46 human tissues, including tubulo-interstitial and glomerular kidney compartments, identified 17 genes differentially expressed in kidney. Fine-mapping highlighted missense driver variants in 11 genes and kidney-specific regulatory variants. These results provide a comprehensive priority list of molecular targets for translational research.

In the middle of the 1990s the interest in Lp(a) vanished after a few badly performed studies almost erased Lp(a) from the map of biological targets. However, since roughly 10 years the interest has begun to grow again mainly for two reasons: first, genetic studies using easily accessible and high-throughput techniques for genotyping of single-nucleotide polymorphisms (SNPs) have allowed large studies in patients with cardiovascular disease and controls to be performed. This strengthened the earlier findings on a copy number variation in the LPA gene and its association with cardiovascular outcomes. Second, new therapies are on the horizon raising strong and justified hope that in a few years drugs will become available which tremendously lower Lp(a) concentrations. This review article should provide an introduction to the genetic determination of Lp(a) concentrations and considerations whether Lp(a) concentrations or genetic variants are important for the prediction of cardiovascular risk.

The objective of the present study was to identify proteins that contribute to pathophysiology and allow prediction of incident type 2 diabetes or incident prediabetes. We quantified 14 candidate proteins using targeted mass spectrometry in plasma samples of the prospective, population-based German KORA F4/FF4 study (6.5-year follow-up). 892 participants aged 42-81 years were selected using a case-cohort design, including 123 persons with incident type 2 diabetes and 255 persons with incident WHO-defined prediabetes. Prospective associations between protein levels and diabetes, prediabetes as well as continuous fasting and 2 h glucose, fasting insulin and insulin resistance were investigated using regression models adjusted for established risk factors. The best predictive panel of proteins on top of a non-invasive risk factor model or on top of HbA1c, age, and sex was selected. Mannan-binding lectin serine peptidase (MASP) levels were positively associated with both incident type 2 diabetes and prediabetes. Adiponectin was inversely associated with incident type 2 diabetes. MASP, adiponectin, apolipoprotein A-IV, apolipoprotein C-II, C-reactive protein, and glycosylphosphatidylinositol specific phospholipase D1 were associated with individual continuous outcomes. The combination of MASP, apolipoprotein E (apoE) and adiponectin improved diabetes prediction on top of both reference models, while prediabetes prediction was improved by MASP plus CRP on top of the HbA1c model. In conclusion, our mass spectrometric approach revealed a novel association of MASP with incident type 2 diabetes and incident prediabetes. In combination, MASP, adiponectin and apoE improved type 2 diabetes prediction beyond non-invasive risk factors or HbA1c, age and sex.

Background: Epidemiological evidence on the influence of long-term exposure to traffic-related particulate matter (TPM 10 ) on heart rate variability (HRV) is weak., Objective: To evaluate the association of long-term exposure (10 years) with TPM 10 on the regulation of the autonomic cardiovascular system and heart rate dynamics (HRD) in an aging general population, as well as potential modifying effects by the a priori selected factors sex, smoking status, obesity, and gene variation in selected glutathione S-transferases (GSTs)., Methods: We analyzed data from 1593 SAPALDIA cohort participants aged ≥ 50 years. For each participant, various HRV and HRD parameters were derived from 24-hour electrocardiogram recordings. Each parameter obtained was then used as the outcome variable in multivariable mixed linear regression models in order to evaluate the association with TPM 10 . Potential modifying effects were assessed using interaction terms., Results: No association between long-term exposure to TPM 10 and HRV/HRD was observed in the entire study population. However, HRD changes were found in subjects without cardiovascular morbidity and both HRD and HRV changes in non-obese subjects without cardiovascular morbidity. Subjects without cardiovascular morbidity with homozygous GSTM1 gene deletion appeared to be more susceptible to the effects of TPM 10 ., Conclusion: This study suggests that long-term exposure to TPM 10 triggers adverse changes in the regulation of the cardiovascular system. These adverse effects were more visible in the subjects without cardiovascular disease, in whom the overall relationship between TPM 10 and HRV/HRD could not be masked by underlying morbidities and the potential counteracting effects of related drug treatments., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A high body mass (BMI) index has repeatedly been associated with non-atopic asthma, but the biological mechanism linking obesity to asthma is still poorly understood. We aimed to test the hypothesis that inflammation and/or innate immunity plays a role in the obesity-asthma link. DNA methylome was measured in blood samples of 61 non-atopic participants with asthma and 146 non-atopic participants without asthma (non-smokers for at least 10 years) taking part in the Swiss Cohort Study on Air Pollution and Lung and Heart Diseases in Adults (SAPALDIA) study. Modification by DNA methylation of the association of BMI or BMI change over 10 years with adult-onset asthma was examined at each CpG site and differentially methylated region. Pathway enrichment tests were conducted for genes in a priori curated inflammatory pathways and the NLRP3-IL1B-IL17 axis. The latter was chosen on the basis of previous work in mice. Inflammatory pathways including glucocorticoid/PPAR signaling ( p = 0.0023), MAPK signaling ( p = 0.013), NF-κB signaling ( p = 0.031), and PI3K/AKT signaling ( p = 0.031) were enriched for the effect modification of BMI, while NLRP3-IL1B-IL17 axis was enriched for the effect modification of BMI change over 10 years ( p = 0.046). DNA methylation measured in peripheral blood is consistent with inflammation as a link between BMI and adult-onset asthma and with the NLRP3-IL1B-IL17 axis as a link between BMI change over 10 years and adult-onset asthma in non-atopic participants.