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1.  Upstream Transcription Factor 1 (USF1) allelic variants regulate lipoprotein metabolism in women and USF1 expression in atherosclerotic plaque 
Scientific Reports  2014;4:4650.
Upstream transcription factor 1 (USF1) allelic variants significantly influence future risk of cardiovascular disease and overall mortality in females. We investigated sex-specific effects of USF1 gene allelic variants on serum indices of lipoprotein metabolism, early markers of asymptomatic atherosclerosis and their changes during six years of follow-up. In addition, we investigated the cis-regulatory role of these USF1 variants in artery wall tissues in Caucasians. In the Cardiovascular Risk in Young Finns Study, 1,608 participants (56% women, aged 31.9 ± 4.9) with lipids and cIMT data were included. For functional study, whole genome mRNA expression profiling was performed in 91 histologically classified atherosclerotic samples. In females, serum total, LDL cholesterol and apoB levels increased gradually according to USF1 rs2516839 genotypes TT < CT < CC and rs1556259 AA < AG < GG as well as according to USF1 H3 (GCCCGG) copy number 0 < 1 < 2. Furthermore, the carriers of minor alleles of rs2516839 (C) and rs1556259 (G) of USF1 gene had decreased USF1 expression in atherosclerotic plaques (P = 0.028 and 0.08, respectively) as compared to non-carriers. The genetic variation in USF1 influence USF1 transcript expression in advanced atherosclerosis and regulates levels and metabolism of circulating apoB and apoB-containing lipoprotein particles in sex-dependent manner, but is not a major determinant of early markers of atherosclerosis.
doi:10.1038/srep04650
PMCID: PMC3983598  PMID: 24722012
2.  A genome-wide association study to identify genetic susceptibility loci that modify ductal and lobular postmenopausal breast cancer risk associated with menopausal hormone therapy use: a two-stage design with replication 
Menopausal hormone therapy (MHT) is associated with an elevated risk of breast cancer in postmenopausal women. To identify genetic loci that modify breast cancer risk related to MHT use in postmenopausal women, we conducted a two-stage genome-wide association study (GWAS) with replication. In stage I, we performed a case-only GWAS in 731 invasive breast cancer cases from the German case-control study Mammary Carcinoma Risk Factor Investigation (MARIE). The 1,200 single nucleotide polymorphisms (SNPs) showing the lowest P values for interaction with current MHT use (within 6 months prior to breast cancer diagnosis), were carried forward to stage II, involving pooled case-control analyses including additional MARIE subjects (1,375 cases, 1,974 controls) as well as 795 cases and 764 controls of a Swedish case-control study. A joint P value was calculated for a combined analysis of stages I and II. Replication of the most significant interaction of the combined stage I and II was performed using 5,795 cases and 5,390 controls from nine studies of the Breast Cancer Association Consortium (BCAC). The combined stage I and II yielded five SNPs on chromosomes 2, 7, and 18 with joint P values <6 × 10−6 for effect modification of current MHT use. The most significant interaction was observed for rs6707272 (P = 3 × 10−7) on chromosome 2 but was not replicated in the BCAC studies (P = 0.21). The potentially modifying SNPs are in strong linkage disequilibrium with SNPs in TRIP12 and DNER on chromosome 2 and SETBP1 on chromosome 18, previously linked to carcinogenesis. However, none of the interaction effects reached genome-wide significance. The inability to replicate the top SNP × MHT interaction may be due to limited power of the replication phase. Our study, however, suggests that there are unlikely to be SNPs that interact strongly enough with MHT use to be clinically significant in European women.
doi:10.1007/s10549-013-2443-z
PMCID: PMC3781176  PMID: 23423446
Postmenopausal breast cancer risk; Menopausal hormone therapy; Polymorphisms; Gene-environment interaction; Genome-wide association study; Case-only study
3.  Identification of Serum Metabolites Associated With Risk of Type 2 Diabetes Using a Targeted Metabolomic Approach 
Diabetes  2013;62(2):639-648.
Metabolomic discovery of biomarkers of type 2 diabetes (T2D) risk may reveal etiological pathways and help to identify individuals at risk for disease. We prospectively investigated the association between serum metabolites measured by targeted metabolomics and risk of T2D in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam (27,548 adults) among all incident cases of T2D (n = 800, mean follow-up 7 years) and a randomly drawn subcohort (n = 2,282). Flow injection analysis tandem mass spectrometry was used to quantify 163 metabolites, including acylcarnitines, amino acids, hexose, and phospholipids, in baseline serum samples. Serum hexose; phenylalanine; and diacyl-phosphatidylcholines C32:1, C36:1, C38:3, and C40:5 were independently associated with increased risk of T2D and serum glycine; sphingomyelin C16:1; acyl-alkyl-phosphatidylcholines C34:3, C40:6, C42:5, C44:4, and C44:5; and lysophosphatidylcholine C18:2 with decreased risk. Variance of the metabolites was largely explained by two metabolite factors with opposing risk associations (factor 1 relative risk in extreme quintiles 0.31 [95% CI 0.21–0.44], factor 2 3.82 [2.64–5.52]). The metabolites significantly improved T2D prediction compared with established risk factors. They were further linked to insulin sensitivity and secretion in the Tübingen Family study and were partly replicated in the independent KORA (Cooperative Health Research in the Region of Augsburg) cohort. The data indicate that metabolic alterations, including sugar metabolites, amino acids, and choline-containing phospholipids, are associated early on with a higher risk of T2D.
doi:10.2337/db12-0495
PMCID: PMC3554384  PMID: 23043162
4.  Impact of common regulatory single-nucleotide variants on gene expression profiles in whole blood 
Genome-wide association studies (GWASs) have uncovered susceptibility loci for a large number of complex traits. Functional interpretation of candidate genes identified by GWAS and confident assignment of the causal variant still remains a major challenge. Expression quantitative trait (eQTL) mapping has facilitated identification of risk loci for quantitative traits and might allow prioritization of GWAS candidate genes. One major challenge of eQTL studies is the need for larger sample numbers and replication. The aim of this study was to evaluate the robustness and reproducibility of whole-blood eQTLs in humans and test their value in the identification of putative functional variants involved in the etiology of complex traits. In the current study, we performed comphrehensive eQTL mapping from whole blood. The discovery sample included 322 Caucasians from a general population sample (KORA F3). We identified 363 cis and 8 trans eQTLs after stringent Bonferroni correction for multiple testing. Of these, 98.6% and 50% of cis and trans eQTLs, respectively, could be replicated in two independent populations (KORA F4 (n=740) and SHIP-TREND (n=653)). Furthermore, we identified evidence of regulatory variation for SNPs previously reported to be associated with disease loci (n=59) or quantitative trait loci (n=20), indicating a possible functional mechanism for these eSNPs. Our data demonstrate that eQTLs in whole blood are highly robust and reproducible across studies and highlight the relevance of whole-blood eQTL mapping in prioritization of GWAS candidate genes in humans.
doi:10.1038/ejhg.2012.106
PMCID: PMC3522194  PMID: 22692066
gene expression; eQTL; GWAS; whole blood
5.  High density genotyping study identifies four new susceptibility loci for atopic dermatitis 
Nature genetics  2013;45(7):808-812.
Atopic dermatitis is a common inflammatory skin disease with a strong heritable component. Pathogenetic models consider keratinocyte differentiation defects and immune alterations as scaffolds1, and recent data indicate a role for autoreactivity in at least a subgroup of patients2. With filaggrin (FLG) a major locus causing a skin barrier deficiency was identified3. To better define risk variants and identify additional susceptibility loci, we densely genotyped 2,425 German cases and 5,449 controls using the Immunochip array, followed by replication in 7,196 cases and 15,480 controls from Germany, Ireland, Japan and China. We identified 4 new susceptibility loci for atopic dermatitis and replicated previous associations. This brings the number of atopic dermatitis risk loci reported in individuals of European ancestry to 11. We estimate that these susceptibility loci together account for 14.4% of the heritability for atopic dermatitis.
doi:10.1038/ng.2642
PMCID: PMC3797441  PMID: 23727859
6.  Human metabolic individuality in biomedical and pharmaceutical research 
Nature  2011;477(7362):10.1038/nature10354.
SUMMARY
Genome-wide association studies (GWAS) have identified many risk loci for complex diseases, but effect sizes are typically small and information on the underlying biological processes is often lacking. Associations with metabolic traits as functional intermediates can overcome these problems and potentially inform individualized therapy. Here we report a comprehensive analysis of genotype-dependent metabolic phenotypes using a GWAS with non-targeted metabolomics. We identified 37 genetic loci associated with blood metabolite concentrations, of which 25 exhibit effect sizes that are unusually high for GWAS and account for 10-60% of metabolite levels per allele copy. Our associations provide new functional insights for many disease-related associations that have been reported in previous studies, including cardiovascular and kidney disorders, type 2 diabetes, cancer, gout, venous thromboembolism, and Crohn’s disease. Taken together our study advances our knowledge of the genetic basis of metabolic individuality in humans and generates many new hypotheses for biomedical and pharmaceutical research.
doi:10.1038/nature10354
PMCID: PMC3832838  PMID: 21886157
7.  Plasma Metabolomics Reveal Alterations of Sphingo- and Glycerophospholipid Levels in Non-Diabetic Carriers of the Transcription Factor 7-Like 2 Polymorphism rs7903146 
PLoS ONE  2013;8(10):e78430.
Aims/Hypothesis
Polymorphisms in the transcription factor 7-like 2 (TCF7L2) gene have been shown to display a powerful association with type 2 diabetes. The aim of the present study was to evaluate metabolic alterations in carriers of a common TCF7L2 risk variant.
Methods
Seventeen non-diabetic subjects carrying the T risk allele at the rs7903146 TCF7L2 locus and 24 subjects carrying no risk allele were submitted to intravenous glucose tolerance test and euglycemic-hyperinsulinemic clamp. Plasma samples were analysed for concentrations of 163 metabolites through targeted mass spectrometry.
Results
TCF7L2 risk allele carriers had a reduced first-phase insulin response and normal insulin sensitivity. Under fasting conditions, carriers of TCF7L2 rs7903146 exhibited a non-significant increase of plasma sphingomyelins (SMs), phosphatidylcholines (PCs) and lysophosphatidylcholines (lysoPCs) species. A significant genotype effect was detected in response to challenge tests in 6 SMs (C16:0, C16:1, C18:0, C18:1, C24:0, C24:1), 5 hydroxy-SMs (C14:1, C16:1, C22:1, C22:2, C24:1), 4 lysoPCs (C14:0, C16:0, C16:1, C17:0), 3 diacyl-PCs (C28:1, C36:6, C40:4) and 4 long-chain acyl-alkyl-PCs (C40:2, C40:5, C44:5, C44:6).
Discussion
Plasma metabolomic profiling identified alterations of phospholipid metabolism in response to challenge tests in subjects with TCF7L2 rs7903146 genotype. This may reflect a genotype-mediated link to early metabolic abnormalities prior to the development of disturbed glucose tolerance.
doi:10.1371/journal.pone.0078430
PMCID: PMC3813438  PMID: 24205231
8.  Joint Analysis of Individual Participants’ Data from 17 Studies on the Association of the IL6 Variant -174G>C with Circulating Glucose Levels, Interleukin-6 Levels, and Body-Mass Index 
Annals of medicine  2009;41(2):128-138.
Background
Several studies have investigated associations between the -174G>C polymorphism (rs1800795) of the IL6-gene, but presented inconsistent results.
Aims
This joint analysis aimed to clarify whether IL6 -174G>C was associated with type 2 diabetes mellitus (T2DM) related quantitative phenotypes.
Methods
Individual-level data from all studies of the IL6-T2DM consortium on Caucasian subjects with available BMI were collected. As study-specific estimates did not show heterogeneity (P>0.1), they were combined by using the inverse-variance fixed-effect model.
Results
The main analysis included 9440, 7398, 24,117, or 5659 nondiabetic and manifest T2DM subjects for fasting glucose, 2-hour glucose, BMI or circulating interleukin-6 levels, respectively. IL6 -174 C-allele carriers had significantly lower fasting glucose (−0.091mmol/L, P=0.014). There was no evidence for association between IL6 -174G>C and BMI or interleukin-6. In an additional analysis of 641 subjects known to develop T2DM later on, the IL6 -174 CC-genotype was associated with higher baseline interleukin-6 (+0.75pg/mL, P=0.004), which was consistent with higher interleukin-6 in the 966 manifest T2DM subjects (+0.50pg/mL, P=0.044).
Conclusions
Our data suggest association between IL6 -174G>C and quantitative glucose, and exploratory analysis indicated modulated interleukin-6 levels in pre-diabetic subjects, being in-line with this SNP’s previously reported T2DM association and a role of circulating interleukin-6 as intermediate phenotype.
doi:10.1080/07853890802337037
PMCID: PMC3801210  PMID: 18752089
blood glucose; body mass index; diabetes mellitus; type 2; epidemiology; molecular; genes; inflammation mediators; interleukin-6; intermediate phenotype; meta-analysis; polymorphism; single nucleotide
9.  Diagnosing Fatty Liver Disease: A Comparative Evaluation of Metabolic Markers, Phenotypes, Genotypes and Established Biomarkers 
PLoS ONE  2013;8(10):e76813.
Background
To date, liver biopsy is the only means of reliable diagnosis for fatty liver disease (FLD). Owing to the inevitable biopsy-associated health risks, however, the development of valid noninvasive diagnostic tools for FLD is well warranted.
Aim
We evaluated a particular metabolic profile with regard to its ability to diagnose FLD and compared its performance to that of established phenotypes, conventional biomarkers and disease-associated genotypes.
Methods
The study population comprised 115 patients with ultrasound-diagnosed FLD and 115 sex- and age-matched controls for whom the serum concentration was measured of 138 different metabolites, including acylcarnitines, amino acids, biogenic amines, hexose, phosphatidylcholines (PCs), lyso-PCs and sphingomyelins. Established phenotypes, biomarkers, disease-associated genotypes and metabolite data were included in diagnostic models for FLD using logistic regression and partial least-squares discriminant analysis. The discriminative power of the ensuing models was compared with respect to area under curve (AUC), integrated discrimination improvement (IDI) and by way of cross-validation (CV).
Results
Use of metabolic markers for predicting FLD showed the best performance among all considered types of markers, yielding an AUC of 0.8993. Additional information on phenotypes, conventional biomarkers or genotypes did not significantly improve this performance. Phospholipids and branched-chain amino acids were most informative for predicting FLD.
Conclusion
We show that the inclusion of metabolite data may substantially increase the power to diagnose FLD over that of models based solely upon phenotypes and conventional biomarkers.
doi:10.1371/journal.pone.0076813
PMCID: PMC3793954  PMID: 24130792
10.  A genomewide perspective of genetic variation in human metabolism 
Nature genetics  2009;42(2):137-141.
Serum metabolite concentrations provide a direct readout of biological processes in the human body, and are associated with disorders such as cardiovascular and metabolic diseases. Here we present a genome-wide association study with 163 metabolic traits using 1809 participants from the KORA population, followed up in the TwinsUK cohort with 422 participants. In eight out of nine replicated loci (FADS1, ELOVL2, ACADS, ACADM, ACADL, SPTLC3, ETFDH, SLC16A9) the genetic variant is located in or near enzyme or solute carrier coding genes, where the associating metabolic traits match the proteins’ function. Many of these loci are located in rate limiting steps of important enzymatic reactions. Use of metabolite concentration ratios as proxies for enzymatic reaction rates reduces the variance and yields robust statistical associations with p-values between 3×10−24 and 6.5×10−179. These loci explained 5.6% to 36.3% of the observed variance. For several loci, associations with clinically relevant parameters have previously been reported.
doi:10.1038/ng.507
PMCID: PMC3773904  PMID: 20037589
11.  Epigenetics meets metabolomics: an epigenome-wide association study with blood serum metabolic traits 
Human Molecular Genetics  2013;23(2):534-545.
Previously, we reported strong influences of genetic variants on metabolic phenotypes, some of them with clinical relevance. Here, we hypothesize that DNA methylation may have an important and potentially independent effect on human metabolism. To test this hypothesis, we conducted what is to the best of our knowledge the first epigenome-wide association study (EWAS) between DNA methylation and metabolic traits (metabotypes) in human blood. We assess 649 blood metabolic traits from 1814 participants of the Kooperative Gesundheitsforschung in der Region Augsburg (KORA) population study for association with methylation of 457 004 CpG sites, determined on the Infinium HumanMethylation450 BeadChip platform. Using the EWAS approach, we identified two types of methylome–metabotype associations. One type is driven by an underlying genetic effect; the other type is independent of genetic variation and potentially driven by common environmental and life-style-dependent factors. We report eight CpG loci at genome-wide significance that have a genetic variant as confounder (P = 3.9 × 10−20 to 2.0 × 10−108, r2 = 0.036 to 0.221). Seven loci display CpG site-specific associations to metabotypes, but do not exhibit any underlying genetic signals (P = 9.2 × 10−14 to 2.7 × 10−27, r2 = 0.008 to 0.107). We further identify several groups of CpG loci that associate with a same metabotype, such as 4-vinylphenol sulfate and 4-androsten-3-beta,17-beta-diol disulfate. In these cases, the association between CpG-methylation and metabotype is likely the result of a common external environmental factor, including smoking. Our study shows that analysis of EWAS with large numbers of metabolic traits in large population cohorts are, in principle, feasible. Taken together, our data suggest that DNA methylation plays an important role in regulating human metabolism.
doi:10.1093/hmg/ddt430
PMCID: PMC3869358  PMID: 24014485
12.  A genome-wide association study of atopic dermatitis identifies loci with overlapping effects on asthma and psoriasis 
Human Molecular Genetics  2013;22(23):4841-4856.
Atopic dermatitis (AD) is the most common dermatological disease of childhood. Many children with AD have asthma and AD shares regions of genetic linkage with psoriasis, another chronic inflammatory skin disease. We present here a genome-wide association study (GWAS) of childhood-onset AD in 1563 European cases with known asthma status and 4054 European controls. Using Illumina genotyping followed by imputation, we generated 268 034 consensus genotypes and in excess of 2 million single nucleotide polymorphisms (SNPs) for analysis. Association signals were assessed for replication in a second panel of 2286 European cases and 3160 European controls. Four loci achieved genome-wide significance for AD and replicated consistently across all cohorts. These included the epidermal differentiation complex (EDC) on chromosome 1, the genomic region proximal to LRRC32 on chromosome 11, the RAD50/IL13 locus on chromosome 5 and the major histocompatibility complex (MHC) on chromosome 6; reflecting action of classical HLA alleles. We observed variation in the contribution towards co-morbid asthma for these regions of association. We further explored the genetic relationship between AD, asthma and psoriasis by examining previously identified susceptibility SNPs for these diseases. We found considerable overlap between AD and psoriasis together with variable coincidence between allergic rhinitis (AR) and asthma. Our results indicate that the pathogenesis of AD incorporates immune and epidermal barrier defects with combinations of specific and overlapping effects at individual loci.
doi:10.1093/hmg/ddt317
PMCID: PMC3820131  PMID: 23886662
13.  Metabolite profiling reveals new insights into the regulation of serum urate in humans 
Metabolomics  2013;10:141-151.
Serum urate, the final breakdown product of purine metabolism, is causally involved in the pathogenesis of gout, and implicated in cardiovascular disease and type 2 diabetes. Serum urate levels highly differ between men and women; however the underlying biological processes in its regulation are still not completely understood and are assumed to result from a complex interplay between genetic, environmental and lifestyle factors. In order to describe the metabolic vicinity of serum urate, we analyzed 355 metabolites in 1,764 individuals of the population-based KORA F4 study and constructed a metabolite network around serum urate using Gaussian Graphical Modeling in a hypothesis-free approach. We subsequently investigated the effect of sex and urate lowering medication on all 38 metabolites assigned to the network. Within the resulting network three main clusters could be detected around urate, including the well-known pathway of purine metabolism, as well as several dipeptides, a group of essential amino acids, and a group of steroids. Of the 38 assigned metabolites, 25 showed strong differences between sexes. Association with uricostatic medication intake was not only confined to purine metabolism but seen for seven metabolites within the network. Our findings highlight pathways that are important in the regulation of serum urate and suggest that dipeptides, amino acids, and steroid hormones are playing a role in its regulation. The findings might have an impact on the development of specific targets in the treatment and prevention of hyperuricemia.
Electronic supplementary material
The online version of this article (doi:10.1007/s11306-013-0565-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s11306-013-0565-2
PMCID: PMC3890072  PMID: 24482632
Gaussian Graphical Modeling; Metabolite network; Pathway reconstruction; Allopurinol; Uric acid; Purine metabolism
14.  Posttraumatic Stress Disorder and Not Depression Is Associated with Shorter Leukocyte Telomere Length: Findings from 3,000 Participants in the Population-Based KORA F4 Study 
PLoS ONE  2013;8(7):e64762.
Background
A link between severe mental stress and shorter telomere length (TL) has been suggested. We analysed the impact of Posttraumatic Stress Disorder (PTSD) on TL in the general population and postulated a dose-dependent TL association in subjects suffering from partial PTSD compared to full PTSD.
Methods
Data are derived from the population-based KORA F4 study (2006–2008), located in southern Germany including 3,000 individuals (1,449 men and 1,551 women) with valid and complete TL data. Leukocyte TL was measured using a quantitative PCR-based technique. PTSD was assessed in a structured interview and by applying the Posttraumatic Diagnostic Scale (PDS) and the Impact of Event Scale (IES). A total of 262 (8.7%) subjects qualified for having partial PTSD and 51 (1.7%) for full PTSD. To assess the association of PTSD with the average TL, linear regression analyses with adjustments for potential confounding factors were performed.
Results
The multiple model revealed a significant association between partial PTSD and TL (beta = −0.051, p = 0.009) as well as between full PTSD and shorter TL (beta = −0.103, p = 0.014) indicating shorter TL on average for partial and full PTSD. An additional adjustment for depression and depressed mood/exhaustion gave comparable beta estimations.
Conclusions
Participants with partial and full PTSD had significantly shorter leukocyte TL than participants without PTSD. The dose-dependent variation in TL of subjects with partial and full PTSD exceeded the chronological age effect, and was equivalent to an estimated 5 years in partial and 10 years in full PTSD of premature aging.
doi:10.1371/journal.pone.0064762
PMCID: PMC3700974  PMID: 23843935
15.  Tobacco Smoking Leads to Extensive Genome-Wide Changes in DNA Methylation 
PLoS ONE  2013;8(5):e63812.
Environmental factors such as tobacco smoking may have long-lasting effects on DNA methylation patterns, which might lead to changes in gene expression and in a broader context to the development or progression of various diseases. We conducted an epigenome-wide association study (EWAs) comparing current, former and never smokers from 1793 participants of the population-based KORA F4 panel, with replication in 479 participants from the KORA F3 panel, carried out by the 450K BeadChip with genomic DNA obtained from whole blood. We observed wide-spread differences in the degree of site-specific methylation (with p-values ranging from 9.31E-08 to 2.54E-182) as a function of tobacco smoking in each of the 22 autosomes, with the percent of variance explained by smoking ranging from 1.31 to 41.02. Depending on cessation time and pack-years, methylation levels in former smokers were found to be close to the ones seen in never smokers. In addition, methylation-specific protein binding patterns were observed for cg05575921 within AHRR, which had the highest level of detectable changes in DNA methylation associated with tobacco smoking (–24.40% methylation; p = 2.54E-182), suggesting a regulatory role for gene expression. The results of our study confirm the broad effect of tobacco smoking on the human organism, but also show that quitting tobacco smoking presumably allows regaining the DNA methylation state of never smokers.
doi:10.1371/journal.pone.0063812
PMCID: PMC3656907  PMID: 23691101
16.  Effects of smoking and smoking cessation on human serum metabolite profile: results from the KORA cohort study 
BMC Medicine  2013;11:60.
Background
Metabolomics helps to identify links between environmental exposures and intermediate biomarkers of disturbed pathways. We previously reported variations in phosphatidylcholines in male smokers compared with non-smokers in a cross-sectional pilot study with a small sample size, but knowledge of the reversibility of smoking effects on metabolite profiles is limited. Here, we extend our metabolomics study with a large prospective study including female smokers and quitters.
Methods
Using targeted metabolomics approach, we quantified 140 metabolite concentrations for 1,241 fasting serum samples in the population-based Cooperative Health Research in the Region of Augsburg (KORA) human cohort at two time points: baseline survey conducted between 1999 and 2001 and follow-up after seven years. Metabolite profiles were compared among groups of current smokers, former smokers and never smokers, and were further assessed for their reversibility after smoking cessation. Changes in metabolite concentrations from baseline to the follow-up were investigated in a longitudinal analysis comparing current smokers, never smokers and smoking quitters, who were current smokers at baseline but former smokers by the time of follow-up. In addition, we constructed protein-metabolite networks with smoking-related genes and metabolites.
Results
We identified 21 smoking-related metabolites in the baseline investigation (18 in men and six in women, with three overlaps) enriched in amino acid and lipid pathways, which were significantly different between current smokers and never smokers. Moreover, 19 out of the 21 metabolites were found to be reversible in former smokers. In the follow-up study, 13 reversible metabolites in men were measured, of which 10 were confirmed to be reversible in male quitters. Protein-metabolite networks are proposed to explain the consistent reversibility of smoking effects on metabolites.
Conclusions
We showed that smoking-related changes in human serum metabolites are reversible after smoking cessation, consistent with the known cardiovascular risk reduction. The metabolites identified may serve as potential biomarkers to evaluate the status of smoking cessation and characterize smoking-related diseases.
doi:10.1186/1741-7015-11-60
PMCID: PMC3653729  PMID: 23497222
metabolic network; metabolomics; molecular epidemiology; smoking; smoking cessation
17.  Identification and MS-assisted interpretation of genetically influenced NMR signals in human plasma 
Genome Medicine  2013;5(2):13.
Nuclear magnetic resonance spectroscopy (NMR) provides robust readouts of many metabolic parameters in one experiment. However, identification of clinically relevant markers in 1H NMR spectra is a major challenge. Association of NMR-derived quantities with genetic variants can uncover biologically relevant metabolic traits. Using NMR data of plasma samples from 1,757 individuals from the KORA study together with 655,658 genetic variants, we show that ratios between NMR intensities at two chemical shift positions can provide informative and robust biomarkers. We report seven loci of genetic association with NMR-derived traits (APOA1, CETP, CPS1, GCKR, FADS1, LIPC, PYROXD2) and characterize these traits biochemically using mass spectrometry. These ratios may now be used in clinical studies.
doi:10.1186/gm417
PMCID: PMC3706909  PMID: 23414815
18.  Gene Set of Nuclear-Encoded Mitochondrial Regulators Is Enriched for Common Inherited Variation in Obesity 
PLoS ONE  2013;8(2):e55884.
There are hints of an altered mitochondrial function in obesity. Nuclear-encoded genes are relevant for mitochondrial function (3 gene sets of known relevant pathways: (1) 16 nuclear regulators of mitochondrial genes, (2) 91 genes for oxidative phosphorylation and (3) 966 nuclear-encoded mitochondrial genes). Gene set enrichment analysis (GSEA) showed no association with type 2 diabetes mellitus in these gene sets. Here we performed a GSEA for the same gene sets for obesity. Genome wide association study (GWAS) data from a case-control approach on 453 extremely obese children and adolescents and 435 lean adult controls were used for GSEA. For independent confirmation, we analyzed 705 obesity GWAS trios (extremely obese child and both biological parents) and a population-based GWAS sample (KORA F4, n = 1,743). A meta-analysis was performed on all three samples. In each sample, the distribution of significance levels between the respective gene set and those of all genes was compared using the leading-edge-fraction-comparison test (cut-offs between the 50th and 95th percentile of the set of all gene-wise corrected p-values) as implemented in the MAGENTA software. In the case-control sample, significant enrichment of associations with obesity was observed above the 50th percentile for the set of the 16 nuclear regulators of mitochondrial genes (pGSEA,50 = 0.0103). This finding was not confirmed in the trios (pGSEA,50 = 0.5991), but in KORA (pGSEA,50 = 0.0398). The meta-analysis again indicated a trend for enrichment (pMAGENTA,50 = 0.1052, pMAGENTA,75 = 0.0251). The GSEA revealed that weak association signals for obesity might be enriched in the gene set of 16 nuclear regulators of mitochondrial genes.
doi:10.1371/journal.pone.0055884
PMCID: PMC3568071  PMID: 23409076
19.  Eight genetic loci associated with variation in lipoprotein-associated phospholipase A2 mass and activity and coronary heart disease: meta-analysis of genome-wide association studies from five community-based studies 
European Heart Journal  2011;33(2):238-251.
Aims
Lipoprotein-associated phospholipase A2 (Lp-PLA2) generates proinflammatory and proatherogenic compounds in the arterial vascular wall and is a potential therapeutic target in coronary heart disease (CHD). We searched for genetic loci related to Lp-PLA2 mass or activity by a genome-wide association study as part of the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium.
Methods and results
In meta-analyses of findings from five population-based studies, comprising 13 664 subjects, variants at two loci (PLA2G7, CETP) were associated with Lp-PLA2 mass. The strongest signal was at rs1805017 in PLA2G7 [P = 2.4 × 10−23, log Lp-PLA2 difference per allele (beta): 0.043]. Variants at six loci were associated with Lp-PLA2 activity (PLA2G7, APOC1, CELSR2, LDL, ZNF259, SCARB1), among which the strongest signals were at rs4420638, near the APOE–APOC1–APOC4–APOC2 cluster [P = 4.9 × 10−30; log Lp-PLA2 difference per allele (beta): −0.054]. There were no significant gene–environment interactions between these eight polymorphisms associated with Lp-PLA2 mass or activity and age, sex, body mass index, or smoking status. Four of the polymorphisms (in APOC1, CELSR2, SCARB1, ZNF259), but not PLA2G7, were significantly associated with CHD in a second study.
Conclusion
Levels of Lp-PLA2 mass and activity were associated with PLA2G7, the gene coding for this protein. Lipoprotein-associated phospholipase A2 activity was also strongly associated with genetic variants related to low-density lipoprotein cholesterol levels.
doi:10.1093/eurheartj/ehr372
PMCID: PMC3258449  PMID: 22003152
Genome-wide association; Inflammation; Lipoprotein-associated phospholipase A2
20.  A Variant In the Abo Gene Explains the Variation in Soluble E-Selectin Levels—Results from Dense Genotyping in Two Independent Populations 
PLoS ONE  2012;7(12):e51441.
Background
Elevated soluble (s) E-selectin levels have been associated with various cardiovascular diseases. Recently, genetic variants in the ABO blood group have been related to E-selectin levels in a small cohort of patients with type 1 diabetes. We evaluated whether this association is reproducible in two large samples of Caucasians.
Methodology/ Principal Findings
Data of the present study was drawn from the population-based MONICA/KORA Augsburg study (n = 1,482) and the patients-based LURIC study (n = 1,546). A high-density genotyping array (50K IBC Chip) containing single-nucleotide polymorphisms (SNPs) from E-selectin candidate genes selected on known biology of E-selectin metabolism, mouse genetic studies, and human genetic association studies, was used for genotyping. Linear regression analyses with adjustment for age and sex (and survey in KORA) were applied to assess associations between gene variants and sE-selectin concentrations. A number of 12 SNPs (in KORA) and 13 SNPs (in LURIC), all from the ABO blood group gene, were significantly associated with the log-transformed concentration of E-selectin. The strongest association was observed for rs651007 with a change of log-transformed sE-selectin per one copy of the minor allele of −0.37 ng/ml (p = 1.87×10−103) in KORA and −0.35 ng/ml (p = 5.11×10−84) in LURIC. Inclusion of rs651007 increased the explained sE-selectin variance by 0.256 in KORA and 0.213 in LURIC. All SNPs had minor allele frequencies above 20% showing a substantial gene variation.
Conclusions/ Significance
Our findings in two independent samples indicate that the genetic variants at the ABO locus affect sE-selectin levels. Since distinct genome-wide association studies linked the ABO gene with myocardial infarction (MI) in the presence of coronary atherosclerosis and with coronary artery disease, these findings may not only enhance our understanding of adhesion molecule biology, but may also provide a focus for several novel research avenues.
doi:10.1371/journal.pone.0051441
PMCID: PMC3532506  PMID: 23300549
21.  Mutation screen in the GWAS derived obesity gene SH2B1 including functional analyses of detected variants 
BMC Medical Genomics  2012;5:65.
Background
The SH2B1 gene (Src-homology 2B adaptor protein 1 gene) is a solid candidate gene for obesity. Large scale GWAS studies depicted markers in the vicinity of the gene; animal models suggest a potential relevance for human body weight regulation.
Methods
We performed a mutation screen for variants in the SH2B1 coding sequence in 95 extremely obese children and adolescents. Detected variants were genotyped in independent childhood and adult study groups (up to 11,406 obese or overweight individuals and 4,568 controls). Functional implications on STAT3 mediated leptin signalling of the detected variants were analyzed in vitro.
Results
We identified two new rare mutations and five known SNPs (rs147094247, rs7498665, rs60604881, rs62037368 and rs62037369) in SH2B1. Mutation g.9483C/T leads to a non-synonymous, non-conservative exchange in the beta (βThr656Ile) and gamma (γPro674Ser) splice variants of SH2B1. It was additionally detected in two of 11,206 (extremely) obese or overweight children, adolescents and adults, but not in 4,506 population-based normal-weight or lean controls. The non-coding mutation g.10182C/A at the 3’ end of SH2B1 was only detected in three obese individuals. For the non-synonymous SNP rs7498665 (Thr484Ala) we observed nominal over-transmission of the previously described risk allele in 705 obesity trios (nominal p = 0.009, OR = 1.23) and an increased frequency of the same allele in 359 cases compared to 429 controls (nominal p = 0.042, OR = 1.23). The obesity risk-alleles at Thr484Ala and βThr656Ile/γPro674Ser had no effect on STAT3 mediated leptin receptor signalling in splice variants β and γ.
Conclusion
The rare coding mutation βThr656Ile/γPro674Ser (g.9483C/T) in SH2B1 was exclusively detected in overweight or obese individuals. Functional analyzes did not reveal impairments in leptin signalling for the mutated SH2B1.
doi:10.1186/1755-8794-5-65
PMCID: PMC3544595  PMID: 23270367
SH2B1; Obesity; BMI; rs7498665; Mutation screen
22.  SNCA Variants Are Associated with Increased Risk for Multiple System Atrophy 
Annals of neurology  2009;65(5):610-614.
To test whether the synucleinopathies Parkinson’s disease and multiple system atrophy (MSA) share a common genetic etiology, we performed a candidate single nucleotide polymorphism (SNP) association study of the 384 most associated SNPs in a genome-wide association study of Parkinson’s disease in 413 MSA cases and 3,974 control subjects. The 10 most significant SNPs were then replicated in additional 108 MSA cases and 537 controls. SNPs at the SNCA locus were significantly associated with risk for increased risk for the development of MSA (combined p = 5.5 × 1012; odds ratio 6.2).
doi:10.1002/ana.21685
PMCID: PMC3520128  PMID: 19475667
23.  Variants in MTNR1B influence fasting glucose levels 
Prokopenko, Inga | Langenberg, Claudia | Florez, Jose C | Saxena, Richa | Soranzo, Nicole | Thorleifsson, Gudmar | Loos, Ruth J F | Manning, Alisa K | Jackson, Anne U | Aulchenko, Yurii | Potter, Simon C | Erdos, Michael R | Sanna, Serena | Hottenga, Jouke-Jan | Wheeler, Eleanor | Kaakinen, Marika | Lyssenko, Valeriya | Chen, Wei-Min | Ahmadi, Kourosh | Beckmann, Jacques S | Bergman, Richard N | Bochud, Murielle | Bonnycastle, Lori L | Buchanan, Thomas A | Cao, Antonio | Cervino, Alessandra | Coin, Lachlan | Collins, Francis S | Crisponi, Laura | de Geus, Eco J C | Dehghan, Abbas | Deloukas, Panos | Doney, Alex S F | Elliott, Paul | Freimer, Nelson | Gateva, Vesela | Herder, Christian | Hofman, Albert | Hughes, Thomas E | Hunt, Sarah | Illig, Thomas | Inouye, Michael | Isomaa, Bo | Johnson, Toby | Kong, Augustine | Krestyaninova, Maria | Kuusisto, Johanna | Laakso, Markku | Lim, Noha | Lindblad, Ulf | Lindgren, Cecilia M | McCann, Owen T | Mohlke, Karen L | Morris, Andrew D | Naitza, Silvia | Orrù, Marco | Palmer, Colin N A | Pouta, Anneli | Randall, Joshua | Rathmann, Wolfgang | Saramies, Jouko | Scheet, Paul | Scott, Laura J | Scuteri, Angelo | Sharp, Stephen | Sijbrands, Eric | Smit, Jan H | Song, Kijoung | Steinthorsdottir, Valgerdur | Stringham, Heather M | Tuomi, Tiinamaija | Tuomilehto, Jaakko | Uitterlinden, André G | Voight, Benjamin F | Waterworth, Dawn | Wichmann, H-Erich | Willemsen, Gonneke | Witteman, Jacqueline C M | Yuan, Xin | Zhao, Jing Hua | Zeggini, Eleftheria | Schlessinger, David | Sandhu, Manjinder | Boomsma, Dorret I | Uda, Manuela | Spector, Tim D | Penninx, Brenda WJH | Altshuler, David | Vollenweider, Peter | Jarvelin, Marjo Riitta | Lakatta, Edward | Waeber, Gerard | Fox, Caroline S | Peltonen, Leena | Groop, Leif C | Mooser, Vincent | Cupples, L Adrienne | Thorsteinsdottir, Unnur | Boehnke, Michael | Barroso, Inês | Van Duijn, Cornelia | Dupuis, Josée | Watanabe, Richard M | Stefansson, Kari | McCarthy, Mark I | Wareham, Nicholas J | Meigs, James B | Abecasis, Gonçalo R
Nature genetics  2008;41(1):77-81.
To identify previously unknown genetic loci associated with fasting glucose concentrations, we examined the leading association signals in ten genome-wide association scans involving a total of 36,610 individuals of European descent. Variants in the gene encoding melatonin receptor 1B (MTNR1B) were consistently associated with fasting glucose across all ten studies. The strongest signal was observed at rs10830963, where each G allele (frequency 0.30 in HapMap CEU) was associated with an increase of 0.07 (95% CI = 0.06-0.08) mmol/l in fasting glucose levels (P = 3.2 = × 10−50) and reduced beta-cell function as measured by homeostasis model assessment (HOMA-B, P = 1.1 × 10−15). The same allele was associated with an increased risk of type 2 diabetes (odds ratio = 1.09 (1.05-1.12), per G allele P = 3.3 × 10−7) in a meta-analysis of 13 case-control studies totaling 18,236 cases and 64,453 controls. Our analyses also confirm previous associations of fasting glucose with variants at the G6PC2 (rs560887, P = 1.1 × 10−57) and GCK (rs4607517, P = 1.0 × 10−25) loci.
doi:10.1038/ng.290
PMCID: PMC2682768  PMID: 19060907
24.  Analyzing Illumina Gene Expression Microarray Data from Different Tissues: Methodological Aspects of Data Analysis in the MetaXpress Consortium 
PLoS ONE  2012;7(12):e50938.
Microarray profiling of gene expression is widely applied in molecular biology and functional genomics. Experimental and technical variations make meta-analysis of different studies challenging. In a total of 3358 samples, all from German population-based cohorts, we investigated the effect of data preprocessing and the variability due to sample processing in whole blood cell and blood monocyte gene expression data, measured on the Illumina HumanHT-12 v3 BeadChip array.
Gene expression signal intensities were similar after applying the log2 or the variance-stabilizing transformation. In all cohorts, the first principal component (PC) explained more than 95% of the total variation. Technical factors substantially influenced signal intensity values, especially the Illumina chip assignment (33–48% of the variance), the RNA amplification batch (12–24%), the RNA isolation batch (16%), and the sample storage time, in particular the time between blood donation and RNA isolation for the whole blood cell samples (2–3%), and the time between RNA isolation and amplification for the monocyte samples (2%). White blood cell composition parameters were the strongest biological factors influencing the expression signal intensities in the whole blood cell samples (3%), followed by sex (1–2%) in both sample types. Known single nucleotide polymorphisms (SNPs) were located in 38% of the analyzed probe sequences and 4% of them included common SNPs (minor allele frequency >5%). Out of the tested SNPs, 1.4% significantly modified the probe-specific expression signals (Bonferroni corrected p-value<0.05), but in almost half of these events the signal intensities were even increased despite the occurrence of the mismatch. Thus, the vast majority of SNPs within probes had no significant effect on hybridization efficiency.
In summary, adjustment for a few selected technical factors greatly improved reliability of gene expression analyses. Such adjustments are particularly required for meta-analyses.
doi:10.1371/journal.pone.0050938
PMCID: PMC3517598  PMID: 23236413
25.  Genetic Variation in the Epidermal Transglutaminase Genes Is Not Associated with Atopic Dermatitis 
PLoS ONE  2012;7(11):e49694.
Background
Atopic dermatitis (AD) is a common chronic inflammatory skin disorder where epidermal barrier dysfunction is a major factor in the pathogenesis. The identification of AD susceptibility genes related to barrier dysfunction is therefore of importance. The epidermal transglutaminases (TGM1, TGM3 and TGM5) encodes essential cross-linking enzymes in the epidermis.
Objective
To determine whether genetic variability in the epidermal transglutaminases contributes to AD susceptibility.
Methods
Forty-seven single nucleotide polymorphisms (SNPs) in the TGM1, TGM3 and TGM5 gene region were tested for genetic association with AD, independently and in relation to FLG genotype, using a pedigree disequilibrium test (PDT) in a Swedish material consisting of 1753 individuals from 539 families. In addition, a German case-control material, consisting of 533 AD cases and 1996 controls, was used for in silico analysis of the epidermal TGM regions. Gene expression of the TGM1, TGM3 and TGM5 gene was investigated by relative quantification with Real Time PCR (qRT-PCR). Immunohistochemical (IHC) analysis was performed to detect TG1, TG3 and TG5 protein expression in the skin of patients and healthy controls.
Results
PDT analysis identified a significant association between the TGM1 SNP rs941505 and AD with allergen-specific IgE in the Swedish AD family material. However, the association was not replicated in the German case-control material. No significant association was detected for analyzed SNPs in relation to FLG genotype. TG1, TG3 and TG5 protein expression was detected in AD skin and a significantly increased TGM3 mRNA expression was observed in lesional skin by qRT-PCR.
Conclusion
Although TGM1 and TGM3 may be differentially expressed in AD skin, the results from the genetic analysis suggest that genetic variation in the epidermal transglutaminases is not an important factor in AD susceptibility.
doi:10.1371/journal.pone.0049694
PMCID: PMC3506648  PMID: 23189155

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