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1.  The LIFE-Adult-Study: objectives and design of a population-based cohort study with 10,000 deeply phenotyped adults in Germany 
BMC Public Health  2015;15:691.
The LIFE-Adult-Study is a population-based cohort study, which has recently completed the baseline examination of 10,000 randomly selected participants from Leipzig, a major city with 550,000 inhabitants in the east of Germany. It is the first study of this kind and size in an urban population in the eastern part of Germany. The study is conducted by the Leipzig Research Centre for Civilization Diseases (LIFE). Our objective is to investigate prevalences, early onset markers, genetic predispositions, and the role of lifestyle factors of major civilization diseases, with primary focus on metabolic and vascular diseases, heart function, cognitive impairment, brain function, depression, sleep disorders and vigilance dysregulation, retinal and optic nerve degeneration, and allergies.
The study covers a main age range from 40-79 years with particular deep phenotyping in elderly participants above the age of 60. The baseline examination was conducted from August 2011 to November 2014. All participants underwent an extensive core assessment programme (5-6 h) including structured interviews, questionnaires, physical examinations, and biospecimen collection. Participants over 60 underwent two additional assessment programmes (3-4 h each) on two separate visits including deeper cognitive testing, brain magnetic resonance imaging, diagnostic interviews for depression, and electroencephalography.
The participation rate was 33 %. The assessment programme was accepted well and completely passed by almost all participants. Biomarker analyses have already been performed in all participants. Genotype, transcriptome and metabolome analyses have been conducted in subgroups. The first follow-up examination will commence in 2016.
PMCID: PMC4509697  PMID: 26197779
Cohort study; Epidemiology; Population based; Diseases of civilization
2.  Dissecting the genetics of the human transcriptome identifies novel trait-related trans-eQTLs and corroborates the regulatory relevance of non-protein coding loci† 
Human Molecular Genetics  2015;24(16):4746-4763.
Genetics of gene expression (eQTLs or expression QTLs) has proved an indispensable tool for understanding biological pathways and pathomechanisms of trait-associated SNPs. However, power of most genome-wide eQTL studies is still limited. We performed a large eQTL study in peripheral blood mononuclear cells of 2112 individuals increasing the power to detect trans-effects genome-wide. Going beyond univariate SNP-transcript associations, we analyse relations of eQTLs to biological pathways, polygenetic effects of expression regulation, trans-clusters and enrichment of co-localized functional elements. We found eQTLs for about 85% of analysed genes, and 18% of genes were trans-regulated. Local eSNPs were enriched up to a distance of 5 Mb to the transcript challenging typically implemented ranges of cis-regulations. Pathway enrichment within regulated genes of GWAS-related eSNPs supported functional relevance of identified eQTLs. We demonstrate that nearest genes of GWAS-SNPs might frequently be misleading functional candidates. We identified novel trans-clusters of potential functional relevance for GWAS-SNPs of several phenotypes including obesity-related traits, HDL-cholesterol levels and haematological phenotypes. We used chromatin immunoprecipitation data for demonstrating biological effects. Yet, we show for strongly heritable transcripts that still little trans-chromosomal heritability is explained by all identified trans-eSNPs; however, our data suggest that most cis-heritability of these transcripts seems explained. Dissection of co-localized functional elements indicated a prominent role of SNPs in loci of pseudogenes and non-coding RNAs for the regulation of coding genes. In summary, our study substantially increases the catalogue of human eQTLs and improves our understanding of the complex genetic regulation of gene expression, pathways and disease-related processes.
PMCID: PMC4512630  PMID: 26019233
3.  Identification of a novel SERPINA-1 mutation causing alpha-1 antitrypsin deficiency in a patient with severe bronchiectasis and pulmonary embolism 
Deficiency in the serine protease inhibitor, alpha-1 antitrypsin (AAT), is known to cause emphysema and liver disease. Other manifestations, including airway disease or skin disorders, have also been described. A 44-year-old woman presented to our emergency department with dyspnea and respiratory insufficiency. She had never smoked, and had been diagnosed with COPD 9 years earlier. Three months previously, she had suffered a pulmonary embolism. Chest computed tomography scan revealed severe cystic bronchiectasis with destruction of the lung parenchyma. The sweat test was normal and there was no evidence of the cystic fibrosis transmembrane conductance regulator (CFTR) mutation. Capillary zone electrophoresis showed a decrease of alpha-1 globin band and AAT levels were below the quantification limit (<25 mg/dL). No S or Z mutation was identified, but sequencing analysis found a homozygous cytosine and adenine (CA) insertion in exon 2 of the SERPINA-1 gene, probably leading to a dysfunctional protein (PI Null/Null). This mutation has not been previously identified. The atypical presentation of the patient, with severe cystic bronchiectasis, highlights AAT deficiency as a differential diagnosis in bronchiectasis. Further, awareness should be raised regarding a possible increased risk of thromboembolism associated with AAT deficiency.
PMCID: PMC4428364  PMID: 26005342
alpha-1 antitrypsin deficiency; bronchiectasis; SERPINA-1 mutation; pulmonary embolism
4.  Inflammatory Cytokines in General and Central Obesity and Modulating Effects of Physical Activity 
PLoS ONE  2015;10(3):e0121971.
Chronic systemic inflammation in obesity originates from local immune responses in visceral adipose tissue. However, assessment of a broad range of inflammation-mediating cytokines and their relationship to physical activity and adipometrics has scarcely been reported to date.
To characterize the profile of a broad range of pro- and anti-inflammatory cytokines and the impact of physical activity and energy expenditure in individuals with general obesity, central obesity, and non-obese subjects.
Design, Setting, and Participants
A cross-sectional study comprising 117 obese patients (body mass index (BMI) ≥ 30) and 83 non-obese community-based volunteers.
Main Outcomes Measures
Serum levels of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12, IL-13, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ and tumor necrosis factor (TNF)-α were measured. Physical activity and energy expenditure (MET) were assessed with actigraphy. Adipometrics comprised BMI, weight, abdominal-, waist- and hip-circumference, waist to hip ratio (WHR), and waist-to-height-ratio (WHtR).
General obesity was associated with significantly elevated levels of IL-5, IL-10, IL-12, IL-13, IFN-γ and TNF-α, central obesity with significantly elevated IL-5, IL-10, IL-12, IL-13 and IFN-γ-levels. In participants with general obesity, levels of IL-4, IL-10 and IL-13 were significantly elevated in participants with low physical activity, even when controlled for BMI which was negatively associated with physical acitivity. Cytokines significantly correlated with adipometrics, particularly in obese participants.
Results confirm up-regulation of certain pro- and anti-inflammatory cytokines in obesity. In obese subjects, physical activity may lower levels and thus reduce pro-inflammatory effects of cytokines that may link obesity, insulin resistance and diabetes.
PMCID: PMC4363366  PMID: 25781614
5.  Comparison of Whole Blood RNA Preservation Tubes and Novel Generation RNA Extraction Kits for Analysis of mRNA and MiRNA Profiles 
PLoS ONE  2014;9(12):e113298.
Whole blood expression profiling is frequently performed using PAXgene (Qiagen) or Tempus (Life Technologies) tubes. Here, we compare 6 novel generation RNA isolation protocols with respect to RNA quantity, quality and recovery of mRNA and miRNA.
3 PAXgene and 3 Tempus Tubes were collected from participants of the LIFE study with (n = 12) and without (n = 35) acute myocardial infarction (AMI). RNA was extracted with 4 manual protocols from Qiagen (PAXgene Blood miRNA Kit), Life Technologies (MagMAX for Stabilized Blood Tubes RNA Isolation Kit), and Norgen Biotek (Norgen Preserved Blood RNA Purification Kit I and Kit II), and 2 (semi-)automated protocols on the QIAsymphony (Qiagen) and MagMAX Express-96 Magnetic Particle Processor (Life Technologies). RNA quantity and quality was determined. For biological validation, RNA from 12 representative probands, extracted with all 6 kits (n = 72), was reverse transcribed and mRNAs (matrix metalloproteinase 9, arginase 1) and miRNAs (miR133a, miR1), shown to be altered by AMI, were analyzed.
RNA yields were highest using the Norgen Kit I with Tempus Tubes and lowest using the Norgen Kit II with PAXgene. The disease status was the second major determinant of RNA yields (LIFE-AMI 11.2 vs. LIFE 6.7 µg, p<0.001) followed by the choice of blood collection tube. (Semi-)automation reduced overall RNA extraction time but did not generally reduce hands-on-time. RNA yields and quality were comparable between manual and automated extraction protocols. mRNA expression was not affected by collection tubes and RNA extraction kits but by RT/qPCR reagents with exception of the Norgen Kit II, which led to mRNA depletion. For miRNAs, expression differences related to collection tubes (miR30b), RNA isolation (Norgen Kit II), and RT/qRT reagents (miR133a) were observed.
We demonstrate that novel generation RNA isolation kits significantly differed with respect to RNA recovery and affected miRNA but not mRNA expression profiles.
PMCID: PMC4254602  PMID: 25469788
6.  Variability of linezolid concentrations after standard dosing in critically ill patients: a prospective observational study 
Critical Care  2014;18(4):R148.
Severe infections in intensive care patients show high morbidity and mortality rates. Linezolid is an antimicrobial drug frequently used in critically ill patients. Recent data indicates that there might be high variability of linezolid serum concentrations in intensive care patients receiving standard doses. This study was aimed to evaluate whether standard dosing of linezolid leads to therapeutic serum concentrations in critically ill patients.
In this prospective observational study, 30 critically ill adult patients with suspected infections received standard dosing of 600 mg linezolid intravenously twice a day. Over 4 days, multiple serum samples were obtained from each patient, in order to determine the linezolid concentrations by liquid chromatography tandem mass spectrometry.
A high variability of serum linezolid concentrations was observed (range of area under the linezolid concentration time curve over 24 hours (AUC24) 50.1 to 453.9 mg/L, median 143.3 mg*h/L; range of trough concentrations (Cmin) < 0.13 to 14.49 mg/L, median 2.06 mg/L). Furthermore, potentially subtherapeutic linezolid concentrations over 24 hours and at single time points (defined according to the literature as AUC24 < 200 mg*h/L and Cmin < 2 mg/L) were observed for 63% and 50% of the patients, respectively. Finally, potentially toxic levels (defined as AUC24 > 400 mg*h/L and Cmin > 10 mg/L) were observed for 7 of the patients.
A high variability of linezolid serum concentrations with a substantial percentage of potentially subtherapeutic levels was observed in intensive care patients. The findings suggest that therapeutic drug monitoring of linezolid might be helpful for adequate dosing of linezolid in critically ill patients.
Trial registration NCT01793012. Registered 24 January 2013.
PMCID: PMC4227093  PMID: 25011656
7.  Altered Expression of Raet1e, a Major Histocompatibility Complex Class 1–Like Molecule, Underlies the Atherosclerosis Modifier Locus Ath11 10b 
Circulation research  2013;113(9):1054-1064.
Quantitative trait locus mapping of an intercross between C57.Apoe−/− and FVB.Apoe−/− mice revealed an atherosclerosis locus controlling aortic root lesion area on proximal chromosome 10, Ath11. In a previous work, subcongenic analysis showed Ath11 to be complex with proximal (10a) and distal (10b) regions.
To identify the causative genetic variation underlying the atherosclerosis modifier locus Ath11 10b.
Methods and Results
We now report subcongenic J, which narrows the 10b region to 5 genes, Myb, Hbs1L, Aldh8a1, Sgk1, and Raet1e. Sequence analysis of these genes revealed no amino acid coding differences between the parental strains. However, comparing aortic expression of these genes between F1.Apoe−/− Chr10SubJ(B/F) and F1.Apoe−/− Chr10SubJ(F/F) uncovered a consistent difference only for Raet1e, with decreased, virtually background, expression associated with increased atherosclerosis in the latter. The key role of Raet1e was confirmed by showing that transgene-induced aortic overexpression of Raet1e in F1.Apoe−/− Chr10SubJ(F/F) mice decreased atherosclerosis. Promoter reporter constructs comparing C57 and FVB sequences identified an FVB mutation in the core of the major aortic transcription start site abrogating activity.
This nonbiased approach has revealed Raet1e, a major histocompatibility complex class 1–like molecule expressed in lesional aortic endothelial cells and macrophage-rich regions, as a novel atherosclerosis gene and represents one of the few successes of the quantitative trait locus strategy in complex diseases.
PMCID: PMC3938025  PMID: 23948654
atherosclerosis; gene expression; genetic susceptibility; mice; mouse model; quantitative trait loci
8.  A multi-split mapping algorithm for circular RNA, splicing, trans-splicing and fusion detection 
Genome Biology  2014;15(2):R34.
Numerous high-throughput sequencing studies have focused on detecting conventionally spliced mRNAs in RNA-seq data. However, non-standard RNAs arising through gene fusion, circularization or trans-splicing are often neglected. We introduce a novel, unbiased algorithm to detect splice junctions from single-end cDNA sequences. In contrast to other methods, our approach accommodates multi-junction structures. Our method compares favorably with competing tools for conventionally spliced mRNAs and, with a gain of up to 40% of recall, systematically outperforms them on reads with multiple splits, trans-splicing and circular products. The algorithm is integrated into our mapping tool segemehl (
PMCID: PMC4056463  PMID: 24512684
9.  Secretory Phospholipase A2-IIA and Cardiovascular Disease 
Holmes, Michael V. | Simon, Tabassome | Exeter, Holly J. | Folkersen, Lasse | Asselbergs, Folkert W. | Guardiola, Montse | Cooper, Jackie A. | Palmen, Jutta | Hubacek, Jaroslav A. | Carruthers, Kathryn F. | Horne, Benjamin D. | Brunisholz, Kimberly D. | Mega, Jessica L. | van Iperen, Erik P.A. | Li, Mingyao | Leusink, Maarten | Trompet, Stella | Verschuren, Jeffrey J.W. | Hovingh, G. Kees | Dehghan, Abbas | Nelson, Christopher P. | Kotti, Salma | Danchin, Nicolas | Scholz, Markus | Haase, Christiane L. | Rothenbacher, Dietrich | Swerdlow, Daniel I. | Kuchenbaecker, Karoline B. | Staines-Urias, Eleonora | Goel, Anuj | van 't Hooft, Ferdinand | Gertow, Karl | de Faire, Ulf | Panayiotou, Andrie G. | Tremoli, Elena | Baldassarre, Damiano | Veglia, Fabrizio | Holdt, Lesca M. | Beutner, Frank | Gansevoort, Ron T. | Navis, Gerjan J. | Mateo Leach, Irene | Breitling, Lutz P. | Brenner, Hermann | Thiery, Joachim | Dallmeier, Dhayana | Franco-Cereceda, Anders | Boer, Jolanda M.A. | Stephens, Jeffrey W. | Hofker, Marten H. | Tedgui, Alain | Hofman, Albert | Uitterlinden, André G. | Adamkova, Vera | Pitha, Jan | Onland-Moret, N. Charlotte | Cramer, Maarten J. | Nathoe, Hendrik M. | Spiering, Wilko | Klungel, Olaf H. | Kumari, Meena | Whincup, Peter H. | Morrow, David A. | Braund, Peter S. | Hall, Alistair S. | Olsson, Anders G. | Doevendans, Pieter A. | Trip, Mieke D. | Tobin, Martin D. | Hamsten, Anders | Watkins, Hugh | Koenig, Wolfgang | Nicolaides, Andrew N. | Teupser, Daniel | Day, Ian N.M. | Carlquist, John F. | Gaunt, Tom R. | Ford, Ian | Sattar, Naveed | Tsimikas, Sotirios | Schwartz, Gregory G. | Lawlor, Debbie A. | Morris, Richard W. | Sandhu, Manjinder S. | Poledne, Rudolf | Maitland-van der Zee, Anke H. | Khaw, Kay-Tee | Keating, Brendan J. | van der Harst, Pim | Price, Jackie F. | Mehta, Shamir R. | Yusuf, Salim | Witteman, Jaqueline C.M. | Franco, Oscar H. | Jukema, J. Wouter | de Knijff, Peter | Tybjaerg-Hansen, Anne | Rader, Daniel J. | Farrall, Martin | Samani, Nilesh J. | Kivimaki, Mika | Fox, Keith A.A. | Humphries, Steve E. | Anderson, Jeffrey L. | Boekholdt, S. Matthijs | Palmer, Tom M. | Eriksson, Per | Paré, Guillaume | Hingorani, Aroon D. | Sabatine, Marc S. | Mallat, Ziad | Casas, Juan P. | Talmud, Philippa J.
This study sought to investigate the role of secretory phospholipase A2 (sPLA2)-IIA in cardiovascular disease.
Higher circulating levels of sPLA2-IIA mass or sPLA2 enzyme activity have been associated with increased risk of cardiovascular events. However, it is not clear if this association is causal. A recent phase III clinical trial of an sPLA2 inhibitor (varespladib) was stopped prematurely for lack of efficacy.
We conducted a Mendelian randomization meta-analysis of 19 general population studies (8,021 incident, 7,513 prevalent major vascular events [MVE] in 74,683 individuals) and 10 acute coronary syndrome (ACS) cohorts (2,520 recurrent MVE in 18,355 individuals) using rs11573156, a variant in PLA2G2A encoding the sPLA2-IIA isoenzyme, as an instrumental variable.
PLA2G2A rs11573156 C allele associated with lower circulating sPLA2-IIA mass (38% to 44%) and sPLA2 enzyme activity (3% to 23%) per C allele. The odds ratio (OR) for MVE per rs11573156 C allele was 1.02 (95% confidence interval [CI]: 0.98 to 1.06) in general populations and 0.96 (95% CI: 0.90 to 1.03) in ACS cohorts. In the general population studies, the OR derived from the genetic instrumental variable analysis for MVE for a 1-log unit lower sPLA2-IIA mass was 1.04 (95% CI: 0.96 to 1.13), and differed from the non-genetic observational estimate (OR: 0.69; 95% CI: 0.61 to 0.79). In the ACS cohorts, both the genetic instrumental variable and observational ORs showed a null association with MVE. Instrumental variable analysis failed to show associations between sPLA2 enzyme activity and MVE.
Reducing sPLA2-IIA mass is unlikely to be a useful therapeutic goal for preventing cardiovascular events.
PMCID: PMC3826105  PMID: 23916927
cardiovascular diseases; drug development; epidemiology; genetics; Mendelian randomization; ACS, acute coronary syndrome(s); CI, confidence interval; LDL-C, low-density lipoprotein cholesterol; MI, myocardial infarction; MVE, major vascular events; OR, odds ratio; RCT, randomized clinical trial; SNP, single-nucleotide polymorphism; sPLA2, secretory phospholipase A2
10.  Alu Elements in ANRIL Non-Coding RNA at Chromosome 9p21 Modulate Atherogenic Cell Functions through Trans-Regulation of Gene Networks 
PLoS Genetics  2013;9(7):e1003588.
The chromosome 9p21 (Chr9p21) locus of coronary artery disease has been identified in the first surge of genome-wide association and is the strongest genetic factor of atherosclerosis known today. Chr9p21 encodes the long non-coding RNA (ncRNA) antisense non-coding RNA in the INK4 locus (ANRIL). ANRIL expression is associated with the Chr9p21 genotype and correlated with atherosclerosis severity. Here, we report on the molecular mechanisms through which ANRIL regulates target-genes in trans, leading to increased cell proliferation, increased cell adhesion and decreased apoptosis, which are all essential mechanisms of atherogenesis. Importantly, trans-regulation was dependent on Alu motifs, which marked the promoters of ANRIL target genes and were mirrored in ANRIL RNA transcripts. ANRIL bound Polycomb group proteins that were highly enriched in the proximity of Alu motifs across the genome and were recruited to promoters of target genes upon ANRIL over-expression. The functional relevance of Alu motifs in ANRIL was confirmed by deletion and mutagenesis, reversing trans-regulation and atherogenic cell functions. ANRIL-regulated networks were confirmed in 2280 individuals with and without coronary artery disease and functionally validated in primary cells from patients carrying the Chr9p21 risk allele. Our study provides a molecular mechanism for pro-atherogenic effects of ANRIL at Chr9p21 and suggests a novel role for Alu elements in epigenetic gene regulation by long ncRNAs.
Author Summary
Chromosome 9p21 is the strongest genetic factor for coronary artery disease and encodes the long non-coding RNA (ncRNA) ANRIL. Here, we show that increased ANRIL expression mediates atherosclerosis risk through trans-regulation of gene networks leading to pro-atherogenic cellular properties, such as increased proliferation and adhesion. ANRIL may act as a scaffold, guiding effector-proteins to chromatin. These functions depend on an Alu motif present in ANRIL RNA and mirrored several thousand-fold in the genome. Alu elements are a family of primate-specific short interspersed repeat elements (SINEs) and have been linked with genetic disease. Current models propose that either exonisation of Alu elements or changes of cis-regulation of adjacent genes are the underlying disease mechanisms. Our work extends the function of Alu transposons to regulatory components of long ncRNAs with a central role in epigenetic trans-regulation. Furthermore, it implies a pivotal role for Alu elements in genetically determined vascular disease and describes a plausible molecular mechanism for a pro-atherogenic function of ANRIL at chromosome 9p21.
PMCID: PMC3701717  PMID: 23861667
11.  Reduced Food Intake and Body Weight in Mice Deficient for the G Protein-Coupled Receptor GPR82 
PLoS ONE  2011;6(12):e29400.
G protein-coupled receptors (GPCR) are involved in the regulation of numerous physiological functions. Therefore, GPCR variants may have conferred important selective advantages during periods of human evolution. Indeed, several genomic loci with signatures of recent selection in humans contain GPCR genes among them the X-chromosomally located gene for GPR82. This gene encodes a so-called orphan GPCR with unknown function. To address the functional relevance of GPR82 gene-deficient mice were characterized. GPR82-deficient mice were viable, reproduced normally, and showed no gross anatomical abnormalities. However, GPR82-deficient mice have a reduced body weight and body fat content associated with a lower food intake. Moreover, GPR82-deficient mice showed decreased serum triacylglyceride levels, increased insulin sensitivity and glucose tolerance, most pronounced under Western diet. Because there were no differences in respiratory and metabolic rates between wild-type and GPR82-deficient mice our data suggest that GPR82 function influences food intake and, therefore, energy and body weight balance. GPR82 may represent a thrifty gene most probably representing an advantage during human expansion into new environments.
PMCID: PMC3247265  PMID: 22216272
12.  Rationale and Design of the Leipzig (LIFE) Heart Study: Phenotyping and Cardiovascular Characteristics of Patients with Coronary Artery Disease 
PLoS ONE  2011;6(12):e29070.
We established the Leipzig (LIFE) Heart Study, a biobank and database of patients with different stages of coronary artery disease (CAD) for studies of clinical, metabolic, cellular and genetic factors of cardiovascular diseases.
The Leipzig (LIFE) Heart Study (NCT00497887) is an ongoing observational angiographic study including subjects with different entities of CAD. Cohort 1, patients undergoing first-time diagnostic coronary angiography due to suspected stable CAD with previously untreated coronary arteries. Cohort 2, patients with acute myocardial infarction (MI) requiring percutaneous revascularization. Cohort 3, patients with known left main coronary artery disease (LMCAD).
We present preliminary results of demographics and phenotyping based on a 4-years analysis of a total of 3,165 subjects. Cohort 1 (n = 2,274) shows the typical distribution of elective coronary angiography cohorts with 43% cases with obstructive CAD and 37% normal angiograms. Cohorts 2 and 3 consist of 590 and 301 subjects, respectively, adding patients with severe forms of CAD. The suitability of the database and biobank to perform association studies was confirmed by replication of the CAD susceptibility locus on chromosome 9p21 (OR per allele: 1.55 (any CAD), 1.54 (MI), 1.74 (LMCAD), p<10−6, respectively). A novel finding was that patients with LMCAD had a stronger association with 9p21 than patients with obstructive CAD without LMCAD (OR 1.22, p = 0.042). In contrast, 9p21 did not associate with myocardial infarction in excess of stable CAD.
The Leipzig (LIFE) Heart Study provides a basis to identify molecular targets related to atherogenesis and associated metabolic disorders. The study may contribute to an improvement of individual prediction, prevention, and treatment of CAD.
PMCID: PMC3245257  PMID: 22216169
13.  Adipogenesis and insulin sensitivity in obesity are regulated by retinoid-related orphan receptor gamma 
EMBO Molecular Medicine  2011;3(11):637-651.
Obesity is a well-known risk factor for the development of secondary complications such as type 2 diabetes. However, only a part of the obese population develops secondary metabolic disorders. Here, we identify the transcription factor retinoid-related orphan receptor gamma (RORγ) as a negative regulator of adipocyte differentiation through expression of its newly identified target gene matrix metalloproteinase 3. In vivo differentiation of adipocyte progenitor cells from Rorγ-deficient mice is enhanced and obese Rorγ−/− mice show decreased adipocyte sizes. These small adipocytes are highly insulin sensitive, leading to an improved control of circulating free fatty acids. Ultimately, Rorγ−/− mice are protected from hyperglycemia and insulin resistance in the state of obesity. In adipose stromal-vascular fraction from obese human subjects, Rorγ expression is correlated with adipocyte size and negatively correlated with adipogenesis and insulin sensitivity. Taken together, our findings identify RORγ as a factor, which controls adipogenesis as well as adipocyte size and modulates insulin sensitivity in obesity. RORγ might therefore serve as a novel pharmaceutical target to treat obesity-associated insulin resistance.
PMCID: PMC3377107  PMID: 21853531
adipogenesis; matrix metalloproteinase 3; obesity; retinoid-related orphan receptor gamma; type 2 diabetes
14.  The mouse atherosclerosis locus at chromosome 10 (Ath11) acts early in lesion formation with subcongenic strains delineating two narrowed regions 
Ath11, an atherosclerosis susceptibility locus on proximal chromosome 10 (0–21cM) revealed in a cross between apolipoprotein E deficient C57BL/6 (B6) and FVB mice, was recently confirmed in congenic mice. The objectives of this study were to assess how Ath11 affects lesion development and morphology, to determine aortic gene expression in congenics, and to narrow the congenic interval.
Methods and Results
Assessing lesion area over time in congenic mice showed that homozygosity for the FVB allele increased lesion area at 6 weeks persisting through to 24 weeks of age. Staining of aortic root sections at 16 weeks did not reveal obvious differences between congenics. Aortic expression-array analysis at 6 weeks revealed 97 >2 fold regulated genes, including one gene in the QTL interval, Aldh8a1, and two gene clusters regulated by Hnf4α and Esr1. Analysis of lesion area in 11 subcongenic strains revealed two narrowed regions, 10a (21 genes) acting in females and 10b (7 genes) acting in both genders.
Ath11 appears to act early in lesion formation with significant effects on aortic gene expression. This QTL is genetically complex containing a female specific region 10a from 0 to 7.3 Mb, and a gender independent region 10b from 20.1 to 21.9 Mb.
PMCID: PMC2917113  PMID: 20466976
Atherosclerosis; QTL; Ath11; congenics; subcongenics
15.  Lack of Association between Adiponectin Levels and Atherosclerosis in Mice 
Adiponectin is an adipocyte-derived, secreted protein that is implicated in the protection against a cluster of related metabolic disorders. Mice lacking adiponectin display impaired hepatic insulin sensitivity and respond only partially to PPARγ agonists. Adiponectin has been associated with anti-inflammatory and anti-atherogenic properties, however, the direct involvement of adiponectin on the atherogenic process has not been studied.
Methods and Results
We crossed adiponectin knockout mice (Adn−/−) or mice with chronically elevated adiponectin levels (AdnTg) into the low-density lipoprotein receptor null (Ldlr−/−) and the apoliprotein E null (Apoe−/−) mouse models. Adiponectin levels did not correlate with a suppression of the atherogenic process. Plaque volume in the aortic root, cholesterol accumulation in the aorta and plaque morphology under various dietary conditions were not affected by circulating adiponectin levels. In light of the strong associations reported for adiponectin with cardiovascular disease in humans, the lack of a phenotype in gain- and loss-of-function studies in mice may suggests lack of causation for adiponectin in inhibiting the build up of atherosclerotic lesions.
These data indicate that the actions of adiponectin on the cardiovascular system are complex and multifaceted, with a minimal direct impact on atherosclerotic plaque formation in preclinical rodent models.
PMCID: PMC2896306  PMID: 20299691
Adiponectin; atherosclerosis; LDL receptor knockout mice
16.  Highly Elevated Serum Hepcidin in Patients with Acute Myeloid Leukemia prior to and after Allogeneic Hematopoietic Cell Transplantation: Does This Protect from Excessive Parenchymal Iron Loading? 
Advances in Hematology  2011;2011:491058.
Hepcidin is upregulated by inflammation and iron. Inherited (HFE genotype) and treatment-related factors (blood units (BU), Iron overload) affecting hepcidin (measured by C-ELISA) were studied in 42 consecutive patients with AML prior to and after allogeneic hematopoietic cell transplantation (HCT). Results. Elevated serum ferritin pre- and post-HCT was present in all patients. Median hepcidin pre- and post-HCT of 358 and 398 ng/mL, respectively, were elevated compared to controls (median 52 ng/mL) (P < .0001). Liver and renal function, prior chemotherapies, and conditioning had no impact on hepcidin. Despite higher total BU after HCT compared to pretransplantation (P < .0005), pre- and posttransplant ferritin and hepcidin were similar. BU influenced ferritin (P = .001) and hepcidin (P = .001). No correlation of pre- or posttransplant hepcidin with pretransplant ferritin was found. HFE genotype did not influence hepcidin. Conclusions. Hepcidin is elevated in AML patients pre- and post-HCT due to transfusional iron-loading suggesting that hepcidin synthesis remains intact despite chemotherapy and HCT.
PMCID: PMC3112503  PMID: 21687645
17.  Association of FTO variants with BMI and fat mass in the self-contained population of Sorbs in Germany 
The association between common variants in the FTO gene with weight, adiposity and body mass index (BMI) has now been widely replicated. Although the causal variant has yet to be identified, it most likely maps within a 47 kb region of intron 1 of FTO. We performed a genome-wide association study in the Sorbian population and evaluated the relationships between FTO variants and BMI and fat mass in this isolate of Slavonic origin resident in Germany. In a sample of 948 Sorbs, we could replicate the earlier reported associations of intron 1 SNPs with BMI (eg, P-value=0.003, β=0.02 for rs8050136). However, using genome-wide association data, we also detected a second independent signal mapping to a region in intron 2/3 about 40–60 kb away from the originally reported SNPs (eg, for rs17818902 association with BMI P-value=0.0006, β=−0.03 and with fat mass P-value=0.0018, β=−0.079). Both signals remain independently associated in the conditioned analyses. In conclusion, we extend the evidence that FTO variants are associated with BMI by putatively identifying a second susceptibility allele independent of that described earlier. Although further statistical analysis of these findings is hampered by the finite size of the Sorbian isolate, these findings should encourage other groups to seek alternative susceptibility variants within FTO (and other established susceptibility loci) using the opportunities afforded by analyses in populations with divergent mutational and/or demographic histories.
PMCID: PMC2987177  PMID: 19584900
FTO; BMI; Sorbs
18.  No Reduction of Atherosclerosis in C-reactive Protein (CRP)-deficient Mice* 
The Journal of Biological Chemistry  2010;286(8):6272-6279.
C-reactive protein (CRP), a phylogenetically highly conserved plasma protein, is the classical acute phase reactant in humans. Upon infection, inflammation, or tissue damage, its plasma level can rise within hours >1000-fold, providing an early, nonspecific disease indicator of prime clinical importance. In recent years, another aspect of CRP expression has attracted much scientific and public attention. Apart from transient, acute phase-associated spikes in plasma concentration, highly sensitive measurements have revealed stable interindividual differences of baseline CRP values in healthy persons. Strikingly, even modest elevations in stable baseline CRP plasma levels have been found to correlate with a significantly increased risk of future cardiovascular disease. These observations have triggered intense controversies about potential atherosclerosis-promoting properties of CRP. To directly assess potential effects of CRP on atherogenesis, we have generated CRP-deficient mice via gene targeting and introduced the inactivated allele into atherosclerosis-susceptible ApoE−/− and LDLR−/− mice, two well established mouse models of atherogenesis. Morphometric analyses of atherosclerotic plaques in CRP-deficient animals revealed equivalent or increased atherosclerotic lesions compared with controls, an experimental result, which does not support a proatherogenic role of CRP. In fact, our data suggest that mouse CRP may even mediate atheroprotective effects, adding a cautionary note to the idea of targeting CRP as therapeutic intervention against progressive cardiovascular disease.
PMCID: PMC3057833  PMID: 21149301
Apolipoproteins; Atherosclerosis; Gene Knock-out; Inflammation; Mouse Genetics; C-reactive Protein; Acute Phase Proteins
19.  A novel strategy using F1-congenic mice for validation of QTLs: Studies at the proximal chromosome 10 atherosclerosis susceptibility locus 
We have previously identified a quantitative trait locus (QTL) for atherosclerosis susceptibility on proximal chromosome 10 (Chr10) (Ath11) in independent crosses of FVB and C57BL/6 (B6) mice on the apolipoprotein E (ApoE−/−) and LDL receptor (LDLR−/−) deficient backgrounds. The aims of the current study were to (1) test a novel strategy for validating QTLs using interval-specific congenic strains that were heterozygous (F1) across the genome, (2) validate the Chr10 QTL and (3) to assess whether the phenotype is transferable by bone marrow transplantation.
Methods and Results
We generated Chr10 (0 to 21cM) interval-specific mice on the F1.ApoE−/− background by crossing congenic FVB.ApoE−/−Chr10B6/FVB with B6.ApoE−/−, and B6.ApoE−/−Chr10B6/FVB with FVB.ApoE−/− mice. Lesion size was significantly larger in the resultant F1.ApoE−/−Chr10FVB/FVB mice compared to F1.ApoE−/−Chr10B6/FVB and F1.ApoE−/−Chr10B6/B6 mice, validating the Chr10 QTL. The effect of the congenic interval was more robust on the F1.ApoE−/− than on the FVB.ApoE−/− and B6.ApoE−/− backgrounds. Bone marrow transplantation in congenic mice showed that the effect of the proximal Chr10 interval was not transferable by bone-marrow derived cells.
A novel strategy of congenic strains on an F1 background proved useful to validate an atherosclerosis susceptibility QTL on mouse proximal Chr10.
PMCID: PMC2738862  PMID: 19251590
Atherosclerosis; congenic mice; chromosome 10; transplantation
20.  Increased ADAM17 mRNA Expression and Activity is Associated with Atherosclerosis Resistance in LDL-Receptor Deficient Mice 
We have previously identified an atherosclerosis quantitative trait locus (QTL) on mouse chromosome (Chr) 12 in an F2-intercross of atherosclerosis-resistant FVB and atherosclerosis-susceptible C57BL/6 (B6) mice on the LDL-receptor deficient background. The aim of the present study was to identify potentially causative genes at this locus.
Methods and Results
Expression QTL (eQTL) analysis of candidate genes in livers of F2-mice revealed that a disintegrin and metalloproteinase 17 (ADAM17) mRNA expression mapped to the physical position of ADAM17 on proximal Chr12 (21.6 Mb, LOD 3.3) and co-localized with the atherosclerosis QTL. The FVB allele was associated with significantly higher ADAM17 mRNA expression (39%) than the B6 allele. Likewise, ADAM17 mRNA levels in the parental strains were significantly elevated in FVB.LDLR−/− compared to B6.LDLR−/− mice in liver, macrophages, and aorta (68%, 58%, and 32% respectively). Reporter gene assays revealed a genetic variant that might explain these expression differences. Moreover, FVB.LDLR−/− macrophages showed 5-fold increased PMA-induced shedding of TNF-alpha and 32% increased release of TNF-receptor I compared to B6.LDLR−/−. The atherosclerosis locus and expression differences were confirmed in Chr12 interval-specific congenic mice.
Our data provide functional evidence for ADAM17 as a candidate gene of atherosclerosis susceptibility at the murine Chr12 QTL.
PMCID: PMC2789234  PMID: 18356551
Atherosclerosis; genetics; ADAM17; mouse models; expression QTL
21.  From genotype to phenotype in human atherosclerosis - recent findings 
Current Opinion in Lipidology  2013;24(5):410-418.
Purpose of review
Since 2007, genome-wide association studies (GWAS) have led to the identification of numerous loci of atherosclerotic cardiovascular disease. The majority of these loci harbor genes previously not known to be involved in atherogenesis. In this review, we summarize the recent progress in understanding the pathophysiology of genetic variants in atherosclerosis.
Recent findings
Fifty-eight loci with P < 10−7 have been identified in GWAS for coronary heart disease and myocardial infarction. Of these, 23 loci (40%) overlap with GWAS loci of classical risk factors such as lipids, blood pressure, and diabetes mellitus, suggesting a potential causal relation. The vast majority of the remaining 35 loci (60%) are at genomic regions where the mechanism in atherogenesis is unclear. Loci most frequently found in independent GWAS were at Chr9p21.3 (ANRIL/CDKN2B-AS1), Chr6p24.1 (PHACTR1), and Chr1p13.3 (CELSR2, PSRC1, MYBPHL, SORT1). Recent work suggests that Chr9p21.3 exerts its effects through epigenetic regulation of target genes, whereas mechanisms at Chr6p24.1 remain obscure, and Chr1p13.3 affects plasma LDL cholesterol.
Novel GWAS loci indicate that our understanding of atherosclerosis is limited and implicate a role of hitherto unknown mechanisms, such as epigenetic gene regulation in atherogenesis.
PMCID: PMC3814939  PMID: 24005217
1p13.3; 6p24.1; 9p21.3; atherosclerosis; GWAS

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