Klaus Stark

New susceptibility locus for coronary artery disease on chromosome 3q22.3

We present a three-stage analysis of genome-wide SNP data in 1,222 German individuals with myocardial infarction and 1,298 controls, in silico replication in three additional genome-wide datasets of coronary artery disease (CAD) and subsequent replication in ∼25,000 subjects. We identified one new CAD risk locus on 3q22.3 in MRAS (P = 7.44 × 10−13; OR = 1.15, 95% CI = 1.11–1.19), and suggestive association with a locus on 12q24.31 near HNF1A-C12orf43 (P = 4.81 × 10−7; OR = 1.08, 95% CI = 1.05–1.11).

Hepatitis E Virus Seroprevalence among Adults, Germany

We assessed hepatitis E virus (HEV) antibody seroprevalence in a sample of the adult population in Germany. Overall HEV IgG prevalence was 16.8% (95% CI 15.6%–17.9%) and increased with age, leveling off at >60 years of age. HEV is endemic in Germany, and the lifetime risk for exposure is high.

The Bacterial Paromomycin Resistance Gene, aphH, as a Dominant Selectable Marker in Volvox carteri

The aminoglycoside antibiotic paromomycin that is highly toxic to the green alga Volvox carteri is efficiently inactivated by aminoglycoside 3-phosphotransferase from Streptomyces rimosus. Therefore, we made constructs in which the bacterial aphH gene encoding this enzyme was combined with Volvox cis-regulatory elements in an attempt to develop a new dominant selectable marker--paromomycin resistance (PmR)--for use in Volvox nuclear transformation. The construct that provided the most efficient transformation was one in which aphH was placed between a chimeric promoter that was generated by fusing the Volvox hsp70 and rbcS3 promoters and the 3 UTR of the Volvox rbcS3 gene. When this plasmid was used in combination with a high-impact biolistic device, the frequency of stable PmR transformants ranged about 15 per 106 target cells. Due to rapid and sharp selection, PmR transformants were readily isolated after six days, which is half the time required for previously used markers. Co-transformation of an unselected marker ranged about 30%. The chimeric aphH gene was stably integrated into the Volvox genome, frequently as tandem multiple copies, and was expressed at a level that made selection of PmR transformants simple and unambiguous. This makes the engineered bacterial aphH gene an efficient dominant selection marker for the transformation and co-transformation of a broad range of V. carteri strains without the recurring need for using auxotrophic recipient strains.

Volvox germline-specific genes that are putative targets of RegA repression encode chloroplast proteins.

Abstract In Volvox carteri, regA acts as a master gene to suppress all germ cell functions in somatic cells. Its product, RegA, has features of a transcriptional repressor. Here we report cDNA sequences representing 15 nuclear genes with properties expected of RegA targets: they are expressed strongly in germ cells and in regA−, but not regA+, somatic cells. Two of them encode polypeptides with no recognizable features, but ten (like three previously sequenced ones) encode chloroplast proteins of known function, and the remaining three encode putative chloroplast proteins of unknown function. This suggests that RegA blocks reproductive development in somatic cells by preventing chloroplast biogenesis, thereby making it impossible for the cells to grow enough to reproduce.

Lack of Evidence for Schmallenberg Virus Infection in Highly Exposed Persons, Germany, 2012

Schmallenberg virus, a novel orthobunyavirus, is spreading among ruminants, especially sheep, throughout Europe. To determine the risk for human infection, we conducted a survey among shepherds to assess possible exposure and symptoms. We also performed serologic and molecular assays. No evidence of transmission to humans was detected.

Large-Scale Candidate Gene Analysis of HDL Particle Features

Background HDL cholesterol (HDL-C) is an established marker of cardiovascular risk with significant genetic determination. However, HDL particles are not homogenous, and refined HDL phenotyping may improve insight into regulation of HDL metabolism. We therefore assessed HDL particles by NMR spectroscopy and conducted a large-scale candidate gene association analysis. Methodology/Principal Findings We measured plasma HDL-C and determined mean HDL particle size and particle number by NMR spectroscopy in 2024 individuals from 512 British Caucasian families. Genotypes were 49,094 SNPs in >2,100 cardiometabolic candidate genes/loci as represented on the HumanCVD BeadChip version 2. False discovery rates (FDR) were calculated to account for multiple testing. Analyses on classical HDL-C revealed significant associations (FDR<0.05) only for CETP (cholesteryl ester transfer protein; lead SNP rs3764261: pu200a=u200a5.6*10−15) and SGCD (sarcoglycan delta; rs6877118: pu200a=u200a8.6*10−6). In contrast, analysis with HDL mean particle size yielded additional associations in LIPC (hepatic lipase; rs261332: pu200a=u200a6.1*10−9), PLTP (phospholipid transfer protein, rs4810479: pu200a=u200a1.7*10−8) and FBLN5 (fibulin-5; rs2246416: pu200a=u200a6.2*10−6). The associations of SGCD and Fibulin-5 with HDL particle size could not be replicated in PROCARDIS (nu200a=u200a3,078) and/or the Womens Genome Health Study (nu200a=u200a23,170). Conclusions We show that refined HDL phenotyping by NMR spectroscopy can detect known genes of HDL metabolism better than analyses on HDL-C.

Common Genetic Variants in ANK2 Modulate QT Interval Results From the KORA Study

Background—Spatial and timely variations in QT interval, even within its normal range, may underlie susceptibility to cardiac arrhythmias and sudden cardiac death. Given its important role in cardiac electrophysiology, we hypothesized that common genetic variation in ankyrin-B gene (ANK2) might modify QT interval length. Methods and Results—The study population consisted of 1188 participants of the World Health Organizational Multinational Monitoring of Trends and Determinants in Cardiovascular Disease (WHO MONICA) general population survey Cooperative Health Research in the Region of Augsburg (KORA S3). Corrected QT interval was calculated using population specific linear regression formulas. A total of 22 single-nucleotide polymorphisms in the genomic region of ANK2 gene were genotyped using TaqMan technology. In a replication study, 6 single nucleotide polymorphisms were genotyped in 3890 individuals from a second population study (KORA S4). The rare variant of the single-nucleotide polymorphism rs6850768 (allele frequency, 0.28) significantly influenced duration of the QT interval, both in KORA S3 and KORA S4 populations. In homozygotes, the shortening of the QT interval was 3.79 ms (95% CI, 1.48 to 5.58; P=0.001 and P=0.0008 for log-additive and dominant model, respectively) in KORA S3 and 2.94 ms (95% CI, 1.11 to 4.77; P=0.001 and P=0.006 for log-additive and dominant genetic model, respectively) in KORA S4. A common 2-locus haplotype (rs11098171-rs6850768; population frequency, 28%) was associated with a QT interval difference of 2.85 ms (permutation; P=0.006) in KORA S3 and 1.23 ms (permutation; P=0.009) in KORA S4. Reverse transcription–polymerase chain reaction expression analysis of the human ANK2 5′ genomic region in the human left ventricular tissue revealed 2 previously unidentified ANK2 5′ exons in the proximity of the identified variants. Conclusions—Common genetic variants juxtaposed with novel exons in the distant 5′ genomic region of ANK2 influence the QT interval length in the general population. These findings support the role of ankyrin-B in normal cardiac electric activity.

Risk factors for sporadic Yersinia enterocolitica infections, Germany 2009-2010

Yersinia enterocolitica is an important cause of acute gastrointestinal disease and post-infectious complications. In Germany, incidence of reported yersiniosis is relatively high compared with other countries of the European Union. Children aged <5 years are most frequently affected. The aim of our study was to identify risk factors for sporadic yersiniosis in Germany. A population-based case-control study was conducted in five federal states of Germany from April 2009 to June 2010. Cases exhibiting gastrointestinal symptoms were notified to the local health department with a Yersinia enterocolitica infection culture-confirmed from stool. Controls were selected from population registries and frequency-matched on age group and state of residency. Cases and controls received a questionnaire on possible risk factors by mail. Multivariable logistic regression modelling was used to identify risk factors and to calculate adjusted odds ratios (aORs). Population attributable fractions (PAFs) were estimated for exposures associated with yersiniosis. We analysed data on 571 case patients and 1798 controls. Consumption of raw minced pork, a dish frequently consumed even by young children in Germany, was the main risk factor for disease (aOR 4·7, 95% confidence interval (CI) 3·5-6·3, PAF 30%). This association varied by age group and, unexpectedly, was strongest for children aged <2 years (aOR 17·5, 95% CI 6·0-51·2). Other independent risk factors included recent preparation of minced pork in the household (aOR 1·4, 95% CI 1·1-1·9, PAF 21%), playing in a sandbox (aOR 1·7, 95% CI 1·3-2·4, PAF 17%), and contact with birds (aOR 1·7, 95% CI 1·1-2·6, PAF 4%). Prevention efforts should specifically target parents and caregivers of young children and focus on the high infection risk associated with consumption of raw minced pork.

Genetic associations with lipoprotein subfractions provide information on their biological nature

Adverse levels of lipoproteins are highly heritable and constitute risk factors for cardiovascular outcomes. Hitherto, genome-wide association studies revealed 95 lipid-associated loci. However, due to the small effect sizes of these associations large sample numbers (>100 000 samples) were needed. Here we show that analyzing more refined lipid phenotypes, namely lipoprotein subfractions, can increase the number of significantly associated loci compared with bulk high-density lipoprotein and low-density lipoprotein analysis in a study with identical sample numbers. Moreover, lipoprotein subfractions provide novel insight into the human lipid metabolism. We measured 15 lipoprotein subfractions (L1-L15) in 1791 samples using (1)H-NMR (nuclear magnetic resonance) spectroscopy. Using cluster analyses, we quantified inter-relationships among lipoprotein subfractions. Additionally, we analyzed associations with subfractions at known lipid loci. We identified five distinct groups of subfractions: one (L1) was only marginally captured by serum lipids and therefore extends our knowledge of lipoprotein biochemistry. During a lipid-tolerance test, L1 lost its special position. In the association analysis, we found that eight loci (LIPC, CETP, PLTP, FADS1-2-3, SORT1, GCKR, APOB, APOA1) were associated with the subfractions, whereas only four loci (CETP, SORT1, GCKR, APOA1) were associated with serum lipids. For LIPC, we observed a 10-fold increase in the variance explained by our regression models. In conclusion, NMR-based fine mapping of lipoprotein subfractions provides novel information on their biological nature and strengthens the associations with genetic loci. Future clinical studies are now needed to investigate their biomedical relevance.

Generation of Highly Purified Human Cardiomyocytes from Peripheral Blood Mononuclear Cell-Derived Induced Pluripotent Stem Cells

Induced pluripotent stem (iPS) cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T, α-actinin and myosin light chain 2 (MLC2V). In a subsequent metabolic selection with lactate, the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%, whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular, atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified, functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling, drug discovery, and regenerative medicine.