Gravitational Force-Induced 3D Chromosomal Conformational Changes Are Associated with Rapid Transcriptional Response in Human T Cells.

The mechanisms underlying gravity perception in mammalian cells are unknown. We have recently discovered that the transcriptome of cells in the immune system, which is the most affected system during a spaceflight, responds rapidly and broadly to altered gravity. To pinpoint potential underlying mechanisms, we compared gene expression and three-dimensional (3D) chromosomal conformational changes in human Jurkat T cells during the short-term gravitational changes in parabolic flight and suborbital ballistic rocket flight experiments. We found that differential gene expression in gravity-responsive chromosomal regions, but not differentially regulated single genes, are highly conserved between different real altered gravity comparisons. These coupled gene expression effects in chromosomal regions could be explained by underlying chromatin structures. Based on a high-throughput chromatin conformation capture (Hi-C) analysis in altered gravity, we found that small chromosomes (chr16-22, with the exception of chr18) showed increased intra- and interchromosomal interactions in altered gravity, whereby large chromosomes showed decreased interactions. Finally, we detected a nonrandom overlap between Hi-C-identified chromosomal interacting regions and gravity-responsive chromosomal regions (GRCRs). We therefore demonstrate the first evidence that gravitational force-induced 3D chromosomal conformational changes are associated with rapid transcriptional response in human T cells. We propose a general model of cellular sensitivity to gravitational forces, where gravitational forces acting on the cellular membrane are rapidly and mechanically transduced through the cytoskeleton into the nucleus, moving chromosome territories to new conformation states and their genes into more expressive or repressive environments, finally resulting in region-specific differential gene expression.

Induction of ICER is superseded by smICER, challenging the impact of ICER under chronic beta-adrenergic stimulation.

ICER (inducible cAMP early repressor) isoforms are transcriptional repressors encoded by the Crem (cAMP responsive element modulator) gene. They were linked to the regulation of a multitude of cellular processes and pathophysiological mechanisms. Here, we show for the first time that two independent induction patterns for CREM repressor isoforms exist in the heart, namely for ICER and smICER (small ICER), which are induced in response to ß-adrenergic stimulation in a transient- and saturation-like manner, respectively. This time-shifted induction pattern, driven by two internal promoters in the Crem gene, leads to the predominant transcription of smIcer after prolonged ß-adrenergic stimulation. Using an ICER knockout mouse model with preserved smICER induction, we show that the transient-like induction of Icer itself has minor effects on gene regulation, cardiac hypertrophy or contractile function in the heart. We conclude that the functions previously linked to ICER may be rather attributed to smICER, also beyond the cardiac background.

Ectopic expression of Snord115 in choroid plexus interferes with editing but not splicing of 5-Ht2c receptor pre-mRNA in mice.

Serotonin 5-HT2C receptor is a G-protein coupled excitatory receptor that regulates several biochemical pathways and has been implicated in obesity, mental state, sleep cycles, autism, neuropsychiatric disorders and neurodegenerative diseases. The activity of 5-HT2CR is regulated via alternative splicing and A to I editing of exon Vb of its pre-mRNA. Snord115 is a small nucleolar RNA that is expressed in mouse neurons and displays an 18-nucleotide base complementary to exon Vb of 5-HT2CR pre-mRNA. For almost two decades this putative guide element of Snord115 has wandered like a ghost through the literature in attempts to elucidate the biological significance of this complementarity. In mice, Snord115 is expressed in neurons and absent in the choroid plexus where, in contrast, 5-Ht2cr mRNA is highly abundant. Here we report the analysis of 5-Ht2cr pre-mRNA posttranscriptional processing via RNA deep sequencing in a mouse model that ectopically expresses Snord115 in the choroid plexus. In contrast to previous reports, our analysis demonstrated that Snord115 does not control alternative splicing of 5-Ht2cr pre-mRNA in vivo. We identified a modest, yet statistically significant reduction of 5-Ht2cr pre-mRNA A to I editing at the major A, B, C and D sites. We suggest that Snord115 and exon Vb of 5Ht2cr pre-mRNA form a double-stranded structure that is subject to ADAR-mediated A to I editing. To the best of our knowledge, this is the first comprehensive Snord115 gain-of-function analysis based on in vivo mouse models.

Rapid coupling between gravitational forces and the transcriptome in human myelomonocytic U937 cells.

The gravitational force has been constant throughout Earth's evolutionary history. Since the cell nucleus is subjected to permanent forces induced by Earth's gravity, we addressed the question, if gene expression homeostasis is constantly shaped by the gravitational force on Earth. We therefore investigated the transcriptome in force-free conditions of microgravity, determined the time frame of initial gravitational force-transduction to the transcriptome and assessed the role of cation channels. We combined a parabolic flight experiment campaign with a suborbital ballistic rocket experiment employing the human myelomonocytic cell line U937 and analyzed the whole gene transcription by microarray, using rigorous controls for exclusion of effects not related to gravitational force and cross-validation through two fully independent research campaigns. Experiments with the wide range ion channel inhibitor SKF-96365 in combination with whole transcriptome analysis were conducted to study the functional role of ion channels in the transduction of gravitational forces at an integrative level. We detected profound alterations in the transcriptome already after 20 s of microgravity or hypergravity. In microgravity, 99.43% of all initially altered transcripts adapted after 5 min. In hypergravity, 98.93% of all initially altered transcripts adapted after 75 s. Only 2.4% of all microgravity-regulated transcripts were sensitive to the cation channel inhibitor SKF-96365. Inter-platform comparison of differentially regulated transcripts revealed 57 annotated gravity-sensitive transcripts. We assume that gravitational forces are rapidly and constantly transduced into the nucleus as omnipresent condition for nuclear and chromatin structure as well as homeostasis of gene expression.

The Transcription Factor ATF4 Promotes Expression of Cell Stress Genes and Cardiomyocyte Death in a Cellular Model of Atrial Fibrillation.

INTRODUCTION: Cardiomyocyte remodelling in atrial fibrillation (AF) has been associated with both oxidative stress and endoplasmic reticulum (ER) stress and is accompanied by a complex transcriptional regulation. Here, we investigated the role the oxidative stress and ER stress responsive bZIP transcription factor ATF4 plays in atrial cardiomyocyte viability and AF induced gene expression. METHODS: HL-1 cardiomyocytes were subjected to rapid field stimulation. Forced expression of ATF4 was achieved by adenoviral gene transfer. Using global gene expression analysis and chromatin immunoprecipitation, ATF4 dependent transcriptional regulation was studied, and tissue specimen of AF patients was analysed by immunohistochemistry. RESULTS: Oxidative stress and ER stress caused a significant reduction in cardiomyocyte viability and were associated with an induction of ATF4. Accordingly, ATF4 was also induced by rapid field stimulation mimicking AF. Forced expression of wild type ATF4 promoted cardiomyocyte death. ATF4 was demonstrated to bind to the promoters of several cell stress genes and to induce the expression of a number of ATF4 dependent stress responsive genes. Moreover, immunohistochemical analyses showed that ATF4 is expressed in the nuclei of cardiomyocytes of tissue specimen obtained from AF patients. CONCLUSION: ATF4 is expressed in human atrial cardiomyocytes and is induced in response to different types of cell stress. High rate electrical field stimulation seems to result in ATF4 induction, and forced expression of ATF4 reduces cardiomyocyte viability.

Stability of gene expression in human T cells in different gravity environments is clustered in chromosomal region 11p15.4.

In the last decades, a plethora of in vitro studies with living human cells contributed a vast amount of knowledge about cellular and molecular effects of microgravity. Previous studies focused mostly on the identification of gravity-responsive genes, whereas a multi-platform analysis at an integrative level, which specifically evaluates the extent and robustness of transcriptional response to an altered gravity environment was not performed so far. Therefore, we investigated the stability of gene expression response in non-activated human Jurkat T lymphocytic cells in different gravity environments through the combination of parabolic flights with a suborbital ballistic rocket and 2D clinostat and centrifuge experiments, using strict controls for excluding all possible other factors of influence. We revealed an overall high stability of gene expression in microgravity and identified olfactory gene expression in the chromosomal region 11p15.4 as particularly robust to altered gravity. We identified that classical reference genes ABCA5, GAPDH, HPRT1, PLA2G4A, and RPL13A were stably expressed in all tested gravity conditions and platforms, while ABCA5 and GAPDH were also known to be stably expressed in U937 cells in all gravity conditions. In summary, 10-20% of all transcripts remained totally unchanged in any gravitational environment tested (between 10-4 and 9 g), 20-40% remained unchanged in microgravity (between 10-4 and 10-2 g) and 97-99% were not significantly altered in microgravity if strict exclusion criteria were applied. Therefore, we suppose a high stability of gene expression in microgravity. Comparison with other stressors suggests that microgravity alters gene expression homeostasis not stronger than other environmental factors.

Characterization of the Genetic Program Linked to the Development of Atrial Fibrillation in CREM-IbΔC-X Mice.

BACKGROUND: Reduced expression of genes regulated by the transcription factors CREB/CREM (cAMP response element-binding protein/modulator) is linked to atrial fibrillation (AF) susceptibility in patients. Cardiomyocyte-directed expression of the inhibitory CREM isoform CREM-IbΔC-X in transgenic mice (TG) leads to spontaneous-onset AF preceded by atrial dilatation and conduction abnormalities. Here, we characterized the altered gene program linked to atrial remodeling and development of AF in CREM-TG mice. METHODS AND RESULTS: Atria of young (TGy, before AF onset) and old (TGo, after AF onset) TG mice were investigated by mRNA microarray profiling in comparison with age-matched wild-type controls (WTy/WTo). Proteomic alterations were profiled in young mice (8 TGy versus 8 WTy). Annotation of differentially expressed genes revealed distinct differences in biological functions and pathways before and after onset of AF. Alterations in metabolic pathways, some linked to altered peroxisome proliferator-activated receptor signaling, muscle contraction, and ion transport were already present in TGy. Electron microscopy revealed significant loss of sarcomeres and mitochondria and increased collagen and glycogen deposition in TG mice. Alterations in electrophysiological pathways became prominent in TGo, concomitant with altered gene expression of K+-channel subunits and ion channel modulators, relevant in human AF. CONCLUSIONS: The most prominent alterations of the gene program linked to CREM-induced atrial remodeling were identified in the expression of genes related to structure, metabolism, contractility, and electric activity regulation, suggesting that CREM transgenic mice are a valuable experimental model for human AF pathophysiology.

Dynamic gene expression response to altered gravity in human T cells.

We investigated the dynamics of immediate and initial gene expression response to different gravitational environments in human Jurkat T lymphocytic cells and compared expression profiles to identify potential gravity-regulated genes and adaptation processes. We used the Affymetrix GeneChip® Human Transcriptome Array 2.0 containing 44,699 protein coding genes and 22,829 non-protein coding genes and performed the experiments during a parabolic flight and a suborbital ballistic rocket mission to cross-validate gravity-regulated gene expression through independent research platforms and different sets of control experiments to exclude other factors than alteration of gravity. We found that gene expression in human T cells rapidly responded to altered gravity in the time frame of 20 s and 5 min. The initial response to microgravity involved mostly regulatory RNAs. We identified three gravity-regulated genes which could be cross-validated in both completely independent experiment missions: ATP6V1A/D, a vacuolar H + -ATPase (V-ATPase) responsible for acidification during bone resorption, IGHD3-3/IGHD3-10, diversity genes of the immunoglobulin heavy-chain locus participating in V(D)J recombination, and LINC00837, a long intergenic non-protein coding RNA. Due to the extensive and rapid alteration of gene expression associated with regulatory RNAs, we conclude that human cells are equipped with a robust and efficient adaptation potential when challenged with altered gravitational environments.

The Beaver's Phylogenetic Lineage Illuminated by Retroposon Reads.

Solving problematic phylogenetic relationships often requires high quality genome data. However, for many organisms such data are still not available. Among rodents, the phylogenetic position of the beaver has always attracted special interest. The arrangement of the beaver's masseter (jaw-closer) muscle once suggested a strong affinity to some sciurid rodents (e.g., squirrels), placing them in the Sciuromorpha suborder. Modern molecular data, however, suggested a closer relationship of beaver to the representatives of the mouse-related clade, but significant data from virtually homoplasy-free markers (for example retroposon insertions) for the exact position of the beaver have not been available. We derived a gross genome assembly from deposited genomic Illumina paired-end reads and extracted thousands of potential phylogenetically informative retroposon markers using the new bioinformatics coordinate extractor fastCOEX, enabling us to evaluate different hypotheses for the phylogenetic position of the beaver. Comparative results provided significant support for a clear relationship between beavers (Castoridae) and kangaroo rat-related species (Geomyoidea) (p < 0.0015, six markers, no conflicting data) within a significantly supported mouse-related clade (including Myodonta, Anomaluromorpha, and Castorimorpha) (p < 0.0015, six markers, no conflicting data).

Rare genetic variants in SMAP1, B3GAT2, and RIMS1 contribute to pediatric venous thromboembolism.

Recent genome-wide association studies (GWAS) have confirmed known risk mutations for venous thromboembolism (VTE) and identified a number of novel susceptibility loci in adults. Here we present a GWAS in 212 nuclear families with pediatric VTE followed by targeted next-generation sequencing (NGS) to identify causative mutations contributing to the association. Three single nucleotide polymorphisms (SNPs) exceeded the threshold for genome-wide significance as determined by permutation testing using 100 000 bootstrap permutations (P < 10-5). These SNPs reside in a region on chromosome 6q13 comprising the genes small ARF GAP1 (SMAP1), an ARF6 guanosine triphosphatase-activating protein that functions in clathrin-dependent endocytosis, and ß-1,3-glucoronyltransferase 2 (B3GAT2), a member of the human natural killer 1 carbohydrate pathway. Rs1304029 and rs2748331 are associated with pediatric VTE with unpermuted/permuted values of P = 1.42 × 10-6/2.0 × 10-6 and P = 6.11 × 10-6/1.8 × 10-5, respectively. Rs2748331 was replicated (P = .00719) in an independent study sample coming from our GWAS on pediatric thromboembolic stroke (combined P = 7.88 × 10-7). Subsequent targeted NGS in 24 discordant sibling pairs identified 17 nonsynonymous coding variants, of which 1 located in SMAP1 and 3 in RIMS1, a member of the RIM family of active zone proteins, are predicted as damaging by Protein Variation Effect Analyzer and/or sorting intolerant from tolerant scores. Three SNPs curtly missed statistical significance in the transmission-disequilibrium test in the full cohort (rs112439957: P = .08326, SMAP1; rs767118962: P = .08326, RIMS1; and rs41265501: P = .05778, RIMS1). In conjunction, our data provide compelling evidence for SMAP1, B3GAT2, and RIMS1 as novel susceptibility loci for pediatric VTE and warrant future functional studies to unravel the underlying molecular mechanisms leading to VTE.

Regulation of ICAM-1 in cells of the monocyte/macrophage system in microgravity.

Cells of the immune system are highly sensitive to altered gravity, and the monocyte as well as the macrophage function is proven to be impaired under microgravity conditions. In our study, we investigated the surface expression of ICAM-1 protein and expression of ICAM-1 mRNA in cells of the monocyte/macrophage system in microgravity during clinostat, parabolic flight, sounding rocket, and orbital experiments. In murine BV-2 microglial cells, we detected a downregulation of ICAM-1 expression in clinorotation experiments and a rapid and reversible downregulation in the microgravity phase of parabolic flight experiments. In contrast, ICAM-1 expression increased in macrophage-like differentiated human U937 cells during the microgravity phase of parabolic flights and in long-term microgravity provided by a 2D clinostat or during the orbital SIMBOX/Shenzhou-8 mission. In nondifferentiated U937 cells, no effect of microgravity on ICAM-1 expression could be observed during parabolic flight experiments. We conclude that disturbed immune function in microgravity could be a consequence of ICAM-1 modulation in the monocyte/macrophage system, which in turn could have a strong impact on the interaction with T lymphocytes and cell migration. Thus, ICAM-1 can be considered as a rapid-reacting and sustained gravity-regulated molecule in mammalian cells.

Prognostic impact of Bcl-2 depends on tumor histology and expression of MALAT-1 lncRNA in non-small-cell lung cancer.

INTRODUCTION: Apoptosis is a crucial pathway in tumor growth and metastatic development. Apoptotic proteins regulate the underlying molecular cascades and are thought to modulate the tumor response to chemotherapy and radiation. However, the prognostic value of the expression of apoptosis regulators in localized non-small-cell lung cancer (NSCLC) is still unclear. METHODS: We investigated the protein expression of apoptosis regulators Bcl-2, Bcl-xl, Mcl-1, and pp32/PHAPI, and the expression of the lncRNA MALAT-1 in tumor samples from 383 NSCLC patients (median age: 65.6 years; 77.5% male; paraffin-embedded tissue microarrays). For statistical analysis correlation tests, Log rank tests and Cox proportional hazard models were applied. RESULTS: Tumor histology was significantly associated with the expression of Bcl-2, Bcl-xl and Mcl-1 (all p < 0.001). Among the tested apoptotic markers only Bcl-2 demonstrated prognostic impact (hazard ratio = 0.64, p = 0.012). For NSCLC patients with non-adenocarcinoma histology, Bcl-2 expression was associated with increased overall survival (p = 0.036). Besides tumor histology, prognostic impact of Bcl-2 was also found to depend on MALAT-1 lncRNA expression. Gene expression analysis of A549 adenocarcinoma cells with differential MALAT-1 lncRNA expression demonstrated an influence on the expression of Bcl-2 and its interacting proteins. CONCLUSIONS: Bcl-2 expression was specifically associated with superior prognosis in localized NSCLC. An interaction of Bcl-2 with MALAT-1 lncRNA expression was revealed, which merits further investigation for risk prediction in resectable NSCLC patients.

A genome-wide association study identifies 6p21 as novel risk locus for dilated cardiomyopathy.

AIMS: Dilated cardiomyopathy (DCM) is one of the leading causes for cardiac transplantations and accounts for up to one-third of all heart failure cases. Since extrinsic and monogenic causes explain only a fraction of all cases, common genetic variants are suspected to contribute to the pathogenesis of DCM, its age of onset, and clinical progression. By a large-scale case-control genome-wide association study we aimed here to identify novel genetic risk loci for DCM. METHODS AND RESULTS: Applying a three-staged study design, we analysed more than 4100 DCM cases and 7600 controls. We identified and successfully replicated multiple single nucleotide polymorphism on chromosome 6p21. In the combined analysis, the most significant association signal was obtained for rs9262636 (P = 4.90 × 10(-9)) located in HCG22, which could again be replicated in an independent cohort. Taking advantage of expression quantitative trait loci (eQTL) as molecular phenotypes, we identified rs9262636 as an eQTL for several closely located genes encoding class I and class II major histocompatibility complex heavy chain receptors. CONCLUSION: The present study reveals a novel genetic susceptibility locus that clearly underlines the role of genetically driven, inflammatory processes in the pathogenesis of idiopathic DCM.

Six sequence variants on chromosome 9p21.3 are associated with a positive family history of myocardial infarction: a multicenter registry.

BACKGROUND: Recent genome-wide association studies have identified several genetic loci linked to coronary artery disease (CAD) and myocardial infarction (MI). The 9p21.3 locus was verified by numerous replication studies to be the first common locus for CAD and MI. In the present study, we investigated whether six single nucleotide polymorphisms (SNP) rs1333049, rs1333040, rs10757274, rs2383206, rs10757278, and rs2383207 representing the 9p21.3 locus were associated with the incidence of an acute MI in patients with the main focus on the familial aggregation of the disease. METHODS: The overall cohort consisted of 976 unrelated male patients presenting with an acute coronary syndrome (ACS) with ST-elevated (STEMI) as well as non-ST-elevated myocardial infarction (NSTEMI). Genotyping data of the investigated SNPs were generated and statistically analyzed in comparison to previously published findings of matchable control cohorts. RESULTS: Statistical evaluation confirmed a highly significant association of all analyzed SNP's with the occurrence of MI (p<0.0001; OR: 1.621-2.039). When only MI patients with a positive family disposition were comprised in the analysis a much stronger association of the accordant risk alleles with incident disease was found with odds ratios up to 2.769. CONCLUSIONS: The findings in the present study confirmed a strong association of the 9p21.3 locus with MI particularly in patients with a positive family history thereby, emphasizing the pathogenic relevance of this locus as a common genetic cardiovascular risk factor.

Genetic variants in the C-reactive protein gene are associated with microangiopathic ischemic stroke.

BACKGROUND AND PURPOSE: C-reactive protein (CRP) is an independent risk factor for cardiovascular disease and ischemic stroke. CRP serum levels are influenced by genetic variation in the CRP gene. Studies investigating the relationship between ischemic stroke and polymorphisms in the CRP gene produced equivocal results. Here we investigate single-nucleotide polymorphisms (SNPs) in the CRP gene in a large German ischemic stroke sample. METHODS: In a case-control design, 1,669 patients with ischemic stroke due to large-artery atherosclerosis, cardioembolism or cerebral microangiopathy were genotyped for 4 haplotype tagging SNPs (rs3093075, rs1205, rs1130864 and rs1800947) in the CRP gene which have been shown to influence CRP serum concentrations. Geographically matched controls were drawn from 2 prospective population-based studies, the Dortmund Health Study and the Study of Health in Pomerania. The genetic association between the SNPs and stroke was assessed using SNP and haplotype approaches. Results were adjusted for covariates by logistic regression. RESULTS: All 4 CRP SNPs reside in one linkage disequilibrium block. None of the SNPs or SNP haplotypes were associated with ischemic stroke as a whole. Three SNPs (rs3093075, rs1130864 and rs1800947) showed a significant association with microangiopathic stroke. A common 4-SNP haplotype was protective while 2 rarer haplotypes conferred susceptibility to microangiopathic stroke. All associations remained significant after adjustment for sex, age, hypertension, diabetes mellitus and hypercholesterolemia and after correction for multiple testing using the 'false discovery rate' method. CONCLUSION: Genetic variation in the CRP gene is associated with microangiopathic but not macroangiopathic or cardioembolic stroke in a large German stroke sample.

Genetic TPH2 variants and the susceptibility for migraine: association of a TPH2 haplotype with migraine without aura.

The serotonergic system plays a major role in the etiology of migraine. The rate-limiting enzyme in serotonin homeostasis and availability is tryptophan hydroxylase (TPH). The TPH2 isoform is responsible for the cerebral serotonin biosynthesis. To investigate the role of genetic variation in TPH2 in the pathogenesis of migraine eight haplotype tagging SNPs covering the whole TPH2 gene where chosen using Haploview and genotyped in 503 migraineurs and 515 healthy controls. Association analysis was performed on a single SNP and haplotype basis using χ² and logistic regression analysis. Single SNP analysis revealed a weak association with migraine, which did not remain after correction for multiple testing. Haplotype analyses revealed association of a haplotype with migraine without aura. Stratification by aura and triptan response did not reveal a positive association with the investigated polymorphisms. These results suggest a possible influence of genetic variation in TPH2 in the pathogenesis of migraine.

Nexilin mutations destabilize cardiac Z-disks and lead to dilated cardiomyopathy.

Z-disks, the mechanical integration sites of heart and skeletal muscle cells, link anchorage of myofilaments to force reception and processing. The key molecules that enable the Z-disk to persistently withstand the extreme mechanical forces during muscle contraction have not yet been identified. Here we isolated nexilin (encoded by NEXN) as a novel Z-disk protein. Loss of nexilin in zebrafish led to perturbed Z-disk stability and heart failure. To evaluate the role of nexilin in human heart failure, we performed a genetic association study on individuals with dilated cardiomyopathy and found several mutations in NEXN associated with the disease. Nexilin mutation carriers showed the same cardiac Z-disk pathology as observed in nexilin-deficient zebrafish. Expression in zebrafish of nexilin proteins encoded by NEXN mutant alleles induced Z-disk damage and heart failure, demonstrating a dominant-negative effect and confirming the disease-causing nature of these mutations. Increasing mechanical strain aggravated Z-disk damage in nexilin-deficient skeletal muscle, implying a unique role of nexilin in protecting Z-disks from mechanical trauma.

Variation in the human soluble epoxide hydrolase gene and risk of restenosis after percutaneous coronary intervention.

BACKGROUND: Restenosis represents the major limiting factor for the long-term efficacy of percutaneous coronary intervention (PCI). Several genetic factors involved in the regulation of the vascular system have been described to play a role in the pathogenesis of restenosis. We investigated whether the EPHX2 K55R polymorphism, previously linked to significantly higher risk for coronary heart disease (CHD), was associated with the occurrence of restenosis after PCI. The association with incident CHD should have been confirmed and a potential correlation of the EPHX2 K55R variant to an increased risk of hypertension was analysed. METHODS: An overall cohort of 706 patients was studied: This cohort comprised of 435 CHD patients who had undergone successful PCI. Follow-up coronary angiography in all patients was performed 6 months after intervention. Another 271 patients in whom CHD had been excluded by coronary angiography served as controls. From each patient EDTA-blood was drawn at the baseline ward round. Genomic DNA was extracted from these samples and genotyping was performed by real-time PCR and subsequent melting curve analysis. RESULTS: In CHD patients 6 month follow-up coronary angiography revealed a restenosis rate of 29.4%, classified as late lumen loss as well as lumen re-narrowing >or= 50%.Statistical analysis showed an equal genotype distribution in restenosis patients and non-restenosis patients (A/A 82.0% and A/G + G/G 18.0% versus A/A 82.1% and A/G + G/G 17.9%). Moreover, neither a significant difference in the genotype distribution of CHD patients and controls nor an association with increased risk of hypertension was found. CONCLUSION: The results of the present study indicate that the EPHX2 K55R polymorphism is not associated with restenosis after PCI, with incidence of CHD, or with an increased risk of hypertension and therefore, can not serve as a predictor for risk of CHD or restenosis after PCI.

Association of the protein Z ATG haplotype with symptomatic nonvascular stroke or thromboembolism in white children: a family-based cohort study.

To clarify the role of protein Z (PZ) in children with stroke/thromboembolism (TE), the present haplotype (HT)-based family study was performed. We genotyped 365 pediatric stroke/TE families (stroke n = 216; TE n = 149) for 4 single nucleotide polymorphisms (SNPs; rs3024718, rs3024731, rs3024772, and rs3024778) to assess the association between genetic variation within a conserved block of linkage disequilibrium harboring the PZ gene and pediatric TE. Association was assessed with use of the transmission disequilibrium test (TDT), corrected for multiple testing (permutation testing: HAPLOVIEW). In addition, PZ antigen was determined and correlated with carriership of PZ haplotypes and the FV G1691A mutation. Rs3024718, rs3024731, and rs3024772 are in tight linkage disequilibrium (LD) and define 4 haplotypes, capturing 97% of the genetic variation for this LD block. HT1 (ATG) was significantly overtransmitted from parents to affected offspring (HT frequency 73.5%, T:U 122:80, chi(2) = 8.791, P = .003). The ATG risk haplotype was significantly correlated with greater PZ antigen levels. Multivariate analysis adjusted for age, sex, established thrombophilias, smoking, fibrinogen, and PZ levels revealed a significant association of the ATG haplotype and TE in children (odds ratio [OR] 1.4; 95% confidence interval [95% CI] 1.08-1.93). Our results suggest that the ATG haplotype of the PZ gene is a genetic marker for symptomatic TE in white German children.