Fiona R. Green

Genetic Variants Associated with Lp(a) Lipoprotein Level and Coronary Disease

BACKGROUND An increased level of Lp(a) lipoprotein has been identified as a risk factor for coronary artery disease that is highly heritable. The genetic determinants of the Lp(a) lipoprotein level and their relevance for the risk of coronary disease are incompletely understood. METHODS We used a novel gene chip containing 48,742 single-nucleotide polymorphisms (SNPs) in 2100 candidate genes to test for associations in 3145 case subjects with coronary disease and 3352 control subjects. Replication was tested in three independent populations involving 4846 additional case subjects with coronary disease and 4594 control subjects. RESULTS Three chromosomal regions (6q26-27, 9p21, and 1p13) were strongly associated with the risk of coronary disease. The LPA locus on 6q26-27 encoding Lp(a) lipoprotein had the strongest association. We identified a common variant (rs10455872) at the LPA locus with an odds ratio for coronary disease of 1.70 (95% confidence interval [CI], 1.49 to 1.95) and another independent variant (rs3798220) with an odds ratio of 1.92 (95% CI, 1.48 to 2.49). Both variants were strongly associated with an increased level of Lp(a) lipoprotein, a reduced copy number in LPA (which determines the number of kringle IV-type 2 repeats), and a small Lp(a) lipoprotein size. Replication studies confirmed the effects of both variants on the Lp(a) lipoprotein level and the risk of coronary disease. A meta-analysis showed that with a genotype score involving both LPA SNPs, the odds ratios for coronary disease were 1.51 (95% CI, 1.38 to 1.66) for one variant and 2.57 (95% CI, 1.80 to 3.67) for two or more variants. After adjustment for the Lp(a) lipoprotein level, the association between the LPA genotype score and the risk of coronary disease was abolished. CONCLUSIONS We identified two LPA variants that were strongly associated with both an increased level of Lp(a) lipoprotein and an increased risk of coronary disease. Our findings provide support for a causal role of Lp(a) lipoprotein in coronary disease.

Genome-wide mapping of susceptibility to coronary artery disease identifies a novel replicated locus on chromosome 17.

Coronary artery disease (CAD) is a leading cause of death world-wide, and most cases have a complex, multifactorial aetiology that includes a substantial heritable component. Identification of new genes involved in CAD may inform pathogenesis and provide new therapeutic targets. The PROCARDIS study recruited 2,658 affected sibling pairs (ASPs) with onset of CAD before age 66 y from four European countries to map susceptibility loci for CAD. ASPs were defined as having CAD phenotype if both had CAD, or myocardial infarction (MI) phenotype if both had a MI. In a first study, involving a genome-wide linkage screen, tentative loci were mapped to Chromosomes 3 and 11 with the CAD phenotype (1,464 ASPs), and to Chromosome 17 with the MI phenotype (739 ASPs). In a second study, these loci were examined with a dense panel of grid-tightening markers in an independent set of families (1,194 CAD and 344 MI ASPs). This replication study showed a significant result on Chromosome 17 (MI phenotype; p = 0.009 after adjustment for three independent replication tests). An exclusion analysis suggests that further genes of effect size λsib > 1.24 are unlikely to exist in these populations of European ancestry. To our knowledge, this is the first genome-wide linkage analysis to map, and replicate, a CAD locus. The region on Chromosome 17 provides a compelling target within which to identify novel genes underlying CAD. Understanding the genetic aetiology of CAD may lead to novel preventative and/or therapeutic strategies.

Interaction between Single Nucleotide Polymorphisms in Selenoprotein P and Mitochondrial Superoxide Dismutase Determines Prostate Cancer Risk

Selenium may affect prostate cancer risk via its plasma carrier selenoprotein P which shows dramatically reduced expression in prostate cancer tumors and cell lines. The selenoprotein P (SEPP1) Ala234 single nucleotide polymorphism (SNP) allele is associated with lower plasma selenoprotein P in men, reducing the concentration/activity of other antioxidant selenoproteins. Selenium status also modifies the effect of the mitochondrial superoxide dismutase (SOD2) SNP Ala16Val on prostate cancer risk. We investigated the relationship of these SNPs with prostate cancer risk. DNA from 2,975 cases and 1,896 age-matched controls from the population-based Prostate Cancer in Sweden study were genotyped using TaqMan assays. Cases were designated aggressive or nonaggressive prostate cancers at diagnosis by clinical criteria. Association with prostate cancer was investigated by logistic regression; gene-gene interaction using a general linear model. The mean plasma selenium concentration measured in 169 controls was relatively low (76.0 +/- 17.2 microg/L). SNP genotype distributions were in Hardy-Weinberg equilibrium. SOD2-Ala16+ men were at a greater risk of prostate cancer [odds ratios (OR), 1.19; 95% confidence intervals (CI), 1.03-1.37] compared with SOD2-Val16 homozygotes. Men homozygous for SEPP1-Ala234 who were also SOD2-Ala16+ had a higher risk of prostate cancer (OR, 1.43; 95% CI, 1.17-1.76) and aggressive prostate cancer (OR, 1.60; 95% CI, 1.22-2.09) than those who were SOD2-Val16 homozygotes (interaction, prostate cancer P = 0.05; aggressive prostate cancer P = 0.01). This interaction was stronger in ever-smokers: SOD2-Ala16+ men homozygous for SEPP1-Ala234 had an almost doubled risk of prostate cancer (OR, 1.97; 95% CI, 1.33-2.91; interaction P = 0.001). In a low-selenium population, SOD2-Ala16+ men homozygous for SEPP1-Ala234 are at an increased risk of prostate cancer/aggressive prostate cancer especially if ever-smokers, because they are likely to produce more mitochondrial H(2)O(2) that they cannot remove, thereby promoting prostate tumor cell proliferation and migration.

Selenoprotein Gene Nomenclature

The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4, and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine sulfoxide reductase B1), and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15-kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV), and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing, and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.

Selenoprotein Gene Variants, Toenail Selenium Levels, and Risk for Advanced Prostate Cancer

Lower selenium levels have been associated with increased risk of prostate cancer (PCa), and genetic variation in the selenoprotein genes selenoprotein P (SEPP1) and glutathione peroxidase 1 (GPX1) is thought to modify this relationship. We investigated whether the association between toenail selenium levels and advanced PCa risk in the prospective Netherlands Cohort Study is modified by common genetic variation in SEPP1 and GPX1. Toenail clippings were used to determine selenium levels and to isolate DNA for genotyping. This case-cohort study, which included 817 case subjects with advanced PCa and 1048 subcohort members, was analyzed with Cox regression models. All statistical tests were two-sided. Three genetic variants were associated with advanced (stage III/IV or IV) PCa risk: SEPP1 rs7579 (lower risk; P trend = .01), GPX1 rs17650792 (higher risk; P trend = .03), and GPX1 rs1800668 (lower risk; P trend = .005). Toenail selenium levels were inversely associated with advanced PCa risk, independently of common genetic variation in SEPP1 and GPX1.

Heme Oxygenase (HO)-1 Induction Prevents Endoplasmic Reticulum Stress-Mediated Endothelial Cell Death and Impaired Angiogenic Capacity

Graphical abstract Figure. No Caption available. ABSTRACT Most of diabetic cardiovascular complications are attributed to endothelial dysfunction and impaired angiogenesis. Endoplasmic Reticulum (ER) and oxidative stresses were shown to play a pivotal role in the development of endothelial dysfunction in diabetes. Hemeoxygenase‐1 (HO‐1) was shown to protect against oxidative stress in diabetes; however, its role in alleviating ER stress‐induced endothelial dysfunction remains not fully elucidated. We aim here to test the protective role of HO‐1 against high glucose‐mediated ER stress and endothelial dysfunction and understand the underlying mechanisms with special emphasis on oxidative stress, inflammation and cell death. Human Umbilical Vein Endothelial Cells (HUVECs) were grown in either physiological or intermittent high concentrations of glucose for 5 days in the presence or absence of Cobalt (III) Protoporphyrin IX chloride (CoPP, HO‐1 inducer) or 4‐Phenyl Butyric Acid (PBA, ER stress inhibitor). Using an integrated cellular and molecular approach, we then assessed ER stress and inflammatory responses, in addition to apoptosis and angiogenic capacity in these cells. Our results show that HO‐1 induction prevented high glucose‐mediated increase of mRNA and protein expression of key ER stress markers. Cells incubated with high glucose exhibited high levels of oxidative stress, activation of major inflammatory and apoptotic responses [nuclear factor (NF)‐&kgr;B and c‐Jun N‐terminal kinase (JNK)] and increased rate of apoptosis; however, cells pre‐treated with CoPP or PBA were fully protected. In addition, high glucose enhanced caspases 3 and 7 cleavage and activity and augmented cleaved poly ADP ribose polymerase (PARP) expression whereas HO‐1 induction prevented these effects. Finally, HO‐1 induction and ER stress inhibition prevented high glucose‐induced reduction in NO release and impaired the angiogenic capacity of HUVECs, and enhanced vascular endothelial growth factor (VEGF)‐A expression. Altogether, we show here the critical role of ER stress‐mediated cell death in diabetes‐induced endothelial dysfunction and impaired angiogenesis and underscore the role of HO‐1 induction as a key therapeutic modulator for ER stress response in ischemic disorders and diabetes. Our results also highlight the complex interplay between ER stress response and oxidative stress.

Genetic risk markers for post-angioplasty restenosis: what should we expect?

Genetic risk markers for coronary artery disease and associated phenotypes, such as restenosis after angioplasty, have the potential to be valuable to the individual, but even more so in facilitating an understanding of causal factors in the disease, and thereby the development of novel preventative and therapeutic strategies. In this issue of Clinical Science, Völzke and co-workers were unable to show association of a panel of candidate polymorphisms with restenosis, but their study has highlighted the need for even larger studies, as well as the potential benefits of finding causal genetic variation.

183 Heme Oxygenase (HO)-1 Induction Prevents Endoplasmic Reticulum Stress-Mediated Endothelial Cell Death and Dysfunction

Diabetes is a widely spread metabolic disorder and is intimately associated with major micro- and macro-vascular complications. Several previous studies including ours highlight the complex interplay between Endoplasmic Reticulum (ER) stress and oxidative stress in the pathogenesis of cardiovascular complications associated with obesity and diabetes. Hemeoxygenase-1 (HO-1) induction has been shown to protect against oxidative stress in diabetes, however the underlying molecular mechanisms have not yet been fully elucidated. We aim in this project to test the hypothesis that HO-1 induction will protect against high glucose-mediated ER stress and oxidative stress in endothelial cells and will enhance cell survival. Endothelial cells, EA.hy926 cell line and Human Umbilical Vein Endothelial cells (HUVECs), were cultured in physiological or high concentrations of glucose to mimic diabetic environment in the presence of cobalt protoporphyrin IX (CoPP, HO-1 inducer), 4-phenylbutyrate (PBA, chemical chaperone to inhibit ER stress), Tauroursodeoxycholic acid (TUDCA, another chemical chaperone to inhibit ER stress) or vehicle. Then, ER stress response was assessed (PCR and western blot). The productions of reactive oxygen species (ROS) and Nitric Oxide (NO) were analysed by flow cytometer and Griess assay, respectively. Furthermore, we analysed apoptosis and caspase 3/7activity in endothelial cells. Expression of inflammatory cytokines was also assessed by PCR. High glucose treatment in endothelial cells induced an increase of mRNA expression of several ER stress response markers (BIP, CHOP, ATF4) in addition to an increase in ROS production and a reduction in NO release. Interestingly, the pre-treatment of cells with TUDCA/PBA or CoPP significantly reduced high glucose-mediated ER stress and oxidative stress in cells. In addition, endothelial cells incubated with high glucose exhibited enhanced cell death and increased caspase 3/7 activity while cells pre-treated with either PBA or CoPP were partially or totally protected. The mRNA expression of inflammatory cytokine IL-6 was significantly enhanced in cells incubated with high glucose while this was completely reversed in cells pre-treated with TUDCA or CoPP. Overall, these results highlight the importance of oxidative stress both in initiating or maintaining ER stress response and in mediating ER stress-induced damage and cell death in endothelial cells. This work also underscores the therapeutic potential of HO-1 induction against hyperglycaemia-mediated endothelial dysfunction.

GENETICS OF INTERLEUKIN 6 AND SELENOPROTEIN S THAT HAVE A ROLE IN INFLAMMATION AND CORONARY ARTERY DISEASE

Coronary artery disease (CAD) is inflammatory and caused by genetic/environmental factors. Interleukin (IL) 6 is a pro-inflammatory cytokine implicated in CAD. Selenoprotein S (SelS) has recently been implicated in inflammation and endoplasmic reticulum (ER) stress. Several IL-6 and SelS polymorphisms are associated with increased CAD risk and the aim of this project is to investigate in vitro the mechanisms underlying this. Flow cytometry was used to assess the surface expression of the macrophage differentiation-specific markers CD14 and CD11c, and IL-6r (CD126) by PBMC derived macrophages. mRNA levels were determined by quantitative PCR (QPCR) in lipopolysaccharide (LPS) and IL-1β-stimulated THP-1 cells and in peripheral blood mononuclear cell (PBMC)-derived macrophages from healthy donors. CD11c-hi, CD14-lo expression on macrophage-like versus monocyte-like PBMCs confirmed their differentiation state. Neither differentiation state nor inflammatory stimulation affected expression of IL-6 or SelS in THP-1 or PBMCs. IL-6 was significantly upregulated in LPS-stimulated THP-1 and PBMC-derived macrophages, consistent with the literature. SelS was upregulated by LPS only in differentiated THP-1 cells. ER stress proteins CHOP (C/EBP homologous protein) and BiP (IgH chain binding protein) were not induced by IL-1β or LPS. CD126 (IL-6r) expression was very low on PBMC-derived macrophages, suggesting that classical IL-6 signalling may not occur in these PBMCs. In conclusion, this PBMC model of inflammation has been validated, and suggests a lack of classical IL-6 signalling; further analysis by IL-6 and SelS genotype will reveal underlying molecular mechanisms of CAD risk association.

TILING ARRAY SHOWS LOW ANRIL, HIGH CDKN2B EXPRESSION ASSOCIATED WITH CHROMOSOME 9P21 CORONARY ARTERY DISEASE (CAD) RISK GENOTYPE

Recent studies suggest that ANRIL expression mediates susceptibility to CAD1 via CDKN2B.2 We used fluorescently-labelled whole-blood RNA, from 20 healthy volunteers genotyped for the CAD-risk-SNP rs2891168, to probe custom-designed Agilent tiling expression microarrays. Raw data were normalized to probe GC content and housekeeping genes. We found that ANRIL exons 1–4 were more abundantly expressed in blood than 5–20, with exons 6, 8, 9, 20 showing low expression. We derived a set of “training” tiling probes from HUVEC cells in which ANRIL expression was attenuated using siRNA against exons 13 and 19. ANRIL expression (probes in exons 5,6,8,13,16,18,19) was reduced by at least 50% and CDKN2B expression (probes in exons 1,2) increased, with no effect on CDKN2A. We confirmed these data using real-time QPCR. Using the tiling probe “training-set”, a probe in exon 16 of ANRIL showed a CAD genotype-specific difference in expression (p<0.001), with the risk-allele lower, and probes in exon 2 of CDKN2B showed higher expression for the risk-allele (p<0.05 and p<0.01). In conclusion, we found by a novel technique that ANRIL expression in whole-blood was CAD-risk-genotype-specific, and confirmed that low ANRIL was correlated with higher CDKN2B, but not CDKN2A expression. These data support recent studies implicating reduced ANRIL expression and reciprocal over-expression of CDKN2B, which is thought to impair TGFβ signalling, in CAD susceptibility.