Family-based association analysis with ordered categorical phenotypes, covariates and interactions

Genetic association analyses of family-based studies with ordered categorical phenotypes are often conducted using methods either for quantitative or for binary traits, which can lead to suboptimal analyses. Here we present an alternative likelihood-based method of analysis for single nucleotide polymorphism (SNP) genotypes and ordered categorical phenotypes in nuclear families of any size. Our approach, which extends our previous work for binary phenotypes, permits straightforward inclusion of covariate, gene-gene and gene-covariate interaction terms in the likelihood, incorporates a simple model for ascertainment and allows for family-specific effects in the hypothesis test. Additionally, our method produces interpretable parameter estimates and valid confidence intervals. We assess the proposed method using simulated data, and apply it to a polymorphism in the c-reactive protein (CRP) gene typed in families collected to investigate human systemic lupus erythematosus. By including sex interactions in the analysis, we show that the polymorphism is associated with anti-nuclear autoantibody (ANA) production in females, while there appears to be no effect in males.

A functional genomics approach reveals novel quantitative trait loci associated with platelet signalling pathways

Platelet response to activation varies widely between individuals but shows interindividual consistency and strong heritability. The genetic basis of this variation has not been properly explored. We therefore systematically measured the effect on function of sequence variation in 97 candidate genes in the collagen and adenosine-diphosphate (ADP) signaling pathways. Resequencing of the genes in 48 European DNA samples nearly doubled the number of known single nucleotide polymorphisms (SNPs) and informed the selection of 1327 SNPs for genotyping in 500 healthy Northern European subjects with known platelet responses to collagen-related peptide (CRP-XL) and ADP. This identified 17 novel associations with platelet function (P < .005) accounting for approximately 46% of the variation in response. Further investigations with platelets of known genotype explored the mechanisms behind some of the associations. SNPs in PEAR1 associated with increased platelet response to CRP-XL and increased PEAR1 protein expression after platelet degranulation. The minor allele of a 3' untranslated region (UTR) SNP (rs2769668) in VAV3 was associated with higher protein expression (P = .03) and increased P-selectin exposure after ADP activation (P = .004). Furthermore the minor allele of the intronic SNP rs17786144 in ITPR1 modified Ca2+ levels after activation with ADP (P < .004). These data provide novel insights into key hubs within platelet signaling networks.

PECAM-1 expression and activity negatively regulate multiple platelet signaling pathways

Platelet endothelial cell adhesion molecule-1 (PECAM-1) inhibits platelet response to collagen and may also inhibit two other major platelet agonists ADP and thrombin although this has been less well explored. We hypothesized that the combined effect of inhibiting these three platelet activating pathways may act to significantly inhibit thrombus formation. We demonstrate a negative relationship between PECAM-1 surface expression and platelet response to cross-linked collagen related peptide (CRP-XL) and ADP, and an inhibitory effect of PECAM-1 clustering on platelet response to CRP-XL, ADP and thrombin. This combined inhibition of multiple signaling pathways results in a marked reduction in thrombus formation. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Platelet 12-lipoxygenase activation via glycoprotein VI: involvement of multiple signaling pathways in agonist control of H(P)ETE synthesis

Lipoxygenases (LOX) contribute to vascular disease and inflammation through generation of bioactive lipids, including 12-hydro(pero xyeicosatetraenoic acid (12-H(P)ETE). The physiological mechanisms that acutely control LOX product generation in mammalian cells are uncharacterized. Human platelets that contain a 12-LOX isoform (p12-LOX) were used to define pathways that activate H(P)ETE synthesis in the vasculature. Collagen and collagen-related peptide (CRP) (1 to 10 g/mL) acutely induced platelet 12-H(P)ETE synthesis. This implicated the collagen receptor glycoprotein VI (GPVI), which signals via the immunoreceptor-based activatory motif (ITAM)- containing FcR chain. Conversely, thrombin only activated at high concentrations ( 0.2 U/mL), whereas U46619 and ADP alone were ineffective. Collagen or CRP-stimulated 12-H(P)ETE generation was inhibited by staurosporine, PP2, wortmannin, BAPTA/AM, EGTA, and L-655238, implicating src-tyrosine kinases, PI3-kinase, Ca2 mobilization, and p12-LOX translocation. In contrast, protein kinase C (PKC) inhibition potentiated 12-H(P)ETE generation. Finally, activation of the immunoreceptor tyrosine-based inhibitory motif (ITIM)– containing platelet endothelial cell adhesion molecule (PECAM-1) inhibited p12-LOX product generation. This study characterizes a receptor-dependent pathway for 12-H(P)ETE synthesis via the collagen receptor GPVI, which is negatively regulated by PECAM-1 and PKC, and demonstrates a novel link between immune receptor signaling and lipid mediator generation in the vasculature. (Circ Res. 2004;94:1598-1605.)

Platelet adhesion to collagen and collagen-related peptide under flow. Roles of the alpha(2)beta(1) integrin, GPVI, and Src tyrosine kinases

Objective - Platelet stimulation by collagen and collagen-related peptides (CRPs) is associated with activation of protein tyrosine kinases. In the present study, we investigated the role of Src family tyrosine kinases in the initial adhesion events of human platelets to collagen and cross-linked CRP. Methods and Results - Under arterial flow conditions, a glycoprotein VI - specific substrate, cross-linked CRP, caused rapid (<2 second) platelet retention and protein tyrosine phosphorylation that were markedly decreased by the Src family kinase inhibitor pyrozolopyrimidine (PP2) or by aggregation inhibitor GRGDSP. CRP-induced platelet retention was transient, and 90% of single platelets or aggregates detached within seconds. PP2, although having no effect on RGD peptide-binding to CRP, completely blocked aggregation and tyrosine phosphorylation of Syk and phospholipase Cγ2 (PLCγ2). In contrast, PP2 weakly (<30%) suppressed firm adhesion to collagen mediated primarily by the alpha(2)beta(1) integrin. Although PP2 prevented activation of Syk and PLCgamma2 in collagen-adherent platelets, tyrosine phosphorylation of several unidentified protein bands persisted, as did autophosphorylation of pp125(FAK). Conclusions - These findings indicate that activation of Src-tyrosine kinases Syk and PLCgamma2 is not required for the initial stable attachment of human platelets to collagen and for FAK autophosphorylation. However, Src-tyrosine kinases are critical for glycoprotein VI - mediated signaling leading to platelet aggregation.

Influence of apolipoprotein E genotype and dietary alpha-tocopherol on redox status and C-reactive protein levels in apolipoprotein E3 and E4 targeted replacement mice

The molecular basis of the positive association between apoE4 genotype and CVD remains unclear. There is direct in vitro evidence indicating that apoE4 is a poorer antioxidant relative to the apoE3 isoform, with some indirect in vivo evidence also available. Therefore it was hypothesised that apoE4 carriers may benefit from alpha-tocopherol (alpha-Toc) supplementation. Targeted replacement mice expressing the human apoE3 and apoE4 were fed with a diet poor (0 mg/kg diet) or rich (200 mg/kg diet) in alpha-Toc for 12 weeks. Neither apoE genotype nor dietary alpha-Toc exerted any effects on the antioxidant defence system, including glutathione, catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase activities. In addition, no differences were observed in mitogen-induced lymphocyte proliferation. alpha-Toc concentrations were modestly higher in plasma and lower in tissues of apoE4 compared with apoE3 mice, with the greatest differences evident in the lung, suggesting that an apoE4 genotype may reduce alpha-Toc delivery to tissues. A tendency towards increased plasma F-2-isoprostanes in apoE4 mice was observed, while liver thiobarbituric acid-reactive substances did not differ between apoE3 and apoE4 mice. In addition, C-reactive protein (CRP) concentrations were reduced in apoE4 mice indicating that this positive effect on CRP may in part negate the increased CVD risk associated with an apoE4 genotype.

Soy-isoflavone-enriched foods and inflammatory biomarkers of cardiovascular disease risk in postmenopausal women: interactions with genotype and equol production

Background: Dietary isoflavones are thought to be cardioprotective because of their structural similarity to estrogen. The reduction of concentrations of circulating inflammatory markers by estrogen may be one of the mechanisms by which premenopausal women are protected against cardiovascular disease. Objective: Our aim was to investigate the effects of isolated soy isoflavones on inflammatory biomarkers [von Willebrand factor, intracellular adhesion molecule 1, vascular cell adhesion molecule 1 (VCAM-1), E-selectin, monocyte chemoattractant protein 1, C-reactive protein (CRP), and endothelin 1 concentrations]. Differences with respect to single-nucleotide polymorphisms in selected genes [estrogen receptor alpha (XbaI and PvuII), estrogen receptor beta [ER beta (AluI) and ER beta[cx] (Tsp5091), endothelial nitric oxide synthase (Glu298Asp), apolipoprotein E (Apo E2, E3, and E4), and cholesteryl ester transfer protein (TaqIB)] and equol production were investigated. Design: One hundred seventeen healthy European postmenopausal women participated in this randomized, double-blind, placebo-controlled, crossover dietary intervention trial. Isoflavone-enriched (genistein-to-daidzein ratio of 2:1;50 mg/d) or placebo cereal bars were consumed for 8 wk, with a washout period of 8 wk between the crossover. Plasma inflammatory factors were measured at 0 and 8 wk of each study arm. Results: Isoflavones improved CRP concentrations [odds ratio (95% Cl) for CRP values >1 mg/L for isoflavone compared with placebo: 0.43 (0.27, 0.69)]; no significant effects of isoflavone treatment on other plasma inflammatory markers were observed. No significant differences in the response to isoflavones were observed according to subgroups of equol production. Differences in the VCAM-1 response to isoflavones and to placebo were found with ER beta AluI genotypes. Conclusion: Isoflavones have beneficial effects on CRP concentrations, but not on other inflammatory biomarkers of cardiovascular disease risk in postmenopausal women, and may improve VCAM-1 in an ER beta gene polymorphic subgroup.

Targeting C-reactive protein for the treatment of cardiovascular disease

Complement-mediated inflammation exacerbates the tissue injury of ischaemic necrosis in heart attacks and strokes, the most common causes of death in developed countries. Large infarct size increases immediate morbidity and mortality and, in survivors of the acute event, larger non-functional scars adversely affect long-term prognosis. There is thus an important unmet medical need for new cardioprotective and neuroprotective treatments. We have previously shown that human C-reactive protein (CRP), the classical acute-phase protein that binds to ligands exposed in damaged tissue and then activates complement(1), increases myocardial and cerebral infarct size in rats subjected to coronary or cerebral artery ligation, respectively(2,3). Rat CRP does not activate rat complement, whereas human CRP activates both rat and human complement(4). Administration of human CRP to rats is thus an excellent model for the actions of endogenous human CRP2,3. Here we report the design, synthesis and efficacy of 1,6-bis(phosphocholine)-hexane as a specific small-molecule inhibitor of CRP. Five molecules of this palindromic compound are bound by two pentameric CRP molecules, crosslinking and occluding the ligand-binding B-face of CRP and blocking its functions. Administration of 1,6-bis(phosphocholine)-hexane to rats undergoing acute myocardial infarction abrogated the increase in infarct size and cardiac dysfunction produced by injection of human CRP. Therapeutic inhibition of CRP is thus a promising new approach to cardioprotection in acute myocardial infarction, and may also provide neuroprotection in stroke. Potential wider applications include other inflammatory, infective and tissue-damaging conditions characterized by increased CRP production, in which binding of CRP to exposed ligands in damaged cells may lead to complement-mediated exacerbation of tissue injury.

Effects of dietary fat modification on oxidative stress and inflammatory markers in the LIPGENE study.

Subjects with the metabolic syndrome (MetS) have enhanced oxidative stress and inflammation. Dietary fat quality has been proposed to be implicated in these conditions. We investigated the impact of four diets distinct in fat quantity and quality on 8-iso-PGF2α (a major F2-isoprostane and oxidative stress indicator), 15-keto-13,14-dihydro-PGF2α (15-keto-dihydro-PGF2α, a major PGF2α metabolite and marker of cyclooxygenase-mediated inflammation) and C-reactive protein (CRP). In a 12-week parallel multicentre dietary intervention study (LIPGENE), 417 volunteers with the MetS were randomly assigned to one of the four diets: two high-fat diets (38 % energy (%E)) rich in SFA or MUFA and two low-fat high-complex carbohydrate diets (28 %E) with (LFHCC n-3) or without (LFHCC) 1·24 g/d of very long chain n-3 fatty acid supplementation. Urinary levels of 8-iso-PGF2α and 15-keto-dihydro-PGF2α were determined by RIA and adjusted for urinary creatinine levels. Serum concentration of CRP was measured by ELISA. Neither concentrations of 8-iso-PGF2α and 15-keto-dihydro-PGF2α nor those of CRP differed between diet groups at baseline (P>0·07) or at the end of the study (P>0·44). Also, no differences in changes of the markers were observed between the diet groups (8-iso-PGF2α, P = 0·83; 15-keto-dihydro-PGF2α, P = 0·45; and CRP, P = 0·97). In conclusion, a 12-week dietary fat modification did not affect the investigated markers of oxidative stress and inflammation among subjects with the MetS in the LIPGENE study.

Glucokinase Regulatory Protein genetic variant interacts with omega-3 PUFA to influence insulin resistance and inflammation in metabolic syndrome

Glucokinase Regulatory Protein (GCKR) plays a central role regulating both hepatic triglyceride and glucose metabolism. Fatty acids are key metabolic regulators, which interact with genetic factors and influence glucose metabolism and other metabolic traits. Omega-3 polyunsaturated fatty acids (n-3 PUFA) have been of considerable interest, due to their potential to reduce metabolic syndrome (MetS) risk. Objective To examine whether genetic variability at the GCKR gene locus was associated with the degree of insulin resistance, plasma concentrations of C-reactive protein (CRP) and n-3 PUFA in MetS subjects. Design Homeostasis model assessment of insulin resistance (HOMA-IR), HOMA-B, plasma concentrations of C-peptide, CRP, fatty acid composition and the GCKR rs1260326-P446L polymorphism, were determined in a cross-sectional analysis of 379 subjects with MetS participating in the LIPGENE dietary cohort. Results Among subjects with n-3 PUFA levels below the population median, carriers of the common C/C genotype had higher plasma concentrations of fasting insulin (P = 0.019), C-peptide (P = 0.004), HOMA-IR (P = 0.008) and CRP (P = 0.032) as compared with subjects carrying the minor T-allele (Leu446). In contrast, homozygous C/C carriers with n-3 PUFA levels above the median showed lower plasma concentrations of fasting insulin, peptide C, HOMA-IR and CRP, as compared with individuals with the T-allele. Conclusions We have demonstrated a significant interaction between the GCKR rs1260326-P446L polymorphism and plasma n-3 PUFA levels modulating insulin resistance and inflammatory markers in MetS subjects. Further studies are needed to confirm this gene-diet interaction in the general population and whether targeted dietary recommendations can prevent MetS in genetically susceptible individuals.

NAADP regulates human platelet function.

Platelets play a vital role in maintaining haemostasis. Human platelet activation depends on Ca2+ release, leading to cell activation, granule secretion and aggregation. NAADP (nicotinic acid-adenine dinucleotide phosphate) is a Ca2+-releasing second messenger that acts on acidic Ca2+ stores and is used by a number of mammalian systems. In human platelets, NAADP has been shown to release Ca2+ in permeabilized human platelets and contribute to thrombin-mediated platelet activation. In the present study, we have further characterized NAADP-mediated Ca2+ release in human platelets in response to both thrombin and the GPVI (glycoprotein VI)-specific agonist CRP (collagen-related peptide). Using a radioligand-binding assay, we reveal an NAADP-binding site in human platelets, indicative of a platelet NAADP receptor. We also found that NAADP releases loaded 45Ca2+ from intracellular stores and that total platelet Ca2+ release is inhibited by the proton ionophore nigericin. Ned-19, a novel cell-permeant NAADP receptor antagonist, competes for the NAADP-binding site in platelets and can inhibit both thrombin- and CRP-induced Ca2+ release in human platelets. Ned-19 has an inhibitory effect on platelet aggregation, secretion and spreading. In addition, Ned-19 extends the clotting time in whole-blood samples. We conclude that NAADP plays an important role in human platelet function. Furthermore, the development of Ned-19 as an NAADP receptor antagonist provides a potential avenue for platelet-targeted therapy and the regulation of thrombosis.

Rhinocetin, a venom-derived integrin-specific antagonist inhibits collagen-induced platelet and endothelial cell functions

Snaclecs are small non-enzymatic proteins present in viper venoms reported to modulate haemostasis of victims through effects on platelets, vascular endothelial and smooth muscle cells. In this study, we have isolated and functionally characterised a snaclec which we named rhinocetin from the venom of West African gaboon viper, Bitis gabonica rhinoceros. Rhinocetin was shown to comprise α and β chains with the molecular masses of 13.5 and 13kDa respectively. Sequence and immunoblot analysis of rhinocetin confirmed this to be a novel snaclec. Rhinocetin inhibited collagen-stimulated activation of human platelets in dose dependent manner, but displayed no inhibitory effects on glycoprotein VI (collagen receptor) selective agonist, CRP-XL-, ADP- or thrombin-induced platelet activation. Rhinocetin antagonised the binding of monoclonal antibodies against the α2 subunit of integrin α2β1 to platelets and coimmunoprecipitation analysis confirmed integrin α2β1 as a target for this venom protein. Rhinocetin inhibited a range of collagen induced platelet functions such as fibrinogen binding, calcium mobilisation, granule secretion, aggregation and thrombus formation. It also inhibited integrin α2β1 dependent functions of human endothelial cells. Together, our data suggest rhinocetin to be a modulator of integrin α2β1 function and thus may provide valuable insights into the role of this integrin in physiological and pathophysiological scenarios including haemostasis, thrombosis and envenomation.

Obesity and body fat classification in the metabolic syndrome: impact on cardiometabolic risk metabotype

Obesity is a key factor in the development of the metabolic syndrome (MetS), which is associated with increased cardiometabolic risk. We investigated whether obesity classification by body mass index (BMI) and body fat percentage (BF%) influences cardiometabolic profile and dietary responsiveness in 486 MetS subjects (LIPGENE dietary intervention study). Anthropometric measures, markers of inflammation and glucose metabolism, lipid profiles, adhesion molecules and haemostatic factors were determined at baseline and after 12 weeks of 4 dietary interventions (high saturated fat (SFA), high monounsaturated fat (MUFA) and 2 low fat high complex carbohydrate (LFHCC) diets, 1 supplemented with long chain n-3 polyunsaturated fatty acids (LC n-3 PUFAs)). 39% and 87% of subjects classified as normal and overweight by BMI were obese according to their BF%. Individuals classified as obese by BMI (± 30 kg/m2) and BF% (± 25% (men) and ± 35% (women)) (OO, n = 284) had larger waist and hip measurements, higher BMI and were heavier (P < 0.001) than those classified as non-obese by BMI but obese by BF% (NOO, n = 92). OO individuals displayed a more pro-inflammatory (higher C reactive protein (CRP) and leptin), pro-thrombotic (higher plasminogen activator inhibitor-1 (PAI-1)), pro-atherogenic (higher leptin/adiponectin ratio) and more insulin resistant (higher HOMA-IR) metabolic profile relative to the NOO group (P < 0.001). Interestingly, tumour necrosis factor alpha (TNF-α) concentrations were lower post-intervention in NOO individuals compared to OO subjects (P < 0.001). In conclusion, assessing BF% and BMI as part of a metabotype may help identify individuals at greater cardiometabolic risk than BMI alone.

Collagen or collagen-related peptide cause (Ca2+)i elevation and increased tyrosine phosphorylation in human megakaryocytes.

Since megakaryocytes are the cellular precursors of platelets we have investigated whether they share responses to platelet agonists, in particular collagen. Although previous studies have reported responses to thrombin in non-human megakaryocytes, through studies of single cell calcium responses and protein tyrosine-phosphorylation we demonstrate for the first time that both isolated human megakaryocytes and CD41/61-positive megakaryocytes derived in culture from CD34+ cells share responses to the platelet agonists collagen, collagen-related peptide and thrombin. The responses to either collagen or CRP were seen only in the most mature megakaryocytes and not in megakaryocyte-like cell lines, suggesting that the response to collagen is a characteristic developed late during megakaryocyte differentiation. These primary cells offer the opportunity to use many molecular and cellular techniques to study and manipulate signalling events in response to platelet receptor agonists, which cannot be performed in the small, anucleate platelet itself.

A collagen-like peptide stimulates tyrosine phosphorylation of syk and phospholipase C gamma2 in platelets independent of the integrin alpha2beta1.

Activation of platelets by collagen is mediated through a tyrosine kinase-dependent pathway that is associated with phosphorylation of the Fc receptor gamma chain, the tyrosine kinase syk, and phospholipase C gamma2 (PLC gamma2). We recently described a collagen-related triple-helical peptide (CRP) with the sequence GCP*(GPP*)GCP*G (single letter amino acid code: P* = hydroxyproline; Morton et al, Biochem J306:337, 1995). The cross-linked peptide is a potent stimulus of platelet activation but, unlike collagen, does not support alpha2beta1-mediated, Mg2+-dependent adhesion, suggesting that its action is independent of the integrin alpha2beta1. This finding suggests the existence of a platelet receptor other than alpha2beta1 that underlies activation. In the present study, we show that CRP stimulates tyrosine phosphorylation of the same pattern of proteins in platelets as collagen, including syk and PLC gamma2. Protein tyrosine phosphorylation induced by CRP is not altered in the absence of Mg2+ or the presence of monoclonal antibodies (MoAbs) to the integrin alpha2beta1 (MoAb 6F1 and MoAb 13), conditions that prevent the interaction of collagen with the integrin. In contrast, phosphorylation of syk and PLC gamma2 by collagen is partially reduced by MoAb 6F1 and MoAb 13 or by removal of Mg2+. This may reflect a direct role of alpha2beta1 in collagen-induced signaling events or an indirect role in which the integrin facilitates the binding of collagen to its signaling receptor. The results show an alpha2beta1-independent pathway of platelet activation by CRP that involves phosphorylation of syk and PLC gamma2. This pathway appears to contribute to platelet activation by collagen.