Paraoxonase 1 (PON1) attenuates diabetes development in mice through its antioxidative properties

Paraoxonase 1 (PON1) is a lipo-lactonase which is associated with HDL and possesses antioxidative properties. Diabetes is characterized by increased oxidative stress and by decreased PON1 activity. We aimed to analyze whether oxidative status and PON1 levels in mouse sera and macrophages could affect streptozotocin (STZ)-induced diabetes development. We have used two models of mice under low oxidative stress: STZ-injected apolipoprotein E-deficient mice supplemented with the antioxidant vitamin E, and P47(phox) knockout mice. In both mice models the decreased serum basal oxidative stress, was associated with a decreased rate of diabetes development, compared with control STZ-injected apolipoprotein E-deficient mice or with C57BL mice respectively. These data suggest that oxidative stress accelerates diabetes development. Next, we analyzed the effect of PON1 on macrophage oxidative stress and on diabetes development in STZ-injected C57BL mice, PON1 knockout mice, and PON1 transgenic mice. PON1 overexpression was associated with decreased diabetes-induced macrophage oxidative stress, decreased diabetes development, and decreased mortality, in comparison to C57BL mice, and even more so when compared to PON1KO mice. We thus concluded that on increasing PON1 expression in mice, diabetes development is attenuated, a phenomenon which could be attributed to the antioxidative properties of PON1, as decrement of oxidative stress significantly attenuated STZ-induced diabetes development.     

Paraoxonase 1 (PON1) status and risk of insecticide exposure

© 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:182–183, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20079     

Beclin 1 deficiency is associated with increased hypoxia-induced angiogenesis

Beclin 1, a tumor suppressor protein, acts as an initiator of autophagy in mammals. Heterozygous disruption of Beclin 1 accelerates tumor growth, but the underlying mechanisms remain unclear. We examined the role of Beclin 1 in tumor proliferation and angiogenesis, using a primary mouse melanoma tumor model. Beclin 1 (Becn1^+/-) hemizygous mice displayed an aggressive tumor growth phenotype with increased angiogenesis under hypoxia, associated with enhanced levels of circulating erythropoietin but not vascular endothelial growth factor, relative to wild-type mice. Using in vivo and ex vivo assays, we demonstrated increased angiogenic activity in Becn1^+/- mice relative to wild-type mice. Endothelial cells from Becn1^+/- mice displayed increased proliferation, migration and tube formation in response to hypoxia relative to wild-type cells. Moreover, Becn1^+/- cells subjected to hypoxia displayed increased hypoxia-inducible factor-2α (HIF-2α) expression relative to HIF-1α. Genetic interference of HIF-2α but not HIF-1α, dramatically reduced hypoxia-inducible proliferation, migration and tube formation in Becn1^+/- endothelial cells. We demonstrated that mice deficient in the autophagic protein Beclin 1 display a pro-angiogenic phenotype associated with the upregulation of HIF-2α and increased erythropoietin production. These results suggest a relationship between Beclin 1 and the regulation of angiogenesis, with implications in tumor growth and development.     

Beclin 1 deficiency is associated with increased hypoxia-induced angiogenesis

Beclin 1, a tumor suppressor protein, acts as an initiator of autophagy in mammals. Heterozygous disruption of Beclin 1 accelerates tumor growth, but the underlying mechanisms remain unclear. We examined the role of Beclin 1 in tumor proliferation and angiogenesis, using a primary mouse melanoma tumor model. Beclin 1 (Becn1 (+/-) ) hemizygous mice displayed an aggressive tumor growth phenotype with increased angiogenesis under hypoxia, associated with enhanced levels of circulating erythropoietin but not vascular endothelial growth factor, relative to wild-type mice. Using in vivo and ex vivo assays, we demonstrated increased angiogenic activity in Becn1 (+/-) mice relative to wild-type mice. Endothelial cells from Becn1 (+/-) mice displayed increased proliferation, migration and tube formation in response to hypoxia relative to wild-type cells. Moreover, Becn1 (+/-) cells subjected to hypoxia displayed increased hypoxia-inducible factor-2α (HIF-2α) expression relative to HIF-1α. Genetic interference of HIF-2α but not HIF-1α, dramatically reduced hypoxia-inducible proliferation, migration and tube formation in Becn1 (+/-) endothelial cells. We demonstrated that mice deficient in the autophagic protein Beclin 1 display a pro-angiogenic phenotype associated with the upregulation of HIF-2α and increased erythropoietin production. These results suggest a relationship between Beclin 1 and the regulation of angiogenesis, with implications in tumor growth and development.     

A mutation in the HFE gene is associated with altered brain iron profiles and increased oxidative stress in mice

Because of the increasing evidence that H63D HFE polymorphism appears in higher frequency in neurodegenerative diseases, we evaluated the neurological consequences of H63D HFE in vivo using mice that carry H67D HFE (homologous to human H63D). Although total brain iron concentration did not change significantly in the H67D mice, brain iron management proteins expressions were altered significantly. The 6-month-old H67D mice had increased HFE and H-ferritin expression. At 12 months, H67D mice had increased H- and L-ferritin but decreased transferrin expression suggesting increased iron storage and decreased iron mobilization. Increased L-ferritin positive microglia in H67D mice suggests that microglia increase iron storage to maintain brain iron homeostasis. The 6-month-old H67D mice had increased levels of GFAP, increased oxidatively modified protein levels, and increased cystine/glutamate antiporter (xCT) and hemeoxygenase-1 (HO-1) expression indicating increased metabolic and oxidative stress. By 12 months, there was no longer increased astrogliosis or oxidative stress. The decrease in oxidative stress at 12 months could be related to an adaptive response by nuclear factor E2-related factor 2 (Nrf2) that regulates antioxidant enzymes expression and is increased in the H67D mice. These findings demonstrate that the H63D HFE impacts brain iron homeostasis, and promotes an environment of oxidative stress and induction of adaptive mechanisms. These data, along with literature reports on humans with HFE mutations provide the evidence to overturn the traditional paradigm that the brain is protected from HFE mutations. The H67D knock-in mouse can be used as a model to evaluate how the H63D HFE mutation contributes to neurodegenerative diseases.     

Injection of paraoxonase 1 (PON1) to mice stimulates their HDL and macrophage antiatherogenicity

We analyzed, for the first time, the effects of recombinant PON1 (rePON1) intraperitoneal injection to C??BL/6 mice on their HDL and macrophage antiatherogenic properties. Thioglycolate-treated mice were injected with either saline (Control), or rePON1 (50 μg/mouse), and 20 H post injection, their blood samples and peritoneal macrophages (MPM) were collected. A significant increase in serum and HDL-PON1 arylesterase and lactonase activities was noted. Similarly, a significant increment, by 3.8 and 2.8 fold, in MPM-PON1 arylesterase and lactonase activities, respectively, as compared to the activities in control MPM was observed. The HDL from rePON1-injected mice was resistant to oxidation by copper ions as compared to control HDL. Furthermore, enrichment of the mouse HDL with rePON1 increased its ability to induce cholesterol efflux from J774A.1 macrophage cell line, and to inhibit macrophage-mediated LDL oxidation. In MPM from rePON1-injected mice vs. control MPM, there was a significant reduction in cholesterol mass, by 42%, in association with inhibition in cellular cholesterol biosynthesis rate, by 33%, and with significant stimulation, by 65%, of human HDL-mediated cholesterol efflux from the cells. We conclude that rePON1 injection to mice improved the mice HDL and MPM antiatherogenic properties, and these effects could probably lead to attenuation of atherosclerosis development.     

Paraoxonase (PON 1) as a biomarker of susceptibility for organophosphate toxicity

Paraoxonase (PON1) is an A-esterase capable of hydrolysing the active metabolites (oxons) of a number of organophosphorus (OP) insecticides such as parathion, diazinon and chlorpyrifos. PON1 activity is highest in liver and plasma, and among animal species significant differences exist, with birds and rabbits displaying very low and high activity, respectively. Human PON1 has two polymorphisms in the coding region (Q192R and L55M) and five polymorphisms in the promoter region. The Q192R polymorphism imparts different catalytic activity toward some OP substrates, while the polymorphism at position -108 (C/T) is the major contributor to differences in the level of PON1 expression. Animal studies have shown that PON1 is an important determinant of OP toxicity, with animal species with a low PON1 activity having an increased sensitivity to OPs. Administration of exogenous PON1 to rats or mice protects them from the toxicity of OPs. PON1 knockout mice display a high sensitivity to the toxicity of diazoxon and chlorpyrifos oxon, but not paraoxon. In vitro assayed catalytic efficiencies of purified PON₁₉₂ isoforms for hydrolysis of specific oxon substrates accurately predict the degree of in vivo protection afforded by each isoform. Low PON1 activity may also contribute to the higher sensitivity of newborns to OP toxicity.     

Prdx1 deficiency in mice promotes tissue specific loss of heterozygosity mediated by deficiency in DNA repair and increased oxidative stress

The loss of the H(2)O(2) scavenger protein encoded by Prdx1 in mice leads to an elevation of reactive oxygen species (ROS) and tumorigenesis of different tissues. Loss of heterozygosity (LOH) mutations could initiate tumorigenesis through loss of tumor suppressor gene function in heterozygous somatic cells. A connection between the severity of ROS and the frequency of LOH mutations in vivo has not been established. Therefore, in this study, we characterized in vivo LOH in ear fibroblasts and splenic T cells of 3-4 month old Prdx1 deficient mice. We found that the loss of Prdx1 significantly elevates ROS amounts in T cells and fibroblasts. The basal amounts of ROS were higher in fibroblasts than in T cells, probably due to a less robust Prdx1 peroxidase activity in the former. Using Aprt as a LOH reporter, we observed an elevation in LOH mutation frequency in fibroblasts, but not in T cells, of Prdx1(-/-) mice compared to Prdx1(+/+) mice. The majority of the LOH mutations in both cell types were derived from mitotic recombination (MR) events. Interestingly, Mlh1, which is known to suppress MR between divergent sequences, was found to be significantly down-regulated in fibroblasts of Prdx1(-/-) mice. Therefore, the combination of elevated ROS amounts and down-regulation of Mlh1 may have contributed to the elevation of MR in fibroblasts of Prdx1(-/-) mice. We conclude that each tissue may have a distinct mechanism through which Prdx1 deficiency promotes tumorigenesis.     

Angiotensin II-induced reduction in exercise capacity is associated with increased oxidative stress in skeletal muscle

Angiotensin II (ANG II)-induced oxidative stress has been known to be involved in the pathogenesis of cardiovascular diseases. We have reported that the oxidative stress in skeletal muscle can limit exercise capacity in mice (16). We thus hypothesized that ANG II could impair the skeletal muscle energy metabolism and limit exercise capacity via enhancing oxidative stress. ANG II (50 ng·kg(-1)·min(-1)) or vehicle was infused into male C57BL/6J mice for 7 days via subcutaneously implanted osmotic minipumps. ANG II did not alter body weight, skeletal muscle weight, blood pressure, cardiac structure, or function. Mice were treadmill tested, and expired gases were analyzed. The work to exhaustion (vertical distance × body weight) and peak oxygen uptake were significantly decreased in ANG II compared with vehicle. In mitochondria isolated from skeletal muscle, ADP-dependent respiration was comparable between ANG II and vehicle, but ADP-independent respiration was significantly increased in ANG II. Furthermore, complex I and III activities were decreased in ANG II. NAD(P)H oxidase activity and superoxide production by lucigenin chemiluminescence were significantly increased in skeletal muscle from ANG II mice. Treatment of ANG II mice with apocynin (10 mmol/l in drinking water), an inhibitor of NAD(P)H oxidase activation, completely inhibited NAD(P)H oxidase activity and improved exercise capacity, mitochondrial respiration, and complex activities in skeletal muscle. ANG II-induced oxidative stress can impair mitochondrial respiration in skeletal muscle and limit exercise capacity.     

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.     

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.     

Severe Vitamin E deficiency exacerbates acute hyperoxic lung injury associated with increased oxidative stress and inflammation

Hyperoxia causes acute lung injury along with an increase of oxidative stress and inflammation. It was hypothesized that vitamin E deficiency might exacerbate acute hyperoxic lung injury. This study used alpha-tocopherol transfer protein knockout (alpha-TTP KO) mice fed a vitamin E-deficient diet (KO E(-) mice) as a model of severe vitamin E deficiency. Compared with wild-type (WT) mice, KO E(-) mice showed a significantly lower survival rate during hyperoxia. After 72 h of hyperoxia, KO E(-) mice had more severe histologic lung damage and higher values of the total cell count and the protein content of bronchoalveolar lavage fluid (BALF) than WT mice. IL-6 mRNA expression in lung tissue and the levels of 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) in both lungs and BALF were higher in KO E(-) mice than in WT mice. It was concluded that severe vitamin E deficiency exacerbates acute hyperoxic lung injury associated with increased oxidative stress or inflammation.     

Severe Vitamin E deficiency exacerbates acute hyperoxic lung injury associated with increased oxidative stress and inflammation

Hyperoxia causes acute lung injury along with an increase of oxidative stress and inflammation. It was hypothesized that vitamin E deficiency might exacerbate acute hyperoxic lung injury. This study used α-tocopherol transfer protein knockout (α-TTP KO) mice fed a vitamin E-deficient diet (KO E(-) mice) as a model of severe vitamin E deficiency. Compared with wild-type (WT) mice, KO E(-) mice showed a significantly lower survival rate during hyperoxia. After 72 h of hyperoxia, KO E(-) mice had more severe histologic lung damage and higher values of the total cell count and the protein content of bronchoalveolar lavage fluid (BALF) than WT mice. IL-6 mRNA expression in lung tissue and the levels of 8-iso-prostaglandin F_2α (8-iso-PGF_2α) in both lungs and BALF were higher in KO E(-) mice than in WT mice. It was concluded that severe vitamin E deficiency exacerbates acute hyperoxic lung injury associated with increased oxidative stress or inflammation.     

Myocardial injury after ischemia-reperfusion in mice deficient in Akt2 is associated with increased cardiac macrophage density

Akt2 protein kinase has been shown to promote cell migration and actin polymerization in several cell types, including macrophages. Because migrating macrophages constitute an important inflammatory response after myocardial ischemia, we determined cardiac macrophage expression after ischemia-reperfusion (I/R) injury and cryo-injury in mice lacking Akt2 (Akt2-KO). At 7 days post-I/R, Akt2-KO cardiac tissues showed an increase in immunohistochemical staining for macrophage markers (Galectin 3 and F4/80) compared with wild-type (WT) mice, indicating macrophage density was increased in the injured Akt2-KO myocardium. This change was time dependent because macrophage density was similar between WT and Akt2-KO myocardium at 3 days post-I/R, but by 7 and 14 days post-I/R, macrophage density was significantly increased in Akt2-KO myocardium. Concomitantly, infarct size was larger and cardiac function was reduced in Akt2-KO mice subjected to I/R. However, when cryo-infarction produced similar infarct sizes in the anterior wall in both WT and Akt2-KO mice, macrophage density remained higher in Akt2-KO mouse myocardium, suggesting Akt2 regulates myocardial macrophage density independent of infarct size. Consistently, bone marrow from Akt2-KO mice enhanced myocardial macrophage density in both C57/B6 WT and Akt2-KO recipient mice. Finally, reciprocal ex-vivo coculturing of macrophages and cardiac myocytes showed that activated Akt2-KO peritoneal macrophages had reduced mobility and adhesion when compared with WT littermate controls. Thus, although Akt-2 KO mice did not affect the initial inflammation response after injury and Akt2 deficiency has been shown to impair cell migration or motility in macrophages, our data suggested a novel mechanism in which increasing retention of Akt2-KO macrophages resulted in increasing cardiac Akt2-KO macrophage density in the myocardial space.     

A biphasic U-shape effect of cellular oxidative stress on the macrophage anti-oxidant paraoxonase 2 (PON2) enzymatic activity

Expression of macrophage paraoxonase 2 (PON2), a cellular lactonase with anti-oxidant and anti-atherogenic properties, was shown to be upregulated under high oxidative stress. The aim of the present study was to analyze the relationship between the extent of cellular oxidative stress in J774A.1 macrophage and PON2 lactonase activity under various levels of oxidation, obtained by cell incubation with either anti-oxidants or oxidants. PON2 activity exhibited a U-shape response curve. In the oxidative stress range below that of control untreated cells, PON2 activity decreased upon increasing macrophage oxidative state, whereas in the range over that of control untreated cells, PON2 activity increased. The biphasic effect of oxidative stress on macrophage PON2 activity could be related to PON2 inactivation (decreased enzymatic activity) under oxidative stress induction at its low range, whereas at high range of oxidative stress, macrophage anti-oxidant compensatory mechanism up-regulates PON2 (increased protein expression), in order to cope with oxidative burden.     

Evidence that oxidative stress is increased in patients with X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a hereditary disorder of peroxisomal metabolism biochemically characterized by the accumulation of very long chain fatty acids (VLCFA), particularly hexacosanoic acid (C26:0) and tetracosanoic acid (C24:0) in different tissues and in biological fluids. The disease is clinically characterized by central and peripheral demyelination and adrenal insufficiency, which is closely related to the increased concentrations of these fatty acids. However, the mechanisms underlying the brain damage in X-ALD are poorly known. Considering that free radical generation is involved in various neurodegenerative disorders, like Parkinson disease, multiple sclerosis and Alzheimer's disease, in the present study we evaluated various oxidative stress parameters, namely chemiluminescence, thiobarbituric acid reactive species (TBA-RS), total radical-trapping antioxidant potential (TRAP), and total antioxidant reactivity (TAR) in plasma of X-ALD patients, as well as the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in erythrocytes and fibroblasts from these patients. It was verified a significant increase of plasma chemiluminescence and TBA-RS, reflecting induction of lipid peroxidation, as well as a decrease of plasma TAR, indicating a deficient capacity to rapidly handle an increase of reactive species. We also observed a significant increase of erythrocytes GPx activity and of catalase and SOD activities in fibroblasts from the patients studied. It is therefore proposed that oxidative stress may be involved in pathophysiology of X-ALD.     

The Role of Paraoxonase 1 (PON1) Gene Polymorphisms in Coronary Artery Disease: A Systematic Review and Meta-Analysis

Although many studies have investigated the association of paraoxonase 1 (PON1) polymorphisms with coronary artery disease (CAD). However, the outcomes were not consistent and remain uncertain. Therefore, it is the need of the hour to analyze the available literature and evaluate the association of PON1 polymorphisms with the CAD. All the relevant studies published in the English language from January 1, 2000, up to September 20, 2020, were identified by searching through various electronic databases. The two researchers independently extracted the information. The data were analyzed by using the MetaGenyo program. The pooled odds ratio was used to find the associations between CAD and PON1 polymorphisms. In the final analysis, we include 10 studies regarding the association of PON1 polymorphisms (rs662 and rs854560) with CAD. Overall, the Q192R polymorphism increased the risk of CAD in the tested genetic models including the homozygote model: OR 1.35, CI 1.02–1.79; allelic model: OR 1.16, CI 1.00–1.33; dominant model: OR 1.25, CI 1.03–1.52. The L55M polymorphism does not significantly associated with CAD in all the tested genetic models including the homozygote model: OR 1.00 CI, 0.64–1.56; allelic model: OR 1.02, 95% CI 0.84–1.23; dominant model: OR 1.08, CI 0.89–1.31. Further analysis showed no publication bias exists in meta-analysis. Our findings suggested that rs662 in the coding region was significantly associated with the CAD however, rs854560 has no significant association with the disease. Nevertheless, in future, there is a need for more studies with a larger sample size which may provide a more definite conclusion.

Study Registration: PROSPERO registration number CRD42020202278

     

Paraoxonase 1 (PON1) polymorphisms, haplotypes and activity in predicting cad risk in North-West Indian Punjabis

Background

Human serum paraoxonase-1 (PON1) prevents oxidation of low density lipoprotein cholesterol (LDL-C) and hydrolyzes the oxidized form, therefore preventing the development of atherosclerosis. The polymorphisms of PON1 gene are known to affect the PON1 activity and thereby coronary artery disease (CAD) risk. As studies are lacking in North-West Indian Punjabi''s, a distinct ethnic group with high incidence of CAD, we determined PON1 activity, genotypes and haplotypes in this population and correlated them with the risk of CAD.

Methodology/Principal Findings

350 angiographically proven (≥70% stenosis) CAD patients and 300 healthy controls were investigated. PON1 activity was determined towards paraoxon (Paraoxonase; PONase) and phenylacetate (Arylesterase; AREase) substrates. In addition, genotyping was carried out by using multiplex PCR, allele specific oligonucleotide –PCR and PCR-RFLP methods and haplotyping was determined by PHASE software. The serum PONase and AREase activities were significantly lower in CAD patients as compared to the controls. All studied polymorphisms except L55M had significant effect on PONase activity. However AREase activity was not affected by them. In a logistic regression model, after adjustment for the conventional risk factors for CAD, QR (OR: 2.73 (1.57–4.72)) and RR (OR, 16.24 (6.41–41.14)) genotypes of Q192R polymorphism and GG (OR: 2.07 (1.02–4.21)) genotype of −162A/G polymorphism had significantly higher CAD risk. Haplotypes L-T-G-Q-C (OR: 3.25 (1.72–6.16)) and L-T-G-R-G (OR: 2.82 (1.01–7.80)) were also significantly associated with CAD.

Conclusions

In conclusion this study shows that CAD patients had lower PONase and AREase activities as compared to the controls. The coding Q192R polymorphism, promoter −162A/G polymorphism and L-T-G-Q-C and L-T-G-R-G haplotypes are all independently associated with CAD.