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The signal sequence polymorphism of the MnSOD gene is associated with the degree of carotid atherosclerosis
Atherosclerosis 168 (2003) 147 /152 www.elsevier.com/locate/atherosclerosis
The signal sequence polymorphism of the MnSOD gene is associated with the degree of carotid atherosclerosis Sakari Kakko a,*, Markku Pa¨iva¨nsalo b, Pirjo Koistinen a, Y. Antero Kesa¨niemi a, Vuokko L. Kinnula a, Markku J. Savolainen a a
Department of Internal Medicine and Biocenter Oulu, University of Oulu, P.O. 5000, FIN-90014 Oulu, Finland b Department of Diagnostic Radiology, University of Oulu, P.O. 5000, FIN-90014 Oulu, Finland Received 10 October 2002; received in revised form 7 February 2003; accepted 18 February 2003
Abstract Redox-state of the cells of vascular walls is an important determinant of atherosclerosis. Manganese superoxide dismutase (MnSOD) is an essential anti-oxidant enzyme working in mitochondria of mammalian cells. A potentially functional amino acid polymorphism (Ala16Val) has been described in the signal sequence of the enzyme. The aim of the current study was to test whether the signal sequence polymorphism of the MnSOD would be associated with the degree of carotid atherosclerosis. The polymorphism was genotyped in a sample of 989 middle-aged hypertensive and control subjects. Carotid atherosclerosis was quantified as intimamedia thickness (IMT) by ultrasound. The signal sequence polymorphism was found to be a minor determinant of carotid IMT explaining 1.3% of the overall variation, the Val allele associated with the higher IMT. In women, a significant interaction with plasma levels of low-density lipoprotein (LDL) cholesterol was detected, since LDL cholesterol levels were positively correlated with carotid IMT only in the carriers of the Val allele and the Val allele was associated with higher IMT only in the subjects with highest plasma levels of LDL cholesterol. In conclusion, the signal sequence polymorphism of the MnSOD gene is a minor determinant of carotid IMT pointing out the importance of redox-balance in the atherogenesis. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Genetic polymorphisms; Atherosclerosis; Quantitative trait locus; Intima-media thickness; Redox-state; Anti-oxidant enzyme
1. Introduction The development of atherosclerosis is a complex process with a network of risk factors, such as hypertension, hyperlipidemia, smoking and genetic predisposition [1,2]. Atherosclerosis could be thought of as a chronic inflammatory disease with the basic abnormality lying in the redox-state of the vascular wall cells [3]. The oxidative stress contributes to atherogenesis e.g. by inducing the expression of various cytokines and adhesion molecules [4 /6], thereby promoting the invasion of inflammatory cells into the vessel wall, which is an early and important event in atherogenesis. Especially, the oxidation of low-density lipoproteins (LDLs) is considered to be a crucial step in atherogenesis [7].
* Corresponding author. Tel.: /358-8-315-4570; fax: /358-8-3154543. E-mail address: [email protected] (S. Kakko).
Superoxide dismutases (SODs) are enzymes catalyzing dismutation of reactive superoxide radicals to hydrogen peroxide and three SODs catalyze this reaction in mammals: manganese superoxide dismutase (MnSOD) on the mitochondria, copper/zinc superoxide dismutase (Cu,Zn-SOD) on the cytosol and extracellular superoxide dismutase (EC-SOD) in the extracellular compartments [8 /10]. Polymorphisms of neither the Cu,Zn-SOD gene nor the EC-SOD gene were associated with the risk of macroangiopathy in patients with type 2 diabetes mellitus [11]. Experimental studies with knockout animals strongly suggest that MnSOD plays a major importance in the oxidant resistance of vital organs [12 / 14]. Single amino acid polymorphism alanine (Ala) to valine (Val) at the 16th amino acid (16th amino acid from the beginning of the signal sequence or /9th amino acid from the first amino acid of the mature protein) of the signal sequence of the MnSOD (Ala16Val) has been suggested to change the secondary structure of the
0021-9150/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0021-9150(03)00091-1
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premature protein and therefore the mitochondrial targeting of the enzyme [15,16]. So far, there are only a few studies linking MnSOD with heart diseases and atherosclerosis. Firstly, MnSOD locus has been linked to the atherogenic lipoprotein phenotype, i.e. the excess of small dense LDL, in humans, in one [17], but not in another study [18]. Overexpression of MnSOD has been shown to protect transgenic mice against myocardial ischemia [19] and, in rabbits, to reverse vascular dysfunction in carotid arteries without atherosclerotic changes, but not in vessels with atherosclerotic plaques [20]. Overexpression of MnSOD inhibits in vitro the oxidation of LDL by endothelial cells [21] and oxidized LDL is able to induce the expression of MnSOD in macrophages [22]. The apoE-deficient mice lacking MnSOD had more severe atherosclerosis compared to the apoE-deficient mice [23]. In addition, the signal sequence polymorphism of the MnSOD gene has been associated with non-familial dilated cardiomyopathy in Japanese subjects [24], but it has not earlier been investigated directly in human atherosclerosis. Carotid atherosclerosis could be measured quantitatively by ultrasonography as intima-media thickness (IMT) [25] and the degree of carotid atherosclerosis defined by IMT is a strong risk factor for myocardial infarction and stroke even after adjustment for traditional risk factors [26]. The purpose of the study was to analyze whether the signal sequence polymorphism of MnSOD gene is associated with the degree of carotid atherosclerosis in the homogenous Finnish sample of hypertensive and control subjects. In addition, smoking, which is likely to increase oxidative stress, and plasma levels of LDL cholesterol, probably associated with the levels of oxidized LDL, were studied for interaction with the association between the MnSOD signal sequence genotypes and carotid IMT.
2. Material and methods 2.1. Material The sample consisted of the control (259 men and 267 women) and hypertensive cohorts (261 men and 258 women) of our the Oulu project elucidating risk of atherosclerosis(OPERA) study. The subjects were from the town of Oulu, aged 40 /61 years and had been selected by the Social Insurance Institution as described earlier [27]. All made an outpatient visit to the research unit for laboratory tests, a physical examination and interviews concerning alcohol consumption, smoking and exercise habits, medication and past medical history. Apart from a classification into current smokers and non-smokers, smoking status was also recorded as pack-years of smoking (1 pack-year /20 cigarettes/day for 1 year). The body mass index (BMI) was calculated
by dividing weight (kg) by height squared (m2). Only those subjects (n/989) having the MnSOD signal sequence polymorphism genotyped and carotid IMT measured were selected for the current sub-study. 2.2. Genotyping of the polymorphism DNA was extracted from peripheral blood leucocytes by a salting-out procedure [28]. The following primer pairs were used for amplification of the MnSOD gene fragment containing the Val16Ala signal sequence polymorphism: 5?AGCCCAGCCGTGCGTAGAC3? and 5?TACTTCTCCTCGGTGACG3?. PCR amplifications were done in 10 ml reaction volumes containing 100 ng of genomic DNA, 2.5 pmol of each primer, 200 mM of each dNTPs, and 0.2 U Dynazyme DNA polymerase (Finnzymes, Espoo, Finland) in the buffer recommended by the manufacturer with 1.5 mM of MgCl2. The samples were denatured for 5 min at 94 8C and subjected to 42 cycles each of 50 s at 94 8C, 50 s at 58 8C, and 1 min at 72 8C, and were finally elongated for 10 min at 72 8C. The reaction products were cleaved using the BsaWI restriction enzyme (New England BioLabs, Beverly, MA), electrophoresed on 2% agarose gel, stained by Gelstar (FMC Bioproducts, Rockland, Maine, USA) and photographed under UV illumination. The length of the BsaWI digested PCR products for the Ala16 allele of MnSOD was 249 and for the Val16 allele 87 and 162 bps. 2.3. IMT The carotid ultrasound examinations and measurements were performed by one trained radiologist as described previously [29]. The parameters measured were IMT, which was defined as the distance between the media /adventitia interface and the lumen /intima interface. Because the near-wall measurements may be difficult to make precisely [25], data from the far wall measurements were used. IMT was measured at five points on each side on the far wall, i.e. at altogether 10 sites: the internal carotid artery (ICA) about 10 mm distal from the flow divider, the bifurcation enlargement (BIF) and three locations of the common carotid artery (CCA), proximal, middle and distal, with about 10/15 mm intervals, the most cranial measuring point being about 10 mm from the bifurcation. The thickest point of the IMT was measured at each site, yet avoiding sites with plaque. The IMT value used in this study was defined as the mean of the measurements in CCA, BIF and ICA. The reproducibility of the measurements was assessed from the video tapes of 31 randomly selected study subjects by one radiologist blinded to the original results. The intrareader variability and Pearson’s correlation coefficient were 3% and 0.97 for the mean IMT.
(4.7) (5.6) (0.86) (0.41) (0.79) (0.30) (0.10) (3.1)* (6.0) (0.89) (0.30) (0.79) (0.72) (0.21)
All the values are expressed as mean (S.D.) or number of subjects. HDL, high density lipoprotein; LDL, low-density lipoprotein. The values are unadjusted. * P B/0.01 for the difference between the homozygous genotypes by Student’s T -test. ** P B/0.05.
134 12 (9%) 28.7 (5.6) 52.0 (6.0) 5.78 (1.09) 1.46 (0.39) 3.50 (0.91) 1.63 (1.21) 0.85 (0.15) 58 5 ( 9%) 28.1 (4.0) 51.3 (6.0) 5.63 (1.00) 1.43 (0.37) 3.40 (0.99) 1.44 (0.87) 0.80 (0.12) 67 2 (3%) 25.9 (4.6) 51.8 (6.1) 5.50 (1.02) 1.59 (0.41) 3.28 (0.86) 1.23 (0.86) 0.84 (0.12)** 107 2 (2%) 26.6 (4.2) 51.8 (6.2) 5.54 (1.09) 1.53 (0.35) 3.32 (1.02) 1.24 (0.69) 0.82 (0.14) 59 0 25.5 51.3 5.40 1.57 3.26 1.05 0.80 72 8 (11%) 29.0 (4.0) 50.5 (6.0) 5.74 (1.01) 1.17 (0.34) 3.51 (0.92) 2.07 (1.34) 0.97 (0.24) 117 14 (12%) 29.9 (4.4) 50.8 (5.4) 5.81 (1.05) 1.16 (0.31) 3.60 (0.90) 2.09 (1.32) 0.93 (0.21) 61 8 (13%) 28.9 (4.7) 50.9 (6.4) 5.73 (0.99) 1.24 (0.28) 3.67 (0.95) 1.68 (1.03) 0.90 (0.20) 64 0 26.0 51.0 5.61 1.23 3.62 1.50 0.95 134 3 (2%) 26.3 (3.4) 51.3 (6.1) 5.80 (1.15) 1.22 (0.31) 3.75 (1.02) 1.53 (0.79) 0.91 (0.22) 54 1 (2%) 27.8 (4.1) 49.7 (6.2) 5.92 (1.14) 1.21 (0.28) 3.81 (1.03) 1.71 (0.87) 0.93 (0.21) Number of subjects Subjects with diabetes BMI (kg/m2) Age (years) Total cholesterol (mmol/l) HDL cholesterol (mmol/l) LDL cholesterol (mmol/l) Triglycerides (mmol/l) IMT (mm)
AlaVal AlaAla AlaAla AlaVal
ValVal AlaAla
AlaVal
ValVal
AlaAla
AlaVal
ValVal
Hypertensive women Control women Hypertensive men
The signal sequence polymorphism of the MnSOD was successfully genotyped and carotid IMT measured in 989 subjects of the OPERA sample. There were 232 subjects with the genotype AlaAla, 492 with the genotype AlaVal and 265 with the genotype ValVal in the sample giving the allele frequencies of 0.483 and 0.517 for the Ala and Val allele, respectively. The genotypes were in Hardy /Weinberg equilibrium. BMI, age, presence of diabetes, lipid and lipoprotein levels, and IMT for the study subjects stratified according to the genotypes of MnSOD signal sequence polymorphism are shown on Table 1. In control women, the ValVal genotype was associated with higher IMT (P /0.03 for the difference between the homozygous genotypes by Student’s T -test). In control men, the Val allele was associated with lower BMI (P /0.008 for the difference between the homozygous genotypes by the Student’s T -test). Table 2 shows the mean IMT values according to the genotypes in the quartiles of LDL cholesterol and according to the smoking status. In women, the ValVal genotype was significantly associated with higher IMT, but only in the highest LDL cholesterol quartile and in those women who had never smoked (P /0.03 and P /0.006, respectively, for the difference between homozygous genotypes by Student’s T -test). In men, the Val allele was associated with high IMT only in current smokers (P /0.05 by Student’s T test for the difference between the homozygous genotypes). There was a significant correlation between
Control men
3. Results
Table 1 Association of the genotypes of MnSOD signal sequence polymorphism with presence of diabetes, BMI, age, lipid and lipoprotein levels and IMT of carotid arteries
The results are given as means (S.D.) or percentages. All the analyses were carried out using the statistical package for social studies (SPSS) version 10.1 (SPSS Inc., Chicago, IL). The differences in lipid and lipoprotein values, BMI and carotid IMT between the different genotype groups were evaluated by Student’s T -test. The general factorial procedure of the General Linear Model (GLM) with Type III sums of squares was used to construct the model to explain the variation in carotid IMT. The genotypes of the signal sequence polymorphism indicated were added as an independent factor into the model without any assumption of the mode of inheritance (i.e. without any combination of genotypes). The h -squared statistical calculation was used to estimate how much of the variation each factor or covariate explained. Covariates included in the analysis were BMI, pack-years of smoking, age, LDL cholesterol and systolic blood pressure and the factor included was the genotype of the signal sequence polymorphism. When sexes were analyzed together, sex was also taken as a factor into the analysis.
ValVal
2.4. Statistical analysis
149 62 2 (3%) 29.2 (5.6) 51.7 (5.6) 5.63 (1.00) 1.39 (0.34) 3.46 (0.88) 1.54 (0.63) 0.83 (0.12)
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S. Kakko et al. / Atherosclerosis 168 (2003) 147 /152
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Table 2 Association between carotid IMT and the genotypes of signal sequence polymorphism in the quartiles of plasma LDL cholesterol levels and according to smoking history Men
Women
AlaAla
AlaVal
ValVal
AlaAla
AlaVal
ValVal
Q1
n/27 0.86 (0.21)
n/61 0.85 (0.18)
n/38 0.88 (0.16)
n/29 0.78 (0.08)
n/65 0.79 (0.10)
n/32 0.81 (0.11)
Q2
n/31 0.89 (0.21)
n/57 0.90 (0.18)
n/39 0.96 (0.26)
n/35 0.80 (0.11)
n/58 0.85 (0.16)
n/28 0.81 (0.09)
Q3
n/24 0.92 (0.17)
n/68 0.94 (0.20)
n/30 1.03 (0.25)
n/31 0.82 (0.12)
n/54 0.83 (0.16)
n/34 0.83 (0.11)
Q4
n/33 0.98 (0.21)
n/65 0.99 (0.26)
n/29 0.99 (0.20)
n/22 0.81 (0.11)
n/64 0.87 (0.15)
n/35 0.88 (0.14)*
Never smoked
n/36 0.92 (0.17)
n/73 0.90 (0.17)
n/45 0.91 (0.17)
n/76 0.79 (0.11)
n/160 0.84 (0.14)
n/80 0.84 (0.13)**
Ex-smokers
n/44 0.93 (0.23)
n/88 0.90 (0.21)
n/45 0.97 (0.24)
n/13 0.80 (0.11)
n/22 0.80 (0.17)
n/17 0.81 (0.11)
Current smokers
n/35 0.89 (0.20)
n/90 0.95 (0.25)
n/46 0.99 (0.25)*
n/28 0.84 (0.10)
n/59 0.84 (0.14)
n/32 0.84 (0.11)
The unadjusted results are shown n /number subjects and mean (S.D.). Q1 (lowest)-Q4 (highest) are for quartiles based on plasma LDL cholesterol levels and the borders between the quartiles are 3.00, 3.67 and 4.21 mmol/l for the men and 2.78, 3.37 and 4.01 mmol/l for the women. * P B/0.05. ** P B/0.01 for the difference between the homozygous genotypes by Student’s T -test.
plasma LDL cholesterol levels and carotid IMT in men with every genotype of signal sequence polymorphism, whereas the significant correlation was detected only in women carrying the Val allele (Table 3). In GLM analysis, the genotypes of signal sequence polymorphism were significantly (P /0.05) associated with IMT, but the genotypes explained only 1.3% of the overall variation of IMT in both sexes (Table 4). The means of IMT taken from GLM (adjusted for the other factors and covariates mentioned in the Table 4) in men Table 3 Spearman correlation coefficients between IMT and LDLs cholesterol levels separately for men and women and every genotype of MnSOD signal sequence polymorphism
Table 4 General liner models explaining the variation of carotid IMT in the study population Sex
Factor/covariate
Effect size
P- value
Men
Age Pack-years BMI LDL cholesterol Systolic blood pressure MnSOD genotype
8.1% 2.5% 0.0% 6.0% 1.7% 1.3%
B/0.001 B/0.001 0.97 B/0.001 0.003 0.04
Women
Age Pack-years BMI LDL cholesterol Systolic blood pressure MnSOD genotype
5.6% 1.0% 1.8% 0.7% 4.9% 1.3%
B/0.001 0.03 0.003 0.07 B/0.001 0.05
BMI, body mass index. LDL, low-density lipoproteins. Sex
Genotype
n
Correlation coefficient
P
Men
AlaAla AlaVal ValVal
115 251 136
0.26 0.22 0.24
0.004 B/0.001 0.005
AlaAla AlaVal ValVal
117 241 129
0.09 0.16 0.25
0.36 0.015 0.005
Women
n , number of subjects. P , the P -value for the correlation coefficient.
were 0.92 mm (the AlaAla genotype), 0.92 mm (the AlaVal genotype) and 0.96 mm (the ValVal genotype), and in women 0.80, 0.83 and 0.84 mm, respectively. If sexes were combined, the means were 0.86, 0.87 and 0.90 mm, respectively, (P /0.02). The exclusion of subjects with diabetes did not affect the results.
S. Kakko et al. / Atherosclerosis 168 (2003) 147 /152
4. Discussion Atherosclerosis is a complex process affected by a network of numerous genes and environmental factors [1,2]. Carotid IMT allows a non-invasive and quantitative determination of the degree of atherosclerosis. In the homogenous and large sample of control and hypertensive subjects from Finnish population we were able to show that the MnSOD gene may be a minor locus determining carotid atherosclerosis. The degree of carotid atherosclerosis has been found to be associated with many candidate genes of proteins involved in blood coagulation (prothrombin), lipoprotein metabolism (apolipoprotein E and cholesteryl ester transfer protein), homocysteine metabolism (5,10-methylenetetrahydrofolate reductase), enzymes protecting lipids against oxidation (paraoxonase) and nitric oxide metabolism (endothelial nitric oxide synthase) [30 /35], but the association with MnSOD gene is a new finding. The biological basis for the association between the signal sequence polymorphism of the MnSOD gene and carotid IMT is not known. It has been suggested that the signal sequence polymorphism affects the conformation of the leader sequence and therefore the premature protein with valine at position 16 in the signal sequence is not effectively targeted into the mitochondria and the anti-oxidant balance of the cell is disturbed. Antioxidant capacity in subjects with the Val allele might therefore be diminished leading to increased oxidative stress and increased atherosclerosis e.g. via expression of adhesion molecules or diminished suppression of LDL oxidation [4,6,21]. On the other hand, the MnSOD in subjects with the Val allele might be working inappropriately in the cytosol, instead of mitochondria, and the hydrogen peroxide produced might be converted into highly reactive hydroxyl radical in this cellular compartment. Our results are strongly supported by the results of a recent interesting study [23]. In this study, the mitochondrial DNA damage was positively correlated with the extent of atherosclerosis in aorta samples of both humans and mice. In addition, the apoE-knockout mice deficient in MnSOD had accelerated atherogenesis when compared to apoE-knockout mice with normal MnSOD function. Interestingly, the association between MnSOD genotype and IMT in women was seen only in those subjects with the highest plasma levels of LDL cholesterol. This strong association abolished the correlation between plasma levels of LDL cholesterol and IMT in subjects with the AlaAla genotype. There may be two alternative explanations for this finding. Firstly , the predisposing association of the Val allele is seen only in subjects with the highest LDL cholesterol, i.e. the high levels of LDL cholesterol are most harmful in subjects with diminished antioxidant capacity of MnSOD. Secondly , high levels
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of LDL cholesterol are not harmful in women with the Ala allele since their mitochondrial targeted anti-oxidant capacity is balanced. A biological link for the interaction might be LDL oxidation suppressed by MnSOD [21]. The biological basis for the interaction with smoking and sex (the association between the MnSOD variation and carotid IMT was found only in men currently smoking and in women who had never smoked) is hard to understand and they are based on the analysis of subgroups. These results remained significant also in non-parametric Mann /Whitney U -test, excluding the confounding effect of a few outliers. In conclusion, the MnSOD locus was found to be a significant modifier of carotid atherosclerosis pointing out the importance of oxidative stress in atherosclerosis. However, the association was rather weak explaining only around 1.3% of the variation in carotid IMT compared to previously published estimated heritabilities of carotid IMT varying from 41 to 75% [36 /38].
Acknowledgements The authors gratefully acknowledge the expert technical assistance of Kirsi Kvist-Ma¨kela¨ and Marja-Leena Kyto¨kangas. This study was supported by grants from the Research Council for Health of the Academy of Finland, the Finnish Foundation for Cardiovascular Research, the Sigrid Juselius Foundation and the Paavo Nurmi Foundation.
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The signal sequence polymorphism of the MnSOD gene is associated with the degree of carotid atherosclerosis Sakari Kakko a,*, Markku Pa¨iva¨nsalo b, Pirjo Koistinen a, Y. Antero Kesa¨niemi a, Vuokko L. Kinnula a, Markku J. Savolainen a a
Department of Internal Medicine and Biocenter Oulu, University of Oulu, P.O. 5000, FIN-90014 Oulu, Finland b Department of Diagnostic Radiology, University of Oulu, P.O. 5000, FIN-90014 Oulu, Finland Received 10 October 2002; received in revised form 7 February 2003; accepted 18 February 2003
Abstract Redox-state of the cells of vascular walls is an important determinant of atherosclerosis. Manganese superoxide dismutase (MnSOD) is an essential anti-oxidant enzyme working in mitochondria of mammalian cells. A potentially functional amino acid polymorphism (Ala16Val) has been described in the signal sequence of the enzyme. The aim of the current study was to test whether the signal sequence polymorphism of the MnSOD would be associated with the degree of carotid atherosclerosis. The polymorphism was genotyped in a sample of 989 middle-aged hypertensive and control subjects. Carotid atherosclerosis was quantified as intimamedia thickness (IMT) by ultrasound. The signal sequence polymorphism was found to be a minor determinant of carotid IMT explaining 1.3% of the overall variation, the Val allele associated with the higher IMT. In women, a significant interaction with plasma levels of low-density lipoprotein (LDL) cholesterol was detected, since LDL cholesterol levels were positively correlated with carotid IMT only in the carriers of the Val allele and the Val allele was associated with higher IMT only in the subjects with highest plasma levels of LDL cholesterol. In conclusion, the signal sequence polymorphism of the MnSOD gene is a minor determinant of carotid IMT pointing out the importance of redox-balance in the atherogenesis. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Genetic polymorphisms; Atherosclerosis; Quantitative trait locus; Intima-media thickness; Redox-state; Anti-oxidant enzyme
1. Introduction The development of atherosclerosis is a complex process with a network of risk factors, such as hypertension, hyperlipidemia, smoking and genetic predisposition [1,2]. Atherosclerosis could be thought of as a chronic inflammatory disease with the basic abnormality lying in the redox-state of the vascular wall cells [3]. The oxidative stress contributes to atherogenesis e.g. by inducing the expression of various cytokines and adhesion molecules [4 /6], thereby promoting the invasion of inflammatory cells into the vessel wall, which is an early and important event in atherogenesis. Especially, the oxidation of low-density lipoproteins (LDLs) is considered to be a crucial step in atherogenesis [7].
* Corresponding author. Tel.: /358-8-315-4570; fax: /358-8-3154543. E-mail address: [email protected] (S. Kakko).
Superoxide dismutases (SODs) are enzymes catalyzing dismutation of reactive superoxide radicals to hydrogen peroxide and three SODs catalyze this reaction in mammals: manganese superoxide dismutase (MnSOD) on the mitochondria, copper/zinc superoxide dismutase (Cu,Zn-SOD) on the cytosol and extracellular superoxide dismutase (EC-SOD) in the extracellular compartments [8 /10]. Polymorphisms of neither the Cu,Zn-SOD gene nor the EC-SOD gene were associated with the risk of macroangiopathy in patients with type 2 diabetes mellitus [11]. Experimental studies with knockout animals strongly suggest that MnSOD plays a major importance in the oxidant resistance of vital organs [12 / 14]. Single amino acid polymorphism alanine (Ala) to valine (Val) at the 16th amino acid (16th amino acid from the beginning of the signal sequence or /9th amino acid from the first amino acid of the mature protein) of the signal sequence of the MnSOD (Ala16Val) has been suggested to change the secondary structure of the
0021-9150/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0021-9150(03)00091-1
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premature protein and therefore the mitochondrial targeting of the enzyme [15,16]. So far, there are only a few studies linking MnSOD with heart diseases and atherosclerosis. Firstly, MnSOD locus has been linked to the atherogenic lipoprotein phenotype, i.e. the excess of small dense LDL, in humans, in one [17], but not in another study [18]. Overexpression of MnSOD has been shown to protect transgenic mice against myocardial ischemia [19] and, in rabbits, to reverse vascular dysfunction in carotid arteries without atherosclerotic changes, but not in vessels with atherosclerotic plaques [20]. Overexpression of MnSOD inhibits in vitro the oxidation of LDL by endothelial cells [21] and oxidized LDL is able to induce the expression of MnSOD in macrophages [22]. The apoE-deficient mice lacking MnSOD had more severe atherosclerosis compared to the apoE-deficient mice [23]. In addition, the signal sequence polymorphism of the MnSOD gene has been associated with non-familial dilated cardiomyopathy in Japanese subjects [24], but it has not earlier been investigated directly in human atherosclerosis. Carotid atherosclerosis could be measured quantitatively by ultrasonography as intima-media thickness (IMT) [25] and the degree of carotid atherosclerosis defined by IMT is a strong risk factor for myocardial infarction and stroke even after adjustment for traditional risk factors [26]. The purpose of the study was to analyze whether the signal sequence polymorphism of MnSOD gene is associated with the degree of carotid atherosclerosis in the homogenous Finnish sample of hypertensive and control subjects. In addition, smoking, which is likely to increase oxidative stress, and plasma levels of LDL cholesterol, probably associated with the levels of oxidized LDL, were studied for interaction with the association between the MnSOD signal sequence genotypes and carotid IMT.
2. Material and methods 2.1. Material The sample consisted of the control (259 men and 267 women) and hypertensive cohorts (261 men and 258 women) of our the Oulu project elucidating risk of atherosclerosis(OPERA) study. The subjects were from the town of Oulu, aged 40 /61 years and had been selected by the Social Insurance Institution as described earlier [27]. All made an outpatient visit to the research unit for laboratory tests, a physical examination and interviews concerning alcohol consumption, smoking and exercise habits, medication and past medical history. Apart from a classification into current smokers and non-smokers, smoking status was also recorded as pack-years of smoking (1 pack-year /20 cigarettes/day for 1 year). The body mass index (BMI) was calculated
by dividing weight (kg) by height squared (m2). Only those subjects (n/989) having the MnSOD signal sequence polymorphism genotyped and carotid IMT measured were selected for the current sub-study. 2.2. Genotyping of the polymorphism DNA was extracted from peripheral blood leucocytes by a salting-out procedure [28]. The following primer pairs were used for amplification of the MnSOD gene fragment containing the Val16Ala signal sequence polymorphism: 5?AGCCCAGCCGTGCGTAGAC3? and 5?TACTTCTCCTCGGTGACG3?. PCR amplifications were done in 10 ml reaction volumes containing 100 ng of genomic DNA, 2.5 pmol of each primer, 200 mM of each dNTPs, and 0.2 U Dynazyme DNA polymerase (Finnzymes, Espoo, Finland) in the buffer recommended by the manufacturer with 1.5 mM of MgCl2. The samples were denatured for 5 min at 94 8C and subjected to 42 cycles each of 50 s at 94 8C, 50 s at 58 8C, and 1 min at 72 8C, and were finally elongated for 10 min at 72 8C. The reaction products were cleaved using the BsaWI restriction enzyme (New England BioLabs, Beverly, MA), electrophoresed on 2% agarose gel, stained by Gelstar (FMC Bioproducts, Rockland, Maine, USA) and photographed under UV illumination. The length of the BsaWI digested PCR products for the Ala16 allele of MnSOD was 249 and for the Val16 allele 87 and 162 bps. 2.3. IMT The carotid ultrasound examinations and measurements were performed by one trained radiologist as described previously [29]. The parameters measured were IMT, which was defined as the distance between the media /adventitia interface and the lumen /intima interface. Because the near-wall measurements may be difficult to make precisely [25], data from the far wall measurements were used. IMT was measured at five points on each side on the far wall, i.e. at altogether 10 sites: the internal carotid artery (ICA) about 10 mm distal from the flow divider, the bifurcation enlargement (BIF) and three locations of the common carotid artery (CCA), proximal, middle and distal, with about 10/15 mm intervals, the most cranial measuring point being about 10 mm from the bifurcation. The thickest point of the IMT was measured at each site, yet avoiding sites with plaque. The IMT value used in this study was defined as the mean of the measurements in CCA, BIF and ICA. The reproducibility of the measurements was assessed from the video tapes of 31 randomly selected study subjects by one radiologist blinded to the original results. The intrareader variability and Pearson’s correlation coefficient were 3% and 0.97 for the mean IMT.
(4.7) (5.6) (0.86) (0.41) (0.79) (0.30) (0.10) (3.1)* (6.0) (0.89) (0.30) (0.79) (0.72) (0.21)
All the values are expressed as mean (S.D.) or number of subjects. HDL, high density lipoprotein; LDL, low-density lipoprotein. The values are unadjusted. * P B/0.01 for the difference between the homozygous genotypes by Student’s T -test. ** P B/0.05.
134 12 (9%) 28.7 (5.6) 52.0 (6.0) 5.78 (1.09) 1.46 (0.39) 3.50 (0.91) 1.63 (1.21) 0.85 (0.15) 58 5 ( 9%) 28.1 (4.0) 51.3 (6.0) 5.63 (1.00) 1.43 (0.37) 3.40 (0.99) 1.44 (0.87) 0.80 (0.12) 67 2 (3%) 25.9 (4.6) 51.8 (6.1) 5.50 (1.02) 1.59 (0.41) 3.28 (0.86) 1.23 (0.86) 0.84 (0.12)** 107 2 (2%) 26.6 (4.2) 51.8 (6.2) 5.54 (1.09) 1.53 (0.35) 3.32 (1.02) 1.24 (0.69) 0.82 (0.14) 59 0 25.5 51.3 5.40 1.57 3.26 1.05 0.80 72 8 (11%) 29.0 (4.0) 50.5 (6.0) 5.74 (1.01) 1.17 (0.34) 3.51 (0.92) 2.07 (1.34) 0.97 (0.24) 117 14 (12%) 29.9 (4.4) 50.8 (5.4) 5.81 (1.05) 1.16 (0.31) 3.60 (0.90) 2.09 (1.32) 0.93 (0.21) 61 8 (13%) 28.9 (4.7) 50.9 (6.4) 5.73 (0.99) 1.24 (0.28) 3.67 (0.95) 1.68 (1.03) 0.90 (0.20) 64 0 26.0 51.0 5.61 1.23 3.62 1.50 0.95 134 3 (2%) 26.3 (3.4) 51.3 (6.1) 5.80 (1.15) 1.22 (0.31) 3.75 (1.02) 1.53 (0.79) 0.91 (0.22) 54 1 (2%) 27.8 (4.1) 49.7 (6.2) 5.92 (1.14) 1.21 (0.28) 3.81 (1.03) 1.71 (0.87) 0.93 (0.21) Number of subjects Subjects with diabetes BMI (kg/m2) Age (years) Total cholesterol (mmol/l) HDL cholesterol (mmol/l) LDL cholesterol (mmol/l) Triglycerides (mmol/l) IMT (mm)
AlaVal AlaAla AlaAla AlaVal
ValVal AlaAla
AlaVal
ValVal
AlaAla
AlaVal
ValVal
Hypertensive women Control women Hypertensive men
The signal sequence polymorphism of the MnSOD was successfully genotyped and carotid IMT measured in 989 subjects of the OPERA sample. There were 232 subjects with the genotype AlaAla, 492 with the genotype AlaVal and 265 with the genotype ValVal in the sample giving the allele frequencies of 0.483 and 0.517 for the Ala and Val allele, respectively. The genotypes were in Hardy /Weinberg equilibrium. BMI, age, presence of diabetes, lipid and lipoprotein levels, and IMT for the study subjects stratified according to the genotypes of MnSOD signal sequence polymorphism are shown on Table 1. In control women, the ValVal genotype was associated with higher IMT (P /0.03 for the difference between the homozygous genotypes by Student’s T -test). In control men, the Val allele was associated with lower BMI (P /0.008 for the difference between the homozygous genotypes by the Student’s T -test). Table 2 shows the mean IMT values according to the genotypes in the quartiles of LDL cholesterol and according to the smoking status. In women, the ValVal genotype was significantly associated with higher IMT, but only in the highest LDL cholesterol quartile and in those women who had never smoked (P /0.03 and P /0.006, respectively, for the difference between homozygous genotypes by Student’s T -test). In men, the Val allele was associated with high IMT only in current smokers (P /0.05 by Student’s T test for the difference between the homozygous genotypes). There was a significant correlation between
Control men
3. Results
Table 1 Association of the genotypes of MnSOD signal sequence polymorphism with presence of diabetes, BMI, age, lipid and lipoprotein levels and IMT of carotid arteries
The results are given as means (S.D.) or percentages. All the analyses were carried out using the statistical package for social studies (SPSS) version 10.1 (SPSS Inc., Chicago, IL). The differences in lipid and lipoprotein values, BMI and carotid IMT between the different genotype groups were evaluated by Student’s T -test. The general factorial procedure of the General Linear Model (GLM) with Type III sums of squares was used to construct the model to explain the variation in carotid IMT. The genotypes of the signal sequence polymorphism indicated were added as an independent factor into the model without any assumption of the mode of inheritance (i.e. without any combination of genotypes). The h -squared statistical calculation was used to estimate how much of the variation each factor or covariate explained. Covariates included in the analysis were BMI, pack-years of smoking, age, LDL cholesterol and systolic blood pressure and the factor included was the genotype of the signal sequence polymorphism. When sexes were analyzed together, sex was also taken as a factor into the analysis.
ValVal
2.4. Statistical analysis
149 62 2 (3%) 29.2 (5.6) 51.7 (5.6) 5.63 (1.00) 1.39 (0.34) 3.46 (0.88) 1.54 (0.63) 0.83 (0.12)
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S. Kakko et al. / Atherosclerosis 168 (2003) 147 /152
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Table 2 Association between carotid IMT and the genotypes of signal sequence polymorphism in the quartiles of plasma LDL cholesterol levels and according to smoking history Men
Women
AlaAla
AlaVal
ValVal
AlaAla
AlaVal
ValVal
Q1
n/27 0.86 (0.21)
n/61 0.85 (0.18)
n/38 0.88 (0.16)
n/29 0.78 (0.08)
n/65 0.79 (0.10)
n/32 0.81 (0.11)
Q2
n/31 0.89 (0.21)
n/57 0.90 (0.18)
n/39 0.96 (0.26)
n/35 0.80 (0.11)
n/58 0.85 (0.16)
n/28 0.81 (0.09)
Q3
n/24 0.92 (0.17)
n/68 0.94 (0.20)
n/30 1.03 (0.25)
n/31 0.82 (0.12)
n/54 0.83 (0.16)
n/34 0.83 (0.11)
Q4
n/33 0.98 (0.21)
n/65 0.99 (0.26)
n/29 0.99 (0.20)
n/22 0.81 (0.11)
n/64 0.87 (0.15)
n/35 0.88 (0.14)*
Never smoked
n/36 0.92 (0.17)
n/73 0.90 (0.17)
n/45 0.91 (0.17)
n/76 0.79 (0.11)
n/160 0.84 (0.14)
n/80 0.84 (0.13)**
Ex-smokers
n/44 0.93 (0.23)
n/88 0.90 (0.21)
n/45 0.97 (0.24)
n/13 0.80 (0.11)
n/22 0.80 (0.17)
n/17 0.81 (0.11)
Current smokers
n/35 0.89 (0.20)
n/90 0.95 (0.25)
n/46 0.99 (0.25)*
n/28 0.84 (0.10)
n/59 0.84 (0.14)
n/32 0.84 (0.11)
The unadjusted results are shown n /number subjects and mean (S.D.). Q1 (lowest)-Q4 (highest) are for quartiles based on plasma LDL cholesterol levels and the borders between the quartiles are 3.00, 3.67 and 4.21 mmol/l for the men and 2.78, 3.37 and 4.01 mmol/l for the women. * P B/0.05. ** P B/0.01 for the difference between the homozygous genotypes by Student’s T -test.
plasma LDL cholesterol levels and carotid IMT in men with every genotype of signal sequence polymorphism, whereas the significant correlation was detected only in women carrying the Val allele (Table 3). In GLM analysis, the genotypes of signal sequence polymorphism were significantly (P /0.05) associated with IMT, but the genotypes explained only 1.3% of the overall variation of IMT in both sexes (Table 4). The means of IMT taken from GLM (adjusted for the other factors and covariates mentioned in the Table 4) in men Table 3 Spearman correlation coefficients between IMT and LDLs cholesterol levels separately for men and women and every genotype of MnSOD signal sequence polymorphism
Table 4 General liner models explaining the variation of carotid IMT in the study population Sex
Factor/covariate
Effect size
P- value
Men
Age Pack-years BMI LDL cholesterol Systolic blood pressure MnSOD genotype
8.1% 2.5% 0.0% 6.0% 1.7% 1.3%
B/0.001 B/0.001 0.97 B/0.001 0.003 0.04
Women
Age Pack-years BMI LDL cholesterol Systolic blood pressure MnSOD genotype
5.6% 1.0% 1.8% 0.7% 4.9% 1.3%
B/0.001 0.03 0.003 0.07 B/0.001 0.05
BMI, body mass index. LDL, low-density lipoproteins. Sex
Genotype
n
Correlation coefficient
P
Men
AlaAla AlaVal ValVal
115 251 136
0.26 0.22 0.24
0.004 B/0.001 0.005
AlaAla AlaVal ValVal
117 241 129
0.09 0.16 0.25
0.36 0.015 0.005
Women
n , number of subjects. P , the P -value for the correlation coefficient.
were 0.92 mm (the AlaAla genotype), 0.92 mm (the AlaVal genotype) and 0.96 mm (the ValVal genotype), and in women 0.80, 0.83 and 0.84 mm, respectively. If sexes were combined, the means were 0.86, 0.87 and 0.90 mm, respectively, (P /0.02). The exclusion of subjects with diabetes did not affect the results.
S. Kakko et al. / Atherosclerosis 168 (2003) 147 /152
4. Discussion Atherosclerosis is a complex process affected by a network of numerous genes and environmental factors [1,2]. Carotid IMT allows a non-invasive and quantitative determination of the degree of atherosclerosis. In the homogenous and large sample of control and hypertensive subjects from Finnish population we were able to show that the MnSOD gene may be a minor locus determining carotid atherosclerosis. The degree of carotid atherosclerosis has been found to be associated with many candidate genes of proteins involved in blood coagulation (prothrombin), lipoprotein metabolism (apolipoprotein E and cholesteryl ester transfer protein), homocysteine metabolism (5,10-methylenetetrahydrofolate reductase), enzymes protecting lipids against oxidation (paraoxonase) and nitric oxide metabolism (endothelial nitric oxide synthase) [30 /35], but the association with MnSOD gene is a new finding. The biological basis for the association between the signal sequence polymorphism of the MnSOD gene and carotid IMT is not known. It has been suggested that the signal sequence polymorphism affects the conformation of the leader sequence and therefore the premature protein with valine at position 16 in the signal sequence is not effectively targeted into the mitochondria and the anti-oxidant balance of the cell is disturbed. Antioxidant capacity in subjects with the Val allele might therefore be diminished leading to increased oxidative stress and increased atherosclerosis e.g. via expression of adhesion molecules or diminished suppression of LDL oxidation [4,6,21]. On the other hand, the MnSOD in subjects with the Val allele might be working inappropriately in the cytosol, instead of mitochondria, and the hydrogen peroxide produced might be converted into highly reactive hydroxyl radical in this cellular compartment. Our results are strongly supported by the results of a recent interesting study [23]. In this study, the mitochondrial DNA damage was positively correlated with the extent of atherosclerosis in aorta samples of both humans and mice. In addition, the apoE-knockout mice deficient in MnSOD had accelerated atherogenesis when compared to apoE-knockout mice with normal MnSOD function. Interestingly, the association between MnSOD genotype and IMT in women was seen only in those subjects with the highest plasma levels of LDL cholesterol. This strong association abolished the correlation between plasma levels of LDL cholesterol and IMT in subjects with the AlaAla genotype. There may be two alternative explanations for this finding. Firstly , the predisposing association of the Val allele is seen only in subjects with the highest LDL cholesterol, i.e. the high levels of LDL cholesterol are most harmful in subjects with diminished antioxidant capacity of MnSOD. Secondly , high levels
151
of LDL cholesterol are not harmful in women with the Ala allele since their mitochondrial targeted anti-oxidant capacity is balanced. A biological link for the interaction might be LDL oxidation suppressed by MnSOD [21]. The biological basis for the interaction with smoking and sex (the association between the MnSOD variation and carotid IMT was found only in men currently smoking and in women who had never smoked) is hard to understand and they are based on the analysis of subgroups. These results remained significant also in non-parametric Mann /Whitney U -test, excluding the confounding effect of a few outliers. In conclusion, the MnSOD locus was found to be a significant modifier of carotid atherosclerosis pointing out the importance of oxidative stress in atherosclerosis. However, the association was rather weak explaining only around 1.3% of the variation in carotid IMT compared to previously published estimated heritabilities of carotid IMT varying from 41 to 75% [36 /38].
Acknowledgements The authors gratefully acknowledge the expert technical assistance of Kirsi Kvist-Ma¨kela¨ and Marja-Leena Kyto¨kangas. This study was supported by grants from the Research Council for Health of the Academy of Finland, the Finnish Foundation for Cardiovascular Research, the Sigrid Juselius Foundation and the Paavo Nurmi Foundation.
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