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The S447X variant of lipoprotein lipase gene is associated with metabolic syndrome and lipid levels among Turks
Clinica Chimica Acta 383 (2007) 110 – 115 www.elsevier.com/locate/clinchim
The S447X variant of lipoprotein lipase gene is associated with metabolic syndrome and lipid levels among Turks Evrim Komurcu-Bayrak a , Altan Onat b , Mehves Poda a , Steve E. Humphries c , Jayshree Acharya c , Gülay Hergenc d , Neslihan Coban a , Gunay Can e , Nihan Erginel-Unaltuna a,⁎ a
c
Department of Genetics, Institute for Experimental Medical Research, Istanbul University, Istanbul, Turkey b Department of Cardiology, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey Centre for Cardiovascular Genetics, Royal Free and University College London (UCL) Medical School, Rayne Institute, London, UK d Department of Biology, Yildiz Technical University, Istanbul, Turkey e Department of Public Health, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey Received 19 April 2007; received in revised form 7 May 2007; accepted 7 May 2007 Available online 18 May 2007
Abstract Background: We evaluated the relationship of the lipoprotein lipase (LPL) S447X variant with serum lipid levels and the metabolic syndrome (MS) in the Turkish Adult Risk Factor (TARF) study. This is the first study examining this LPL variant in the Turkish population. Methods: The sample comprised 1586 Turkish adults. Genotyping was performed using the Taqman allelic discrimination assay. Results: The X447 allele frequency was 0.11 (95% CI: 0.10–0.12). X447 allele carriers had significantly higher levels of HDL-C, LDL-C and total cholesterol; and lower fasting glucose, when compared with the SS genotype in females. In men, no significant association with any parameters was seen. The genotypic impact of the S447X variant on lipid levels appears to be modulated by environmental factors, such as cigarette smoking in women. Logistic regression analysis demonstrated a significantly reduced likelihood for metabolic syndrome in female X447 allele carriers ( p = 0.04), after adjustment for age, cigarette smoking, alcohol usage and physical activity grade. Conclusions: In especially Turkish women, compared to non-carriers, carriers of the LPL X447 allele have higher levels of HDL-C, LDL-C and total cholesterol, and show a degree of protection against developing the metabolic syndrome. © 2007 Elsevier B.V. All rights reserved. Keywords: Metabolic syndrome; Lipids; Lipoprotein lipase; S447X variant; Turkish population
1. Introduction Lipoprotein lipase (LPL) is the key enzyme in plasma triglyceride catabolism, regulating fatty acid supply to peripheral tissues [1]. LPL is synthesized in various tissues, including Abbreviations: WHR, Waist–hip ratio; BMI, body mass index; CHD, coronary heart disease; HDL-C, high-density lipoprotein cholesterol; LPL, lipoprotein lipase; OR, odds ratio; MS, metabolic syndrome; TARF, Turkish Adult Risk Factor Study; SD, standard deviation. ⁎ Corresponding author. Istanbul University, Institute for Experimental Medical Research, Department of Genetics, Vakif Gureba Cad. 34080 Sehremini, Istanbul, Turkey. Tel.: +90 212 4142200 33316; fax: +90 212 5324171. E-mail addresses: [email protected] (E. Komurcu-Bayrak), [email protected] (A. Onat), [email protected] (S.E. Humphries), [email protected] (G. Hergenc), [email protected] (G. Can), [email protected] (N. Erginel-Unaltuna). 0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2007.05.007
skeletal muscle, heart, lung, brain. It is also expressed in adipocytes and cells located at atherosclerotic lesions, such as macrophages and smooth muscle cells [1,2]. The most critical location of LPL is the luminal surface of the capillary endothelium [1]. Population- and family-based studies have detected positive linkage and/or association to the LPL gene with insulin resistance [3], obesity [4], dyslipidemia [5] and myocardial infarction [6]. LPL is a well-known regulator of both plasma TG and HDL-C levels. Hence, LPL, and variations of the LPL gene, are good causal candidates for the atherosclerotic process and metabolic syndrome (MS) [3,7]. Of the more than one hundred mutations of the LPL gene identified so far, 20% occur in the noncoding regions [8]. Common variations in the LPL gene have differential effects on plasma lipid concentrations and the development of atherosclerosis [6,7]. The Ser447Ter (S447X) variant
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located in exon 9, results in premature termination of the protein by two amino acids [9]. In several other populations, the X447 allele has been found to be associated with lower plasma triglyceride levels, higher levels of HDL-cholesterol (HDL-C) and reduced risk of coronary heart disease (CHD) [10–14]. There is a variable association between LPL activity and the truncated protein which results from this variation (S447X) depending on how the LPL activity is measured: the pre-heparin LPL activity is strongly associated with the S447X variant but not with the post-heparin LPL activity [15–17]. Some studies have demonstrated clearly that the X447 allele is associated with higher post-heparin LPL activity in patients with CHD [12,14]. Thus, there is strong evidence to suggest that the S447X variant is associated with effects on plasma lipid levels, and that it may confer a protective effect against the development of MS and subsequent CHD. Recent reports indicate that the relationship between the S447X variant and its differential effect on lipid levels in several populations may be due to differences in the prevalence of both genetic and environmental factors [18,19]. The influence of LPL S447X variant on lipid levels and/or MS is unknown for the Turkish population which has a high prevalence of MS [20], as well as a genetic predisposition to low HDL-C levels [21]. The aim of this study was to examine the relation of the S447X variant with lipid levels and MS in a sample of the Turkish Adult Risk Factor Study (TARF) cohort, representative of Turkish adults [22]. 2. Subjects and methods 2.1. Study sample The design and methodology of the Turkish Adult Risk Factor Study (TARF) have been previously described [22]. Briefly, participants were randomly selected from residents of all 7 different regions of Turkey, and attended the 4 surveys 2000 through 2006. Data were obtained for history of the past years via a questionnaire, physical examination of the cardiovascular system and recording of a resting electrocardiogram. An unselected 1586 persons (772 male and 814 female) were examined for their LPL S447X genotype. Study subjects were unrelated and they gave written consent to participate in the study after being informed of its nature. The study protocol was approved by the Ethics Committee of the Istanbul Medical Faculty, Istanbul University.
2.2. Definitions Individuals with metabolic syndrome (MS) were identified when 3 out of the 5 criteria of the National Cholesterol Education Program (ATP III) [23] were met, modified for prediabetes (fasting glucose rather than 110–125 mg/dl [24]) and further for abdominal obesity using as cutpoint ≥95 cm instead of 102 cm in men, as recently assessed in the Turkish Adult Risk Factor study [25,26].
2.3. Measurement of risk factors Never-and former smokers combined (as non-smokers) and smokers formed the categories in cigarette smoking. Anyone consuming alcohol once a week or more was considered as an alcohol user. Weight was measured without shoes in light indoor clothes using a scale. Body mass index was calculated as weight divided by height squared (kg/m2). Waist circumference was measured with a tape (Roche LI95 63B 00), the subject standing and wearing only underwear, at the level midway between the lower rib margin and the iliac crest.
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Blood samples were collected after an 11-hour or longer fasting. Samples were shipped within a few hours on cooled gel packs to Istanbul to be stored at −75 °C, until analyzed at the Yýldýz Technical University. Serum concentrations of total cholesterol, fasting triglycerides, glucose, and HDL-C (directly without precipitation) were determined using enzymatic kits from Roche Diagnostics with a Hitachi 902 autoanalyzer.
2.4. Determination of the LPL S447X genotypes DNA was extracted from peripheral blood leucocytes using a QIAmp® DNA Maxi KIT (Qiagen, Hilden,Germany). Genotyping was performed using the Taqman technology (ABI 7900HT, Applied Biosystems, UK). DNA amplification was set up in 384 well plates (ABGENE Ltd.) Typical 5 μl PCR reaction consisted of 5 ng dried DNA, 2.5 μl Jumpstart TAQ ready mix (Sigma, #D6442), 0.125 μl Assay mix (Taqman probes: VIC-CCA GCC TGA CTT CTTAMRA and FAM-CCA GCC TCA CTT CT-TAMRA; Primers: 5′-TGA AAT GCC ATG ACA AGT CTC TGA A-3′ and 5′-TCA GCT TTA GCC CAG AAT GCT-3′), 2.375 μl distilled water. PCR was carried out on a MBS 384 thermocycler (Thermo Electron, UK) using the following conditions: 95 °C for 5 min, 95 °C for 15 s, 60 °C for 1 min (40 cycles). Allelic discrimination was assessed using the Taqman software.
2.5. Statistical analysis Genotypic and allelic distributions were compared using the chi-squared test. Hardy–Weinberg equilibrium was computed for the expected genotype distribution. Two-tailed t-tests were used to compare continuous variables and expressed as means and standard deviation (S.D.); while categorical variables were compared using the chi-square test. A two-tailed p value of b0.05 was considered statistically significant. Logistic regression models were used to derive maximum likelihood estimates of odds ratios (OR) and associated 95% confidence intervals (CI), and a 95% CI not overlapping 1 was considered statistically significant. Multiple logistic regression analyses were performed with low HDL-C levels as dependent and LPL SS genotype, age, body mass index, intake of alcohol, cigarette smoking, physical activity grade, lipid lowering drugs, and, in women hormonal replacement therapy, status of menopause, and birth control pills usage as independent variables in the models. All statistical analyses were performed using Windows SPSS version 10.0 software.
3. Results The biometric parameters and characteristics of the participants of the TARF study population are shown in Table 1. Study subjects (n = 1586) were genotyped for the S447X variant of the LPL gene. The distribution of the LPL S447X variant in this adult population was 79.5% (n = 1261), 19. 3% (n = 306) and 1.2% (n = 19) for the SS, SX and XX genotypes, respectively. The X447 allele frequency was found to be 0.11 (95% CI: 0.10–0.12). LPL S447X genotype distribution was in Hardy–Weinberg equilibrium. Because the number of individuals with the XX genotype was low, homozygotes for the X447 allele and heterozygotes were grouped as LPL X447 allele carriers for statistical comparisons. The prevalence of X447 allele carriers was 18.3% (n = 30) in 164 patients with coronary heart disease (CHD), and 20.7% (n = 295) in 1422 individuals without CHD ( p = 0.46). Examining associations between CHD and LPL S447X genotypes in males and females separately, the X447 allele carriers were 19.3% (n = 14) and 20.3% (n = 140) in men ( p = 0.53) and 19.3% (n = 16) and 21.2% (n = 155) in women ( p = 0.68), with and without CHD, respectively.
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Table 1 Characteristics of TARF study participants
Age (y) BMI (kg/m2) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Total cholesterol (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) Fasting triglycerides (mg/dl) Fasting glucose (mg/dl) Waist–hip ratio Lipid-lowering medication (%) Hypertension-lowering medication (%) Medication for diabetes (%) Cigarette smoking (%) Alcohol usage (%) Metabolic syndrome (%) Coronary heart disease (%) Diabetes mellitus (%) Hypertension (%) Birth control pills users (%) Menopausal status (%) Hormonal replacement therapy (%)
Females (n = 814)
Males (n = 772)
p
54 ± 11.7 30.4 ± 5.6 131 ± 25.7 81.3 ± 13.3 199.5 ± 40.5 47.6 ± 11.8 122.5 ± 34.5 147.5 ± 89.5 96.5 ± 34.3 0.87 ± 0.01 8.5 35.1 7.0 16.1 1 49.3 10.1 12.5 52.3 3.7 74.8 9.3
54.2 ± 11.7 28.03 ± 10.4 125 ± 21.4 79.3 ± 12.1 188.9 ± 40.3 39.7 ± 10.7 114.9 ± 33.8 173 ± 105.6 98.4 ± 36.4 0.94 ± 0.01 6.4 21.8 8.5 41.8 15.4 45.5 10.5 13.9 41.1
N0.05 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 N0.05 b0.001 N0.05 b0.001 N0.05 b0.001 b0.001 N0.05 N0.05 N0.05 b0.001
p values were obtained by two-tailed t-tests and the chi-square tests.
Although no significant relationship was found for the LPL S447X genotypes with diabetes, hypertension and obesity (BMI ≥ 30 kg/m2) (data not shown), there was a reverse association with metabolic syndrome. The frequency of the X447 allele was lower in the group with (9.7%) than without MS (11.8%) ( p = 0.053). Frequencies of LPL S447X genotypes were 81.6% (n = 613), 17.3% (n = 130) and 1.1% (n = 8) in participants with MS and 77.6% (n = 648), 21.1% (n = 176) and 1.3% (n = 11) in those without MS for the SS, SX and XX, respectively ( p = 0.053). Examining associations between MS and LPL S447X genotypes in males and females separately, the X447 allele carriers were 18.2% (n = 64) and 21.4% (n = 90) in men ( p = 0.276) and 18.5% (n = 74) and 23.5% (n = 97) in women ( p = 0.078), with and without MS, respectively. After adjusting for age, cigarette smoking, alcohol usage and physical
Fig. 1. Estimated mean concentrations of lipids and fasting glucose and waistto-hip ratio (WHR), and LPL S447X genotype in women (a) and in men (b). [ p values obtained by two-tailed t-tests for comparisons between SS genotype and X allele carriers (SX + XX genotypes)].
activity grade, the X447 allele was associated with protection from MS in women. LPL SS homozygotes showed significantly higher odds for the development of MS (1.46, 95% CI; 1.02–2.09) (Table 2). To explore this further, we examined the relationship between the levels of certain anthropometric variables and lipoproteins and S447X genotypes in women and men. Table 3 Multiple logistic regression analysis showing odds ratios (and 95% CI) for low serum HDL-C levels in men and women
Table 2 Multiple logistic regression analysis showing the odds ratios (and 95% CI) for metabolic syndrome in men and women Men
Current smokers Physical act. grade Alcohol usage Age, y LPL, SS
Women
OR (95% CI)
p
OR (95% CI)
p
0.66 (0.48–0.9) 0.72 (0.53–0.97) 0.88 (0.58–1.34) 1.01 (1–1.02) 1.24 (0.86–1.8)
0.009 0.03 0.56 0.15 0.25
0.57 (0.38–0.87) 0.76 (0.5–1.15) 0.24 (0.03–2) 1.04 (1.02–1.05) 1.46 (1.02–2.09)
0.01 0.19 0.19 0.0001 0.04
OR, Odds ratio; CI, confidence interval; current vs. never smokers; physical activity: high = 1, low = 0; alcohol usage: yes = 1, no = 0; S447X: 1 for SS carrier, 0 for SX + XX carrier. Model comprised 750 men and 786 women with 345 and 386 cases of metabolic syndrome, respectively. LPL SX + XX in 149 men and 163 women. p b 0.05 as indicated in bold.
Men
Current smokers Physical act. grade Alcohol usage Age, y Lipid lowering drug usage LPL, SS
Women
OR (95% CI)
p
OR (95% CI)
p
1.34 (0.97–1.86) 0.89 (0.65–1.22) 0.44 (0.29–0.67) 0.97 (0.96–0.99) 1.58 (0.83–3.02) 1.21 (0.84–1.76)
0.08 0.48 0.0001 0.0001 0.17 0.31
0.92 (0.61–1.4) 0.67 (0.45–1.02) 1.53 (0.30–7.79) 0.98 (0.96–0.99) 0.69 (0.41–1.14) 1.58 (1.11–2.25)
0.93 0.05 0.15 0.05 0.56 0.01
OR, Odds ratio; CI, confidence interval; current vs. never smokers; physical activity: high = 1, low = 0; alcohol usage: yes = 1, no = 0; lipid drugs usage: yes = 1, no = 0, S447X: 1 for SS carrier, 0 for SX + XX carrier. Model comprised 750 men and 786 women with 309 and 296 cases of low HDL-C concentrations (b50 mg/dl in women and b40 mg/dl in men), respectively. LPL SX + XX in 149 men and 163 women. In women also adjusted for menopausal status, hormonal replacement therapy and birth control pills usage. p b 0.05 as indicated in bold.
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Table 4 Mean ± SE biometric and biochemical profile distributions in smokers and non-smokers according to LPL S447X genotypes in womena Non-smokers
Total cholesterol (mg/dl) HDL-C (mg/dl) Fasting triglyceride (mg/dl) LDL-C (mg/dl) Fasting glucose (mg/dl) Waist–hip-ratio
Smokers
SS (n = 532)
SX + XX (n = 142)
SS (n = 104)
SX + XX (n = 25)
197.8 ± 1.7 47.1 ± 0.5 146 ± 3.9 121.6 ± 1.5 99.2 ± 1.6 0.87 ± 0.007
210 ± 3.3⁎ 51.1 ± 1 ⁎ 149 ± 7.4 130 ± 3⁎⁎ 92 ± 3.1⁎⁎⁎ 0.86 ± 0.007⁎⁎⁎
199 ± 3.9 46 ± 1.1 157.4 ± 9.9 120.2 ± 3.4 89.5 ± 2.5 0.84 ± 0.007
198.8 ± 33 47.6 ± 2.3 127.7 ± 20.8 124.6 ± 7.1 93 ± 5.1 0.85 ± 0.01
a⁎
p b 0.001, ⁎⁎p = 0.01 ⁎⁎⁎p b 0.05. p values were obtained by age-adjusted univariate tests for comparisons between SS genotype and X allele carriers (SX + XX genotypes).
Although there were no differences in fasting triglycerides, there were statistically significant associations with total cholesterol, HDL-C, LDL-C and fasting glucose levels in women (Fig. 1a). Female X447 carriers had higher serum concentrations of total cholesterol, HDL-C and LDL-C, but lower fasting glucose levels, when compared with the SS genotype. Additionally, they had lower waist-to-hip ratio (WHR) at a borderline significance (p = 0.09). In men, by contrast, each parameter displayed similar levels in the genotypes (SS, SX + XX) (Fig. 1b). To search for a combined effect of environmental factors with the S447X variant in determining low concentrations of HDL-C (b50 mg/dl in women, b 40 mg/dl in men), logistic regression models were used for men and women separately, in which cigarette smoking, alcohol usage, physical activity grade, BMI, lipid-altering medications and age were also included (Table 3). Compared with the X447 allele carriers, LPL SS homozygotes showed significantly higher odds for low HDL-C levels (1.58, 95% CI; 1.11–2.25) among women, but not among men. Multifactorial analysis in women included adjustments also for menopausal status, hormonal replacement therapy and birth control pills usage. In these subjects, the frequency of cigarette smokers was 16.1% in women and 41.8% in men (Table 1), and there was no difference in frequency of the X447 allele between smokers and non-smokers. Statistically significant parameters in smokers and non-smokers by genotypes in females are shown in Table 4. The association of the X447 allele with the previously mentioned risk parameters was significant only in non-smoking females but not in smoking women. 4. Discussion In this study, the association of the LPL S447X variant with blood lipid-levels and MS was examined in a large sample of the TARF study, representative of Turkish adults. It is the first report to describe the genotype distribution of the LPL S447X variant in this population. The key findings are that X447 allele carriers had significantly higher levels of HDL-C, LDL-C, total cholesterol and lower fasting glucose in women. These were reflected in a significantly reduced likelihood of MS among female carriers, with a smaller non-significant effect in men. Non-carriers exhibited a multi-adjusted significantly elevated association for low HDL-C levels in women (OR 1.58)
again with a smaller non-significant effect in men (OR 1.2). The association between the X447 allele and higher levels of HDL-C, LDL-C, total cholesterol and lower of fasting glucose and WHR was essentially confined to non smoking women. It is of relevance to compare the allele frequency of this variant in the Turkish population and others, in view of considerable diversity reported in other populations (7.4–14%) [13,18,27–29]. In Turkish subjects, the LPL S447X variant has an allelic frequency of 11%, which is similar to that reported by others [13,27]. Agreement exists in most studies on the effects of the different LPL-genotypes on blood lipid profile, though controversial results have been reported as to effects on cardiovascular disease. For example, in subjects from Ireland and France the S447X variant was not shown to be associated with the risk of CHD, whereas an intronic polymorphism (HindIII) was found to be influencing this risk, although in German subjects the same intronic polymorphism was not associated with myocardial infarction [5,6]. We chose to examine the S447X variant of the LPL gene, because it is known to have an effect on increased enzyme activity and to result in a protective lipoprotein profile. The X447 allele was associated with no significant reduction in CHD among women, in contrast to an 18% decrease in risk of ischemic heart disease in men in a recent meta-analysis [27]. Our results are not conclusive for this evaluation, because the number of the patients with coronary heart disease in this particular study population (83 women and 81 men) was low. This variant has previously been shown to be associated with alterations in lipoprotein concentrations and various medical conditions [10,11,28]. However, to our knowledge, this study is the first to report a multiple adjusted association between the LPL S447X variant and clinically identified MS. Female carrier participants displayed a risk for MS reduced by about one-third compared to female non-carriers. This association is likely to be mediated by the impact of these genotypes (SX + XX) on higher levels of HDL-C and lower fasting glucose levels in women. It is unclear why this variant was associated with higher LDL- (and total) cholesterol as well, but a similar correlation was reported by Kobayashi and Saito [29] between serum preheparin LPL mass and total cholesterol. Furthermore, a similar genotype association with LDL and total cholesterol was reported in men of the Framingham Offspring Study [13]. It is an unpublished observation in the TARF study that elevated
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LDL-cholesterol levels interact with abdominal obesity in Turkish women to enhance the development of diabetes. This predisposition to LDL elevation may be related to genetically enhanced whole-body insulin sensitivity relative to hepatic insulin resistance in centrally obese Turkish women (but not men). Recent studies suggested that the relationship between the S447X variant and its differential effect on lipid levels may be due to differences in the prevalence of both genetic and environmental factors [18,19]. Hormonal differences between gender and lifestyle characteristics may play important roles in modulating this relationship. Our results clearly show that the X447 allele was significantly associated with increased HDL-C serum levels in females only. Similar gender-specific effects for HDL-C levels have been reported previously [28,29]. By contrast, in the Framingham Offspring study, the X447 allele was shown to be associated with increased total cholesterol, LDL-C and HDL-C levels in males, but not in females [13]. These differences emphasize the importance of analyzing the genetic association for diseases in each gender separately. Environmental factors, such as smoking status, alcohol consumption and physical inactivity are risk factors for lipoprotein disturbances and MS, in part mediated by alteration of the LPL activity. The genotypic impact of the S447X variant on lipid levels have been reported to be modulated by cigarette smoking and alcohol consumption [18,19,30]. Though the lack of a “protective” effect among smoking women may be due to the smaller number of female smokers, we interpret rather that cigarette smoking may have masked the effects of the X447 allele on HDLcholesterol, glucose, triglyceride and waist-to-hip ratio, since differences in risk variables between carriers of opposing alleles were not of similar magnitude or in the same direction. This study is distinct by being the first to report the allelic frequency of the S447X variation in the LPL gene in a large sample of Turkish adults, and to show in female carriers a reduced likelihood for the metabolic syndrome, compared to non-carriers. Acknowledgments This study was supported by the Scientific and Technological Research Council of Turkey (TUBITAK Project number: SBAG-3091). We thank the Turkish Society of Cardiology and the pharmaceutical companies AstraZeneca, Pfizer and SanofiAventis (Istanbul) that have supported financially the Turkish Adult Risk Factor survey. The partial logistic support of the Turkish Ministry of Health is acknowledged. We appreciate the dedicated work of the survey teams. SEH and JA are supported by the British Heart Foundation (RG2005/014). References [1] Zechner R. The tissue-specific expression of lipoprotein lipase: implications for energy and lipoprotein metabolism. Curr Opin Lipidol 1997;8:77–88. [2] Van Eck M, Zimmermann R, Groot PH, Zechner R, Van Berkel TJ. Role of macrophage-derived lipoprotein lipase in lipoprotein metabolism and atherosclerosis. Arterioscler Thromb Vasc Biol 2000;20:E53–62.
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The S447X variant of lipoprotein lipase gene is associated with metabolic syndrome and lipid levels among Turks Evrim Komurcu-Bayrak a , Altan Onat b , Mehves Poda a , Steve E. Humphries c , Jayshree Acharya c , Gülay Hergenc d , Neslihan Coban a , Gunay Can e , Nihan Erginel-Unaltuna a,⁎ a
c
Department of Genetics, Institute for Experimental Medical Research, Istanbul University, Istanbul, Turkey b Department of Cardiology, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey Centre for Cardiovascular Genetics, Royal Free and University College London (UCL) Medical School, Rayne Institute, London, UK d Department of Biology, Yildiz Technical University, Istanbul, Turkey e Department of Public Health, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey Received 19 April 2007; received in revised form 7 May 2007; accepted 7 May 2007 Available online 18 May 2007
Abstract Background: We evaluated the relationship of the lipoprotein lipase (LPL) S447X variant with serum lipid levels and the metabolic syndrome (MS) in the Turkish Adult Risk Factor (TARF) study. This is the first study examining this LPL variant in the Turkish population. Methods: The sample comprised 1586 Turkish adults. Genotyping was performed using the Taqman allelic discrimination assay. Results: The X447 allele frequency was 0.11 (95% CI: 0.10–0.12). X447 allele carriers had significantly higher levels of HDL-C, LDL-C and total cholesterol; and lower fasting glucose, when compared with the SS genotype in females. In men, no significant association with any parameters was seen. The genotypic impact of the S447X variant on lipid levels appears to be modulated by environmental factors, such as cigarette smoking in women. Logistic regression analysis demonstrated a significantly reduced likelihood for metabolic syndrome in female X447 allele carriers ( p = 0.04), after adjustment for age, cigarette smoking, alcohol usage and physical activity grade. Conclusions: In especially Turkish women, compared to non-carriers, carriers of the LPL X447 allele have higher levels of HDL-C, LDL-C and total cholesterol, and show a degree of protection against developing the metabolic syndrome. © 2007 Elsevier B.V. All rights reserved. Keywords: Metabolic syndrome; Lipids; Lipoprotein lipase; S447X variant; Turkish population
1. Introduction Lipoprotein lipase (LPL) is the key enzyme in plasma triglyceride catabolism, regulating fatty acid supply to peripheral tissues [1]. LPL is synthesized in various tissues, including Abbreviations: WHR, Waist–hip ratio; BMI, body mass index; CHD, coronary heart disease; HDL-C, high-density lipoprotein cholesterol; LPL, lipoprotein lipase; OR, odds ratio; MS, metabolic syndrome; TARF, Turkish Adult Risk Factor Study; SD, standard deviation. ⁎ Corresponding author. Istanbul University, Institute for Experimental Medical Research, Department of Genetics, Vakif Gureba Cad. 34080 Sehremini, Istanbul, Turkey. Tel.: +90 212 4142200 33316; fax: +90 212 5324171. E-mail addresses: [email protected] (E. Komurcu-Bayrak), [email protected] (A. Onat), [email protected] (S.E. Humphries), [email protected] (G. Hergenc), [email protected] (G. Can), [email protected] (N. Erginel-Unaltuna). 0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2007.05.007
skeletal muscle, heart, lung, brain. It is also expressed in adipocytes and cells located at atherosclerotic lesions, such as macrophages and smooth muscle cells [1,2]. The most critical location of LPL is the luminal surface of the capillary endothelium [1]. Population- and family-based studies have detected positive linkage and/or association to the LPL gene with insulin resistance [3], obesity [4], dyslipidemia [5] and myocardial infarction [6]. LPL is a well-known regulator of both plasma TG and HDL-C levels. Hence, LPL, and variations of the LPL gene, are good causal candidates for the atherosclerotic process and metabolic syndrome (MS) [3,7]. Of the more than one hundred mutations of the LPL gene identified so far, 20% occur in the noncoding regions [8]. Common variations in the LPL gene have differential effects on plasma lipid concentrations and the development of atherosclerosis [6,7]. The Ser447Ter (S447X) variant
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located in exon 9, results in premature termination of the protein by two amino acids [9]. In several other populations, the X447 allele has been found to be associated with lower plasma triglyceride levels, higher levels of HDL-cholesterol (HDL-C) and reduced risk of coronary heart disease (CHD) [10–14]. There is a variable association between LPL activity and the truncated protein which results from this variation (S447X) depending on how the LPL activity is measured: the pre-heparin LPL activity is strongly associated with the S447X variant but not with the post-heparin LPL activity [15–17]. Some studies have demonstrated clearly that the X447 allele is associated with higher post-heparin LPL activity in patients with CHD [12,14]. Thus, there is strong evidence to suggest that the S447X variant is associated with effects on plasma lipid levels, and that it may confer a protective effect against the development of MS and subsequent CHD. Recent reports indicate that the relationship between the S447X variant and its differential effect on lipid levels in several populations may be due to differences in the prevalence of both genetic and environmental factors [18,19]. The influence of LPL S447X variant on lipid levels and/or MS is unknown for the Turkish population which has a high prevalence of MS [20], as well as a genetic predisposition to low HDL-C levels [21]. The aim of this study was to examine the relation of the S447X variant with lipid levels and MS in a sample of the Turkish Adult Risk Factor Study (TARF) cohort, representative of Turkish adults [22]. 2. Subjects and methods 2.1. Study sample The design and methodology of the Turkish Adult Risk Factor Study (TARF) have been previously described [22]. Briefly, participants were randomly selected from residents of all 7 different regions of Turkey, and attended the 4 surveys 2000 through 2006. Data were obtained for history of the past years via a questionnaire, physical examination of the cardiovascular system and recording of a resting electrocardiogram. An unselected 1586 persons (772 male and 814 female) were examined for their LPL S447X genotype. Study subjects were unrelated and they gave written consent to participate in the study after being informed of its nature. The study protocol was approved by the Ethics Committee of the Istanbul Medical Faculty, Istanbul University.
2.2. Definitions Individuals with metabolic syndrome (MS) were identified when 3 out of the 5 criteria of the National Cholesterol Education Program (ATP III) [23] were met, modified for prediabetes (fasting glucose rather than 110–125 mg/dl [24]) and further for abdominal obesity using as cutpoint ≥95 cm instead of 102 cm in men, as recently assessed in the Turkish Adult Risk Factor study [25,26].
2.3. Measurement of risk factors Never-and former smokers combined (as non-smokers) and smokers formed the categories in cigarette smoking. Anyone consuming alcohol once a week or more was considered as an alcohol user. Weight was measured without shoes in light indoor clothes using a scale. Body mass index was calculated as weight divided by height squared (kg/m2). Waist circumference was measured with a tape (Roche LI95 63B 00), the subject standing and wearing only underwear, at the level midway between the lower rib margin and the iliac crest.
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Blood samples were collected after an 11-hour or longer fasting. Samples were shipped within a few hours on cooled gel packs to Istanbul to be stored at −75 °C, until analyzed at the Yýldýz Technical University. Serum concentrations of total cholesterol, fasting triglycerides, glucose, and HDL-C (directly without precipitation) were determined using enzymatic kits from Roche Diagnostics with a Hitachi 902 autoanalyzer.
2.4. Determination of the LPL S447X genotypes DNA was extracted from peripheral blood leucocytes using a QIAmp® DNA Maxi KIT (Qiagen, Hilden,Germany). Genotyping was performed using the Taqman technology (ABI 7900HT, Applied Biosystems, UK). DNA amplification was set up in 384 well plates (ABGENE Ltd.) Typical 5 μl PCR reaction consisted of 5 ng dried DNA, 2.5 μl Jumpstart TAQ ready mix (Sigma, #D6442), 0.125 μl Assay mix (Taqman probes: VIC-CCA GCC TGA CTT CTTAMRA and FAM-CCA GCC TCA CTT CT-TAMRA; Primers: 5′-TGA AAT GCC ATG ACA AGT CTC TGA A-3′ and 5′-TCA GCT TTA GCC CAG AAT GCT-3′), 2.375 μl distilled water. PCR was carried out on a MBS 384 thermocycler (Thermo Electron, UK) using the following conditions: 95 °C for 5 min, 95 °C for 15 s, 60 °C for 1 min (40 cycles). Allelic discrimination was assessed using the Taqman software.
2.5. Statistical analysis Genotypic and allelic distributions were compared using the chi-squared test. Hardy–Weinberg equilibrium was computed for the expected genotype distribution. Two-tailed t-tests were used to compare continuous variables and expressed as means and standard deviation (S.D.); while categorical variables were compared using the chi-square test. A two-tailed p value of b0.05 was considered statistically significant. Logistic regression models were used to derive maximum likelihood estimates of odds ratios (OR) and associated 95% confidence intervals (CI), and a 95% CI not overlapping 1 was considered statistically significant. Multiple logistic regression analyses were performed with low HDL-C levels as dependent and LPL SS genotype, age, body mass index, intake of alcohol, cigarette smoking, physical activity grade, lipid lowering drugs, and, in women hormonal replacement therapy, status of menopause, and birth control pills usage as independent variables in the models. All statistical analyses were performed using Windows SPSS version 10.0 software.
3. Results The biometric parameters and characteristics of the participants of the TARF study population are shown in Table 1. Study subjects (n = 1586) were genotyped for the S447X variant of the LPL gene. The distribution of the LPL S447X variant in this adult population was 79.5% (n = 1261), 19. 3% (n = 306) and 1.2% (n = 19) for the SS, SX and XX genotypes, respectively. The X447 allele frequency was found to be 0.11 (95% CI: 0.10–0.12). LPL S447X genotype distribution was in Hardy–Weinberg equilibrium. Because the number of individuals with the XX genotype was low, homozygotes for the X447 allele and heterozygotes were grouped as LPL X447 allele carriers for statistical comparisons. The prevalence of X447 allele carriers was 18.3% (n = 30) in 164 patients with coronary heart disease (CHD), and 20.7% (n = 295) in 1422 individuals without CHD ( p = 0.46). Examining associations between CHD and LPL S447X genotypes in males and females separately, the X447 allele carriers were 19.3% (n = 14) and 20.3% (n = 140) in men ( p = 0.53) and 19.3% (n = 16) and 21.2% (n = 155) in women ( p = 0.68), with and without CHD, respectively.
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Table 1 Characteristics of TARF study participants
Age (y) BMI (kg/m2) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Total cholesterol (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) Fasting triglycerides (mg/dl) Fasting glucose (mg/dl) Waist–hip ratio Lipid-lowering medication (%) Hypertension-lowering medication (%) Medication for diabetes (%) Cigarette smoking (%) Alcohol usage (%) Metabolic syndrome (%) Coronary heart disease (%) Diabetes mellitus (%) Hypertension (%) Birth control pills users (%) Menopausal status (%) Hormonal replacement therapy (%)
Females (n = 814)
Males (n = 772)
p
54 ± 11.7 30.4 ± 5.6 131 ± 25.7 81.3 ± 13.3 199.5 ± 40.5 47.6 ± 11.8 122.5 ± 34.5 147.5 ± 89.5 96.5 ± 34.3 0.87 ± 0.01 8.5 35.1 7.0 16.1 1 49.3 10.1 12.5 52.3 3.7 74.8 9.3
54.2 ± 11.7 28.03 ± 10.4 125 ± 21.4 79.3 ± 12.1 188.9 ± 40.3 39.7 ± 10.7 114.9 ± 33.8 173 ± 105.6 98.4 ± 36.4 0.94 ± 0.01 6.4 21.8 8.5 41.8 15.4 45.5 10.5 13.9 41.1
N0.05 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 N0.05 b0.001 N0.05 b0.001 N0.05 b0.001 b0.001 N0.05 N0.05 N0.05 b0.001
p values were obtained by two-tailed t-tests and the chi-square tests.
Although no significant relationship was found for the LPL S447X genotypes with diabetes, hypertension and obesity (BMI ≥ 30 kg/m2) (data not shown), there was a reverse association with metabolic syndrome. The frequency of the X447 allele was lower in the group with (9.7%) than without MS (11.8%) ( p = 0.053). Frequencies of LPL S447X genotypes were 81.6% (n = 613), 17.3% (n = 130) and 1.1% (n = 8) in participants with MS and 77.6% (n = 648), 21.1% (n = 176) and 1.3% (n = 11) in those without MS for the SS, SX and XX, respectively ( p = 0.053). Examining associations between MS and LPL S447X genotypes in males and females separately, the X447 allele carriers were 18.2% (n = 64) and 21.4% (n = 90) in men ( p = 0.276) and 18.5% (n = 74) and 23.5% (n = 97) in women ( p = 0.078), with and without MS, respectively. After adjusting for age, cigarette smoking, alcohol usage and physical
Fig. 1. Estimated mean concentrations of lipids and fasting glucose and waistto-hip ratio (WHR), and LPL S447X genotype in women (a) and in men (b). [ p values obtained by two-tailed t-tests for comparisons between SS genotype and X allele carriers (SX + XX genotypes)].
activity grade, the X447 allele was associated with protection from MS in women. LPL SS homozygotes showed significantly higher odds for the development of MS (1.46, 95% CI; 1.02–2.09) (Table 2). To explore this further, we examined the relationship between the levels of certain anthropometric variables and lipoproteins and S447X genotypes in women and men. Table 3 Multiple logistic regression analysis showing odds ratios (and 95% CI) for low serum HDL-C levels in men and women
Table 2 Multiple logistic regression analysis showing the odds ratios (and 95% CI) for metabolic syndrome in men and women Men
Current smokers Physical act. grade Alcohol usage Age, y LPL, SS
Women
OR (95% CI)
p
OR (95% CI)
p
0.66 (0.48–0.9) 0.72 (0.53–0.97) 0.88 (0.58–1.34) 1.01 (1–1.02) 1.24 (0.86–1.8)
0.009 0.03 0.56 0.15 0.25
0.57 (0.38–0.87) 0.76 (0.5–1.15) 0.24 (0.03–2) 1.04 (1.02–1.05) 1.46 (1.02–2.09)
0.01 0.19 0.19 0.0001 0.04
OR, Odds ratio; CI, confidence interval; current vs. never smokers; physical activity: high = 1, low = 0; alcohol usage: yes = 1, no = 0; S447X: 1 for SS carrier, 0 for SX + XX carrier. Model comprised 750 men and 786 women with 345 and 386 cases of metabolic syndrome, respectively. LPL SX + XX in 149 men and 163 women. p b 0.05 as indicated in bold.
Men
Current smokers Physical act. grade Alcohol usage Age, y Lipid lowering drug usage LPL, SS
Women
OR (95% CI)
p
OR (95% CI)
p
1.34 (0.97–1.86) 0.89 (0.65–1.22) 0.44 (0.29–0.67) 0.97 (0.96–0.99) 1.58 (0.83–3.02) 1.21 (0.84–1.76)
0.08 0.48 0.0001 0.0001 0.17 0.31
0.92 (0.61–1.4) 0.67 (0.45–1.02) 1.53 (0.30–7.79) 0.98 (0.96–0.99) 0.69 (0.41–1.14) 1.58 (1.11–2.25)
0.93 0.05 0.15 0.05 0.56 0.01
OR, Odds ratio; CI, confidence interval; current vs. never smokers; physical activity: high = 1, low = 0; alcohol usage: yes = 1, no = 0; lipid drugs usage: yes = 1, no = 0, S447X: 1 for SS carrier, 0 for SX + XX carrier. Model comprised 750 men and 786 women with 309 and 296 cases of low HDL-C concentrations (b50 mg/dl in women and b40 mg/dl in men), respectively. LPL SX + XX in 149 men and 163 women. In women also adjusted for menopausal status, hormonal replacement therapy and birth control pills usage. p b 0.05 as indicated in bold.
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Table 4 Mean ± SE biometric and biochemical profile distributions in smokers and non-smokers according to LPL S447X genotypes in womena Non-smokers
Total cholesterol (mg/dl) HDL-C (mg/dl) Fasting triglyceride (mg/dl) LDL-C (mg/dl) Fasting glucose (mg/dl) Waist–hip-ratio
Smokers
SS (n = 532)
SX + XX (n = 142)
SS (n = 104)
SX + XX (n = 25)
197.8 ± 1.7 47.1 ± 0.5 146 ± 3.9 121.6 ± 1.5 99.2 ± 1.6 0.87 ± 0.007
210 ± 3.3⁎ 51.1 ± 1 ⁎ 149 ± 7.4 130 ± 3⁎⁎ 92 ± 3.1⁎⁎⁎ 0.86 ± 0.007⁎⁎⁎
199 ± 3.9 46 ± 1.1 157.4 ± 9.9 120.2 ± 3.4 89.5 ± 2.5 0.84 ± 0.007
198.8 ± 33 47.6 ± 2.3 127.7 ± 20.8 124.6 ± 7.1 93 ± 5.1 0.85 ± 0.01
a⁎
p b 0.001, ⁎⁎p = 0.01 ⁎⁎⁎p b 0.05. p values were obtained by age-adjusted univariate tests for comparisons between SS genotype and X allele carriers (SX + XX genotypes).
Although there were no differences in fasting triglycerides, there were statistically significant associations with total cholesterol, HDL-C, LDL-C and fasting glucose levels in women (Fig. 1a). Female X447 carriers had higher serum concentrations of total cholesterol, HDL-C and LDL-C, but lower fasting glucose levels, when compared with the SS genotype. Additionally, they had lower waist-to-hip ratio (WHR) at a borderline significance (p = 0.09). In men, by contrast, each parameter displayed similar levels in the genotypes (SS, SX + XX) (Fig. 1b). To search for a combined effect of environmental factors with the S447X variant in determining low concentrations of HDL-C (b50 mg/dl in women, b 40 mg/dl in men), logistic regression models were used for men and women separately, in which cigarette smoking, alcohol usage, physical activity grade, BMI, lipid-altering medications and age were also included (Table 3). Compared with the X447 allele carriers, LPL SS homozygotes showed significantly higher odds for low HDL-C levels (1.58, 95% CI; 1.11–2.25) among women, but not among men. Multifactorial analysis in women included adjustments also for menopausal status, hormonal replacement therapy and birth control pills usage. In these subjects, the frequency of cigarette smokers was 16.1% in women and 41.8% in men (Table 1), and there was no difference in frequency of the X447 allele between smokers and non-smokers. Statistically significant parameters in smokers and non-smokers by genotypes in females are shown in Table 4. The association of the X447 allele with the previously mentioned risk parameters was significant only in non-smoking females but not in smoking women. 4. Discussion In this study, the association of the LPL S447X variant with blood lipid-levels and MS was examined in a large sample of the TARF study, representative of Turkish adults. It is the first report to describe the genotype distribution of the LPL S447X variant in this population. The key findings are that X447 allele carriers had significantly higher levels of HDL-C, LDL-C, total cholesterol and lower fasting glucose in women. These were reflected in a significantly reduced likelihood of MS among female carriers, with a smaller non-significant effect in men. Non-carriers exhibited a multi-adjusted significantly elevated association for low HDL-C levels in women (OR 1.58)
again with a smaller non-significant effect in men (OR 1.2). The association between the X447 allele and higher levels of HDL-C, LDL-C, total cholesterol and lower of fasting glucose and WHR was essentially confined to non smoking women. It is of relevance to compare the allele frequency of this variant in the Turkish population and others, in view of considerable diversity reported in other populations (7.4–14%) [13,18,27–29]. In Turkish subjects, the LPL S447X variant has an allelic frequency of 11%, which is similar to that reported by others [13,27]. Agreement exists in most studies on the effects of the different LPL-genotypes on blood lipid profile, though controversial results have been reported as to effects on cardiovascular disease. For example, in subjects from Ireland and France the S447X variant was not shown to be associated with the risk of CHD, whereas an intronic polymorphism (HindIII) was found to be influencing this risk, although in German subjects the same intronic polymorphism was not associated with myocardial infarction [5,6]. We chose to examine the S447X variant of the LPL gene, because it is known to have an effect on increased enzyme activity and to result in a protective lipoprotein profile. The X447 allele was associated with no significant reduction in CHD among women, in contrast to an 18% decrease in risk of ischemic heart disease in men in a recent meta-analysis [27]. Our results are not conclusive for this evaluation, because the number of the patients with coronary heart disease in this particular study population (83 women and 81 men) was low. This variant has previously been shown to be associated with alterations in lipoprotein concentrations and various medical conditions [10,11,28]. However, to our knowledge, this study is the first to report a multiple adjusted association between the LPL S447X variant and clinically identified MS. Female carrier participants displayed a risk for MS reduced by about one-third compared to female non-carriers. This association is likely to be mediated by the impact of these genotypes (SX + XX) on higher levels of HDL-C and lower fasting glucose levels in women. It is unclear why this variant was associated with higher LDL- (and total) cholesterol as well, but a similar correlation was reported by Kobayashi and Saito [29] between serum preheparin LPL mass and total cholesterol. Furthermore, a similar genotype association with LDL and total cholesterol was reported in men of the Framingham Offspring Study [13]. It is an unpublished observation in the TARF study that elevated
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LDL-cholesterol levels interact with abdominal obesity in Turkish women to enhance the development of diabetes. This predisposition to LDL elevation may be related to genetically enhanced whole-body insulin sensitivity relative to hepatic insulin resistance in centrally obese Turkish women (but not men). Recent studies suggested that the relationship between the S447X variant and its differential effect on lipid levels may be due to differences in the prevalence of both genetic and environmental factors [18,19]. Hormonal differences between gender and lifestyle characteristics may play important roles in modulating this relationship. Our results clearly show that the X447 allele was significantly associated with increased HDL-C serum levels in females only. Similar gender-specific effects for HDL-C levels have been reported previously [28,29]. By contrast, in the Framingham Offspring study, the X447 allele was shown to be associated with increased total cholesterol, LDL-C and HDL-C levels in males, but not in females [13]. These differences emphasize the importance of analyzing the genetic association for diseases in each gender separately. Environmental factors, such as smoking status, alcohol consumption and physical inactivity are risk factors for lipoprotein disturbances and MS, in part mediated by alteration of the LPL activity. The genotypic impact of the S447X variant on lipid levels have been reported to be modulated by cigarette smoking and alcohol consumption [18,19,30]. Though the lack of a “protective” effect among smoking women may be due to the smaller number of female smokers, we interpret rather that cigarette smoking may have masked the effects of the X447 allele on HDLcholesterol, glucose, triglyceride and waist-to-hip ratio, since differences in risk variables between carriers of opposing alleles were not of similar magnitude or in the same direction. This study is distinct by being the first to report the allelic frequency of the S447X variation in the LPL gene in a large sample of Turkish adults, and to show in female carriers a reduced likelihood for the metabolic syndrome, compared to non-carriers. Acknowledgments This study was supported by the Scientific and Technological Research Council of Turkey (TUBITAK Project number: SBAG-3091). We thank the Turkish Society of Cardiology and the pharmaceutical companies AstraZeneca, Pfizer and SanofiAventis (Istanbul) that have supported financially the Turkish Adult Risk Factor survey. The partial logistic support of the Turkish Ministry of Health is acknowledged. We appreciate the dedicated work of the survey teams. SEH and JA are supported by the British Heart Foundation (RG2005/014). References [1] Zechner R. The tissue-specific expression of lipoprotein lipase: implications for energy and lipoprotein metabolism. Curr Opin Lipidol 1997;8:77–88. [2] Van Eck M, Zimmermann R, Groot PH, Zechner R, Van Berkel TJ. Role of macrophage-derived lipoprotein lipase in lipoprotein metabolism and atherosclerosis. Arterioscler Thromb Vasc Biol 2000;20:E53–62.
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