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Journal of Human Hypertension (2007) 21, 337–339 & 2007 Nature Publishing Group All rights reserved 0950-9240/07 $30.00 www.nature.com/jhh RESEARCH LETTER E and low-density gene polymorphisms in dyslipidemias-associated essential hypertension

Journal of Human Hypertension (2007) 21, 337–339. (DBP)X90 mm Hg; (3) free of secondary causes of doi:10.1038/sj.jhh.1002151; published online 18 January 2007 hypertension through extensive biochemical and clinical examination; (4) without pharmacological treatment for hypertension. The BP of all 236 Hypertension has been strongly linked to a variety normotensives (131 male subjects/105 female sub- of lipoprotein metabolic abnormalities. Polymorph- jects) was below 140/90 mm Hg; all normotensives isms in the genes encoding the matched hypertensives for area, gender and age involved in metabolic abnormalities are the major without familial hypertension history, hepatic dis- target for evaluating the predisposition to the ease, renal insufficiency and diabetes. This study development of hypertension. In this study, two was approved by the Ethics Committee of Chinese candidate genes including (ApoE) Academy of Medical Sciences/Peking Union Medi- and low-density lipoprotein receptor (LDLR) were cal College and informed consent was obtained from selected to examine their possible contribution to each subject. hypertension in a Chinese Han population. Blood samples were drawn for DNA extraction Hypertension is a complex multifactorial and and biochemical examination. Genomic DNA was polygenic disorder that is thought to result from an extracted from white blood cells. Plasma TC, TG and interaction between an individual’s genetic makeup high-density lipoprotein cholestrol (HDLC) concen- and various environmental factors.1 Given that trations were determined enzymatically using com- hypertension is a major risk factor for cardiovascular mercially available kits and auto analyzer, and LDLC diseases, stroke and end-stage renal disease and was estimated using Friedewald’s formula. prevails globally, prevention of hypertension is an Two common polymorphisms including ApoE important public health goal.2 Onepreventiveap- e2/e3/e4 (rs7412/429358) and LDLR C1773T (rs688) proach is to identify disease-susceptibility genes that were selected based on physiological, biochemical involve a specific physiological or cellular function. and genetic studies reported previously. They are There is growing awareness that patients with involved in the regulation of lipoprotein metabo- hypertension tend to have a high prevalence of lism. The amplification of ApoE e2/e3/e4 poly- associated dyslipidemias such as the elevated total morphism was performed with the primers 50-AAC (TC) and (TG), and reduced AAC TGA CCC CGG TGG CG-30 (sense) and 50-ATG low-density lipoprotein cholesterol (LDLC).3 How- GCG CTG AGG CCG CGC TC-30 (antisense). Poly- ever, the exact mechanism leading to these dyslipi- merase chain reaction (PCR) reactions began with an demias remains unknown. It is reasonable to expect initial denaturation at 941C (5 min), then 30 cycles of that multiple genetic and/or physiological safe- 941C (45 s), 651C (45 s) and 721C (45 s), followed by a guards have developed to maintain lipoprotein final extension at 721C (5 min). PCR products were levels within a range of physiologically acceptable restricted with HhaI (Promega, Beijing, China), and levels. To address this issue, we performed an separated on 8% polyacrylamide gels. The C1773T association study for two lipoprotein-related gene polymorphism of LDLR was genotyped according to polymorphisms and hypertension. Our aim was to the previous report.4 evaluate the relationship of these polymorphisms Statistical calculations were performed using SAS with hypertension, as well as blood pressure and 9.1.3 (Institute Inc., Cary, NC, USA). Hardy–Weinberg plasma lipoprotein profiles, both solely and com- (H–W) equilibrium was tested using w2 test. Compar- bined in a Chinese population. isons among different means were calculated using All subjects in this study were of Han Chinese ANOVA. The association between polymorphisms currently residing in Shijiazhuang city. A total of and hypertension was analysed by logistic regression; 269 hypertensives (153 male subjects/116 female odds ratio (OR), and 95% confidence interval were subjects) met the following criteria: (1) onset of calculated. Data were expressed as mean7s.d.; all hypertension before age 65 years; (2) systolic P-values were two-sided; Po0.05 was considered blood pressure (SBP)X140 mm Hg or diastolic BP statistically significant. Research Letter 338 Table 1 The and genotype distributions of ApoE and LDLR in both groups and the comparisons of blood pressure and plasma lipoprotein profiles among different genotypes both solely and combined in hypertensives

Genotype counts (n) Allele counts (n)

ApoE E2/2 E2/3 E2/4 E3/3 E3/4 E4/4 P e2 e3 e4P

Hypertensives 12 80 48 73 52 4 152 278 108 Normotensives 15 118 9 77 16 1 o0.0001w 157 288 27 0.0006z

LDLR CC CT TT P CT P Hypertensives 100 96 73 296 242 Normotensives 127 85 24 o0.0001z 339 133 o0.0001

Hypertensives ApoE genotypes LDLR genotypes

E2/2+E2/3 E3/3 E3/4+E4/4 CC CT TT

BMI (kg/m2) 25.872.6 26.072.4 25.972.7 25.972.3 25.772.2 26.172.4 SBP (mm Hg) 160.6718.1 161.9717.9 161.7714.9 160.6716.9 161.5718.1 162.3715.6 DBP (mm Hg) 97.2710.5 98.5711.7 98.6710.7 96.0711.8 99.4711.4 100.0716.9* TC (mmol/l) 1.770.8 1.770.7 1.671.0 1.670.7 1.670.8 1.771.5 TG (mmol/l) 4.871.0 5.070.8 5.371.0* 4.971.0 4.970.9 5.471.1* LDLC (mmol/l) 3.070.8 3.270.7 3.670.9* 3.170.8 3.270.7 3.570.6* HDLC (mmol/l) 1.571.0 1.470.3 1.370.3 1.770.4 1.571.0 1.370.3*

Hypertensives Genotype combinations

ApoE-LDLR E2/2+E2/3–CC E2/2+E2/3–TT E3/3–CC E3/3–TT E3/4+E4/4–CC E3/4+E4/4–TT

BMI (kg/m2) 25.772.6 25.972.5 26.172.2 25.772.6 26.270.2 26.072.8 SBP (mmHg) 162.5714.4 159.279.9 160.0718.4 159.0720.4 161.8710.3 159.6716.6 DBP (mmHg) 100.7710.2 99.776.1 101.4712.1 97.074.5 98.0710..8 99.2710.3 TC (mmol/l) 1.670.7 1.870.6 1.770.8 1.670.8 1.670.9 1.771.0 (TG (mmol/l) 4.871.1 4.870.9 4.970.7 5.170.3 5.370.7 5.571.1** LDLC (mmol/l) 3.170.9 3.170.4 3.170.8 3.270.4 3.570.7 3.470.9** HDLC (mmol/l) 1.570.3 1.670.3 1.370.3 1.670.2 1.270.1 1.370.4*

Abbreviations: ApoE, apolipoprotein E; BMI, body mass index; DBP, diastolic blood pressure; HDLC, high-density lipoprotein cholestrol; LDLR; low-density lipoprotein receptor, SBP, systolic blood pressure; TC, total cholestrol; TG, total cholestrol. Data are expressed as mean7s.d. wP was calculated by w2 test 6 Â 2 contingency table degree of freedom (df) ¼ 5; zP was calculated by w2 test 3 Â 2 contingency table (df ¼ 2); P was calculated by w2 test 2 Â 2 contingency table (df) ¼ 1. *denotes statistical difference (Po0.05) among different means using ANOVA compared with e3 allele or CC genotype. **denotes statistical difference (Po0.05) among different means using ANOVA compared with E2/2+E2/3-CC genotype combination.

The genotype distributions for the polymorphisms allele, e4 allele carriers showed higher levels of analysed were shown in Table 1 and were in H–W TC and LDLC (all Po0.05). For C1773T, the TT equilibrium except for LDLR C1773T in hyperten- homozygote carriers showed a higher TC and LDLC sives (P ¼ 0.0049). Both ApoE and LDLR genotype levels and a lower HDLC levels than CC homozygote and allele distributions showed significances carriers. The 1773T allele carriers showed higher between the two groups (all Po0.001). The frequency diastolic BP than CC homozygote carriers. With of e2 allele was 33.3% in normotensives and 28.3% respect to genotype combinations, the differences in hypertensives, whereas the frequency of e4 allele between E2/2 þ E2/3ÀCC and E3/4 þ E4/4ÀTT com- was 5.7% in normotensives but high up to 20.0% in binations in terms of plasma TC, LDLC and HDLC hypertensives. The OR for the e4 allele was 4.14, levels were significant (all Po0.05). with 95%CI from 2.66 to 6.44 (Po0.0001) (data not Since the presence of ApoE polymorphism was shown). The frequency of 1773T allele was 45.0% in first described by Utermann et al.,5 some studies hypertensives, which was significantly higher than have suggested that ApoE was functional. Structural in normotensives (28.2%); the OR for 1773T allele defects of ApoE might result in an impaired was 2.08, with 95%CI from 1.60 to 2.71 (Po0.0001) interaction of ApoE containing lipoproteins with (data not shown). its receptors and induce the development of athero- To investigate the effects of ApoE genotypes on genic dyslipidaemias and premature cardiovascular lipoprotein profiles, subjects with the ApoE2/4 diseases.6 ApoE gene knockout mice showed hyper- genotype were excluded from the analysis, because tension and endothelial dysfunction, which sug- of the opposite effects of these two on gested that ApoE might directly contribute to the lipoprotein . Compared with ApoE e3 endothelial function.7 In our study, plasma TC and

Journal of Human Hypertension Research Letter 339 LDLC levels tended to be higher in subjects with e4 isms studied may be potentially useful genetic allele compared with e3 allele similar to a previous markers for hypertension. Further research in study.8 One possible explanation is that e4 allele different populations is required to elucidate the does not carry a supradyl group and therefore, e4 relationship between these polymorphisms and lipoproteins undergo oxidation and are cleared by hypertension, and mechanisms of the ApoE and scavenger receptors more easily than e3 lipopro- LDLR genes and hypertension. teins.9 Thus, we hypothesised that if ApoE involved, the e4 allele would be associated with hypertension W Niu1, X Guo2,YSu2 and C Qiu1 in part via modulating lipoprotein levels. 1The Institute of Basic Medical Sciences, Chinese With regard to LDLR, a recent paper on the male- Academy of Medical Sciences/Peking Union associated hypertension in LDLR-deficient mice Medical College, Beijing, China and suggested that LDLR was not only involved in 2The Clinical Medical Research Center of Hebei metabolism, but also in the unknown pathogenesis General Hospital, Shijiazhuang City, Hebei of hypertension.10 Hegele et al.11 estimated that variation in the LDLR gene accounted for about province, China 2.5% of the variation in plasma LDLC levels. In this E-mail: [email protected] study, our data showed that in hypertensives, TT Published online 18 January 2007 homozygote carriers conferred a higher diastolic BP, TC and LDLC levels and a lower HDLC levels than References other carriers. We can speculate that C1773T variant correlated with diastolic hypertension via modulat- 1 Tanira MO, Al Balushi KA. Genetic variations related ing circulating lipoproteins. As C1773T variant is to hypertension: a review. J Hum Hypertens 2005; 19: 7–19. per se silent at the amino-acid level, there must 2 Izawa H, Yamada Y, Okada T, Tanaka M, Hirayama H, be another functional variant with or flanking the Yokota M. Prediction of genetic risk for hypertension. LDLR gene in linkage disequilibrium. Hypertension 2003; 41: 1035–1040. In this study, C1773T genotype distribution 3 Ames RP. in hypertension: causes and deviated from H–W equilibrium in hypertensives. prevention. Am Heart J 1991; 122: 1219–1224. Deviations from H–W equilibrium, especially in 4 Katsuya T, Baba S, Ishikawa K, Mannami T, Fu Y, affected individuals, can provide evidence for an Inamoto N et al. Epsilon 4 allele of apolipoprotein E association between the marker and disease suscept- gene associates with lower blood pressure in young ibility.12 Given the validity of our study design, this Japanese subjects: the Suita Study. J Hypertens 2002; is yet another indication that C1773T variant is a 20: 2017–2021. 5 Utermann G, Hees M, Steinmetz A. Polymorphism of potentially genetic marker for hypertension. apolipoprotein E and occurrence of dysbetalipoprotein- In summary, our study demonstrated a strong aemia in man. Nature 1977; 269: 604–607. correlation of ApoE and LDLR gene polymorphisms 6 Brewer Jr HB, Zech LA, Gregg RE, Schwartz D, with hypertension and plasma lipoprotein levels in Schaefer EJ. NIH conference. Type III hyperlipoprotei- a Chinese population. Therefore, the polymorph- nemia: diagnosis, molecular defects, pathology, and treatment. Ann Intern Med 1983; 98: 623–640. 7 Yang R, Powell-Braxton L, Ogaoawara AK, Dybdal N, What is known about this topic BuntingS,OhnedaOet al. Hypertension and endothe- K Hypertension is a complex multifactorial and polygenic lial dysfunction in apolipoprotein E knockout mice. disorder, and polymorphisms in susceptibility genes have 1 Arterioscler Thromb Vasc Biol 1999; 19: 2762–2768. been implicated for predisposition to hypertension 8 Bhavani AB, Sastry KB, Reddy NK, Padma T. Lipid K Hypertension has been strongly linked to a variety of profile and apolipoprotein E polymorphism in essen- lipoprotein metabolic abnormalities3 K ApoE and LDLR are functional and involved in the regulation tial hypertension. Indian Heart J 2005; 57: 151–157. of lipoprotein metabolism with unknown mechanism in the 9 Humphries SE, Talmud PJ, Hawe E, Bolla M, Day IN, pathogenesis of hypertension6–10 Miller GJ. Apolipoprotein E4 and coronary heart disease in middle-aged men who smoke: a prospective What this study adds study. Lancet 2001; 358: 115–119. K The distributions of ApoE and LDLR allele and genotype 10 Trieu VN, Uckun FM. Male-associated hypertension in frequencies were significantly different between LDL-R deficient mice. Biochem Biophys Res Commun hypertensives and normotensives 1998; 247: 277–279. K The ApoE e4 allele would be associated with hypertension in part via modulating lipoprotein levels 11 Hegele RA, Brunt JH, Connelly PW. Multiple genetic K The LDLR C1773T variant was associated with diastolic determinants of variation of plasma lipoproteins in hypertension with TT homozygotes conferring a high Alberta Hutterites. Arterioscler Thromb Vasc Biol diastolic BP 1995; 15: 861–871. 12 Wigginton JE, Cutler DJ, Abecasis GR. A note on exact Abbreviations: ApoE, apolipoprotein E; BP, blood pressure; LDLR, tests of Hardy–Weinberg equilibrium. Am J Hum Genet low-density lipoprotein receptor. 2005; 76: 887–893.

Journal of Human Hypertension