Relation between the angiotensinogen (AGT) M235T gene polymorphism and blood pressure in a large, homogeneous study population

(2003) 17, 555 559 & 2003 Nature Publishing Group All rights reserved 0950-9240/03 $25.00 www.nature.com/jhh ORIGINAL ARTICLE Relation between the angiotensinogen (AGT) M235T gene polymorphism and blood pressure in a large, homogeneous study population JR Ortlepp 1, J Metrikat 2, V Mevissen 1, F Schmitz 1, M Albrecht 2, P Maya-Pelzer 2, P Hanrath 1, K Zerres 3 and R Hoffmann 1 1 Medical Clinic I, University Hospital of Aachen, Pauwelsstrasse 30, Aachen, Germany; 2 German Air Force Institute of Aviation Medicine, Furstenfeldbruck, Germany; 3 Institute of Human Genetics, University Hospital of Aachen, Pauwelsstrasse 30, Aachen, Germany The aim of this study was to assess the association of the angiotensinogen M235T polymorphism with arterial blood pressure (BP) at rest and under physical stress in a homogeneous large-scale study population. In all, 1903 men who passed routine medical examination for military flying duty were recruited. BP and heart rate were measured at rest, during, and after bicycle ergometry. Genotyping for the AGT M235T polymorphism was carried out by PCR and RFLP technique. The AGT T235 allele was associated with a significantly higher diastolic BP (n ¼ 1903; MM 81 7 8, MT 83 7 7, TT 83 7 8; P ¼ 0.003). Pulse pressure (PP) at rest differed significantly between AGT genotypes (n ¼ 1903; MM 51 7 10 mmhg, MT 49 7 10 mmhg, TT 49 7 10 mmhg; P ¼ 0.001). During physical activity, BP values showed no significant difference between genotypes. In healthy young men, the AGT T235 allele is significantly associated with elevated diastolic BP but also reduced PP at rest. During physical activity, the AGT polymorphism had no impact on blood pressure, indicating the existence of other counteracting mechanisms, which might balance the influence of this gene. (2003) 17, 555 559. doi:10.1038/sj.jhh.1001587 Keywords: angiotensinogen; AGT; polymorphism; blood pressure; genetics Introduction The regulation of arterial blood pressure (BP) is complex and under the control of different physiological systems. The renin angiotensin aldosterone system (RAAS) is one of the important regulatory systems. Genes encoding products of the RAAS are plausible candidate genes for modifying blood pressure. The angiotensinogen gene (AGT) is one of the few candidates that has been investigated extensively and its genetic variant in exon 2 shows a transition resulting in replacement of methionine by threonine at amino-acid position 235 (M235T). Positive associations between the AGT M235T polymorphism, plasma angiotensinogen levels, and essential arterial hypertension indicate a pathway by which the AGT gene determines arterial BP. 1 5 Correspondence: Dr JR Ortlepp, Medical Clinic I, University Hospital of Aachen, Pauwelsstrasse 30, Aachen 52057, Germany. E-mail: jrortlepp@ukaachen.de Received 6 February 2003; revised 23 April 2003; accepted 25 April 2003 However, studies in African and Asian populations could not equally support these findings, 6 9 indicating the importance of other genetic factors interfering with the AGT gene. Moreover, the allele frequency of the AGT M235T polymorphism varies strongly within the different ethnic groups. One study found a significant increase in T235 allele frequency in patients with hypertension that was restricted to men, 10 thus gender might also influence the effect of the AGT gene. The diagnosis of essential arterial hypertension is based on BP above a defined cutoff value. However, a recent study indicated that individuals with BP values that have been considered to be at the upper limit of normality already have an increased risk of cardiovascular disease. 11 Thus, arterial hypertension should not be considered as a dichotomous diagnosis of having a BP below or above a certain threshold. Similarly, the same seems to be true for genetic determination of arterial hypertension. Many genes might influence the arterial BP without generating an arterial hypertension. It is the concurrence of multiple factors (gene polymorphisms, gender, age, environ-

556 mental conditions), which leads to the manifestation of arterial hypertension. Thus, to study the effect of one gene polymorphism, it might be appropriate to analyse quantitative traits in a homogeneous (homogeneous for: gender, race, occupation, social status, physical fitness, and age) population, rather than categorical traits (like healthy vs arterial hypertension) in heterogeneous populations. The aim of this study was to assess the association of the AGT M235T polymorphism with arterial BP in a large-scale study population of completely healthy young men. Materials and methods Study population The subjects were German male aviators who passed routine medical examination for military flying duties. Physical activity was assessed by standardized questionnaires. Participants were included into the study during routine medical qualification for flying duty. A total of 2000 men were screened and 1903 gave written informed consent (95%) and formed the study population. The study was approved by the local ethical committees. Body mass index Body weight and height were measured between 7 a.m. and 8 a.m. before breakfast, after urination, and undressed, to calculate BMI by division of weight by square of height (kg/m 2 ). Blood pressure and heart rate measurement at rest BP and heart rate (HR) were measured manually by trained personnel after 5 min of rest in supine position in a quiet room between 7 a.m. and 9 a.m. at neutral, ambient temperature. Systolic (SBP), diastolic (DBP), and mean blood pressure (MBP) as well as pulse pressure (PP ¼ SBP DBP) were assessed. DBP was taken as Korotkov V. BP and HR during and after physical exercise, physical fitness (PWC 170) Ergometry was performed in 1750 men. Physical fitness (determined by physical working capacity at an HR of 170 bpm ¼ PWC 170) was assessed by standard cycle ergometry in 451 sitting position and expressed as the achieved performance (watts (W)) per kilogram of body weight (kg) at an actual HR of 170 bpm. Ergometric power was increased every 3 min by steps of 50 W, starting from 100 W up to the point of physical exhaustion or commonly accepted termination criteria. Ergometric performance was monitored by continuous ECG, supervised by an experienced physician. HR, SBP, DBP, MBP, and PP were assessed 3 min, and 6 min during ergometry and 3 min after ergometry. At 3 min ergometry corresponds to 3 min 100 W, 6 min ergometry corresponds to 3 min 100 W plus 3 min 150 W. Genotyping Genomic DNA of patients was extracted from whole blood using the InVitek Blood Kit (InVitek, Berlin, Germany). The AGT M235T polymorphism was tested as described previously. 1 Statistical analysis Continuous data are presented as mean 7 s.d. and categorical data are presented as frequencies. Differences in BP were calculated by ANOVA. A linear regression analysis was performed to evaluate the impact of AGT on pulse pressure. A multivariable analysis was performed including BMI, age, and AGT genotype. Significant differences were defined Table 1 Characteristics of the study population N 1903 Age (year) 33 7 11 Height (cm) 181 7 6 Weight (cm) 79 7 19 BMI (kg/m 2 ) 24.2 7 2.5 SBP at rest (mmhg) 132 7 12 DBP at rest (mmhg) 82 7 7 MBP at rest (mmhg) 99 7 8 PP at rest (mmhg) 50 7 10 HR at rest (bpm) 64 7 12 Physical activity (h/week) 4.5 7 3.2 Physical fitness PWC170 (W/kg) 2.55 7 0.42 SBP at 3 min ergometry (mmhg) 164 7 17 DBP at 3 min ergometry (mmhg) 86 7 9 MBP at 3 min ergometry (mmhg) 112 7 10 PP at 3 min ergometry (mmhg) 78 7 16 HR at 3 min ergometry (bpm) 121 7 15 SBP at 6 min ergometry (mmhg) 188 7 19 DBP at 6 min ergometry (mmhg) 88 7 11 MBP at 6 min ergometry (mmhg) 121 7 11 PP at 6 min ergometry (mmhg) 100 7 18 HR at 6 min ergometry (bpm) 144 7 17 SBP 3 min after ergometry (mmhg) 165 7 19 DBP 3 min after ergometry (mmhg) 72 7 13 MBP 3 min after ergometry (mmhg) 103 7 12 PP 3 min after ergometry (mmhg) 92 7 21 HR 3 min after ergometry (bpm) 118 7 16 Physical fitness (determined by physical working capacity at an HR of 170 bpm=pwc170) was assessed by standard cycle ergometry in 451 sitting position and expressed as the achieved performance (watt (W) per kilogram of body weight (kg) at an actual HR of 170 bpm). Ergometric power was increased every 3 min by steps of 50 W, starting from 100 W up to the point of physical exhaustion or commonly accepted termination criteria. A 3 min ergometry corresponds to 3 min of 100 W, 6 min ergometry corresponds to 3 min of 100 W plus 3 min of 150 W; ergometry was performed in 1750 men. SBP=systolic blood pressure; DBP=diastolic blood pressure, MBP=mean blood pressure; PP=pulse pressure, HR=heart rate, PWC 170=physical working capacity at a heart rate of 170 bpm.

as a two-sided P-valueo0.05. Statistical figures show mean values with 95% confidence interval. All calculations were performed with SPSS for Windows s 10.0. The null hypothesis was that men with different AGT genotypes do not have different BP values. Results Phenotypes The characteristics of the study population are given in Table 1. The study population represents a hyperhealthy population with few obese men and high physical activity and fitness. Genotypes The frequencies of the AGT M235T genotypes and alleles are shown in Table 2. Table 2 Genotype and allele frequencies of the angiotensinogen AGT M235T polymorphism AGT 235 genotype MM (n(%)) 627 (32.9%) TM (n(%)) 1039 (54.6%) TT (n(%)) 237 (12.5%) AGT 235 allele frequency M 0.602 T 0.398 Relation of AGT genotype and BP values As given in Table 3, the AGT genotypes were associated with different DBP and PP at rest. Men homozygous for the T allele had the lowest PP at rest. These findings are further illustrated in Figure 1. During and after physical activity BP values did not differ between the AGT genotypes (Table 3). Multivariable analysis In a multivariable analysis for DBP and PP including age, BMI, and AGT genotype, all variables were significantly associated with DBP (age: F ¼ 139.664, Po0.001; BMI: F ¼ 9.535, P ¼ 0.002; AGT genotype: F ¼ 5.390, P ¼ 0.005) and PP (age: F ¼ 32.001, Po0.001; BMI: F ¼ 29.240, Po0.001; AGT genotype: F ¼ 8.012, Po0.001). Discussion The impact of the angiotensinogen gene (AGT) M235T polymorphism on plasma angiotensinogen levels and essential arterial hypertension has been reported. 1 5 However, the finding of an association could not be repeated in all subsequent studies. 6 9 A recent study found a significant impact of the AGT genotype on arterial blood pressure during exercise in 190 men, participating in the HERITAGE family study. 12 However, the impact of the AGT M235T polymorphism on the regulation of arterial blood 557 SBP mean with 95 %CI [mmhg] MBP mean with 95 %CI [mmhg] 137 136 135 134 133 132 131 130 129 128 127 104 103 102 101 100 99 98 97 96 95 94 P=0.486 P=0.323 DBP mean with 95 %CI [mmhg] PP mean with 95 %CI [mmhg] 88 87 86 85 84 83 82 81 80 79 78 53 52 51 50 49 48 47 46 45 44 43 P=0.003 P=0.001 Figure 1 Relation of the AGT M235T genotype with SBP, DBP, MBP, and PP at rest in a study population of 1903 young male active aviators of the German Armed Forces. SBP ¼ systolic blood pressure; DBP ¼ diastolic blood pressure, MBP ¼ mean blood pressure; PP ¼ pulse pressure.

558 Table 3 Relation of AGT genotype anthropomorphic parameters and blood pressure values at rest, during, and after exercise AGT 235 MM TM TT P N 627 1039 237 Age (year) 33 7 12 33 7 11 32 7 12 0.335 BMI (kg/m 2 ) 24.1 7 2.5 24.2 7 2.5 24.1 7 2.7 0.390 SBP at rest (mmhg) 132 7 12 132 7 12 132 7 12 0.486 DBP at rest (mmhg) 81 7 8 837 7 837 8 0.003 MBP at rest (mmhg) 98 7 8 997 7 997 8 0.323 PP at rest (mmhg) 51 7 10 49 7 10 49 7 10 0.001 HR at rest (bpm) 64 7 12 64 7 11 64 7 12 0.755 Physical activity (h/week) 4.5 7 3.3 4.5 7 3.1 4.6 7 3.1 0.673 Physical fitness PWC170 (W/kg) 2.56 7 0.42 2.55 7 0.43 2.55 7 0.45 0.901 SBP at 3 min ergometry (mmhg) 163 7 17 164 7 16 165 7 17 0.680 DBP at 3 min ergometry (mmhg) 86 7 9 867 9 867 9 0.501 MBP at 3 min ergometry (mmhg) 112 7 10 112 7 9 112 7 10 0.882 PP at 3 min ergometry (mmhg) 77 7 16 78 7 16 79 7 18 0.337 HR at 3 min ergometry (bpm) 121 7 15 120 7 15 121 7 15 0.549 SBP at 6 min ergometry (mmhg) 188 7 20 188 7 19 188 7 20 0.954 DBP at 6 min ergometry (mmhg) 88 7 11 88 7 11 87 7 11 0.379 MBP at 6 min ergometry (mmhg) 121 7 12 122 7 11 121 7 12 0.595 PP at 6 min ergometry (mmhg) 100 7 19 100 7 18 101 7 20 0.805 HR at 6 min ergometry (bpm) 144 7 17 144 7 17 145 7 17 0.799 SBP 3 min after ergometry (mmhg) 164 7 19 165 7 19 164 7 199 0.572 DBP 3 min after ergometry (mmhg) 72 7 13 72 7 13 72 7 13 0.932 MBP 3 min after ergometry (mmhg) 103 7 12 103 7 12 103 7 11 0.703 PP 3 min after ergometry (mmhg) 92 7 21 93 7 21 92 7 22 0.748 HR 3 min after ergometry (bpm) 118 7 16 118 7 16 118 7 15 0.823 Physical fitness (determined by physical working capacity at an HR of 170 bpm=pwc170) was assessed by standard cycle ergometry in 451 sitting position and expressed as the achieved performance (watt (W) per kilogram of body weight (kg) at an actual HR of 170 bpm). Ergometric power was increased every 3 min by steps of 50 W, starting from 100 W up to the point of physical exhaustion or commonly accepted termination criteria. A 3 min ergometry corresponds to 3 min of 100 W, 6 min ergometry corresponds to 3 min of 100 W plus 3 min of 150 W; ergometry was performed in 1750 men. SBP=systolic blood pressure; DBP=diastolic blood pressure, MBP=mean blood pressure; PP=pulse pressure, HR=heart rate, PWC 170=physical working capacity at a heart rate of 170 bpm. pressure remains incompletely defined. This study evaluated the relation of the AGT genotype to arterial BP in almost 2000 male active aviators of the German Armed Forces. The major findings of this study are that the AGT M235T gene polymorphism is significantly associated with increased DBP and reduced PP in men at a relatively young age, whereas SBP did not differ significantly between the AGT genotypes. Even when the F-value for the AGT genotype in the multivariable analysis for blood pressure is low, the effect of the AGT genotype on BP regulation is detectable in a period of life far before the general incidence of arterial hypertension. During physical stress, the influence of the AGT gene polymorphism on blood pressure regulation could not be detected. Other additional yet unknown genetic or nongenetic factors could influence this genotype phenotype relation. We speculate that individuals with the T allele might be more prone to cardiovascular diseases later in life, because of the elevation of DBP very early in life. The effect of reduced PP in this young population remains uncertain. Moreover, a difference in the DBP of 2 mmhg might also have only minor relevance. Owing to the relatively small extent of the effect of the AGT genotype polymorphisms of other genes might be associated with BP regulation. In addition, multiple other polymorphisms within the AGT gene have been described and the combination of these multiple AGT polymorphisms might have a higher influence on BP regulation than just a single polymorphism. Further large studies should therefore investigate the haplotype analysis of several candidate genes of the RAAS. The strength of this study is the number of included subjects and the homogeneity of the examined population. In this study population, all subjects had the same gender, race and nationality, similar age, physical fitness, occupation, annual income, social status, and their principal meals were provided by the military five days a week. Thus, this population was controlled for more exogenic factors than it would be possible for patient or population selected study populations. The limitation of this study is that this cohort is not representative of a regular population. For example, we cannot predict the effect of the AGT gene on blood pressure of females, old men, or

classical hypertensives. Furthermore, because of the cross-sectional character of this study, we cannot prove that gene carriers with relatively high BP will develop cardiovascular disease years or decades later. However, even if the examined military pilots do not match a regular population, they are in particular suitable to serve as an example for genetic environmental interactions because of their highly homogeneous population characteristics. In summary, we conclude that the effect of the AGT M235T gene polymorphism on blood pressure regulation, even to a small extent, is detectable very early in life, far before the onset of arterial hypertension. Acknowledgement This study was supported by the START program of the University Hospital of Aachen. The work resulting in this report was performed under German Federal Ministry of Defense Contract 21L2-S- 200103. The opinions expressed in this paper are those of the authors and do not necessarily reflect the opinions or official policy of the German Federal Ministry of Defense. We gratefully acknowledge the support of the medical assistants of Dep. I and the backing of Dep. III of the German Air Force Institute of Aviation Medicine. JR Ortlepp designed the study, did statistical data analysis, J Metrikat coordinated and realized data collection, M Albrecht and P Maya-Pelzer helped with data collection, V Mevissen, F Schmitz, and K Zerres fulfilled genetic analysis, P Hanrath, K Zerres, and R Hoffmann took part in interpretation of data and critical revision. All authors helped to write the report. This work is part of the doctoral thesis of JM Conflict of Interest Statement: None declared. References 1 Jeunemaitre X et al. Molecular basis of human hypertension: role of angiotensinogen. Cell 1992; 71: 169 180. 2 Caulfield M et al. Linkage of the angiotensinogen gene to essential hypertension. N Engl J Med 1994; 330: 1629 1633. 3 Tiret L et al. Genetic variation at the angiotensinogen locus in relation to high blood pressure and myocardial infarction: the ECTIM Study. J Hypertens 1995; 13: 311 317. 4 Schunkert H et al. The angiotensinogen T235 variant and the use of antihypertensive drugs in a populationbased cohort. Hypertension 1997; 29: 628 633. 5 Kunz R et al. Association between the angiotensinogen 235T-variant and essential hypertension in whites: a systematic review and methodological appraisal. Hypertension 1997; 30: 1331 1337. 6 Rotimi C et al. Hypertension, serum angiotensinogen, and molecular variants of the angiotensinogen gene among Nigerians. Circulation 1997; 95: 2348 2350. 7 Niu T et al. Angiotensinogen gene and hypertension in Chinese. J Clin Invest 1998; 101: 188 194. 8 Hingorami AD et al. Blood pressure and the M235T polymorphism of the angiotensinogen gene. Hypertension 1996; 28: 907 911. 9 Brand E et al. Evaluation of the angiotensinogen locus in human essential hypertension: a European study. Hypertension 1998; 31: 725 729. 10 Tiret L et al. Gene polymorphisms of the renin angiotensin system in relation to hypertension and parental history of myocardial infarction and stroke: the PEGASE study. Projet d Etude des Gene de l Hypertension Arterielle Severe a moderee Essentielle. J Hypertens 1998; 16: 37 44. 11 Vasan RS et al. Impact of high-normal blood pressure on the risk of cardiovascular disease. New Engl J Med 2001; 345: 1291 1297. 12 Rankinen T et al. AGT M235T and ACE ID polymorphisms and exercise blood pressure in the HERITAGE Family Study. Am J Physiol Heart Circ Physiol 2000; 279: H368 H374. 559