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Ambulatory Blood Pressure Monitoring for Detecting the Relation Between Angiotensinogen Gene Polymorphism and Hypertension
AJH 1997; 10:687 – 691
Ambulatory Blood Pressure Monitoring for Detecting the Relation Between Angiotensinogen Gene Polymorphism and Hypertension Ali G. Gharavi, Michael L. Lipkowitz, Joseph A. Diamond, Rima Chamie, and Robert A. Phillips
Compared to office measurements, ambulatory monitoring is a more accurate method of blood pressure ( BP ) characterization and may therefore be useful in a genetics study of hypertension. We studied the relation between the M235T polymorphism of the angiotensinogen gene and hypertension using office and ambulatory ( BP ) measurements. We enrolled untreated subjects ( 33 men and 17 women ) who were referred for evaluation of office BP ú140 / 90 mm Hg on at least two separate occasions. The M235T genotypes of the angiotensinogen gene were determined by polymerase chain reaction ( PCR ) amplification of DNA extracted from peripheral blood leukocytes and digested with BSTU1. The distribution of the genotypes were MM Å 0.22, MT Å 0.44, TT Å 0.34. Based on office measurements, a significant difference in diastolic blood pressure ( BP ) was detected only between the TT and the MT genotype subjects ( office BP:
MM Å 150 { 25 / 97 { 13 mm Hg, MT Å 147 { 23 / 95 { 13 mm Hg, TT Å 161 { 25 / 104 { 15 mm Hg ) . By contrast, with ambulatory BP monitoring, both systolic and diastolic blood pressures were significant higher in TT versus MM and MT ( ambulatory BP, MM Å 138 { 10 / 88 { 9 mm Hg, MT Å 141 { 15 / 89 { 11 mm Hg, TT Å 152 { 18 / 97 { 12 mm Hg ) . Covariate analysis revealed an independent relationship between the M235T genotype and systolic, diastolic, and mean ambulatory BP. Ambulatory monitoring improved the analytic power of our study and allowed detection of a clear and consistent relationship between angiotensinogen polymorphism and hypertension with a relatively small sample size. Am J Hypertens 1997; 10:687 – 691 q 1997 American Journal of Hypertension, Ltd.
enetic studies of essential hypertension usually use office rather than ambulatory blood pressure ( BP ) measurements to characterize phenotype. Office BP measurements are inherently variable and may, therefore, obscure a relationship between a genetic marker and
G
hypertension. Compared to office BP, ambulatory BP monitoring ( ABPM ) accurately defines the level of BP, identifies subjects with white-coat hypertension, 1,2 and may be a more accurate predictor of morbidity and mortality.3 – 5 ABPM measurements may improve the statistical power of a study and allow recruitment
Received October 8, 1996. Accepted January 13, 1997. From the Hypertension Section, Cardiovascular Institute and Division of Nephrology Mount Sinai School of Medicine, New York, New York. This paper was presented in part at the eleventh scientific meeting of the American Society of Hypertension, May 1996, New York,
New York. Address correspondence and reprint requests to Dr. Ali G. Gharavi, MD, Mount Sinai School of Medicine, Hypertension Section, Cardiovascular Institute, Box 1085, One Gustave L. Levy Place, New York, NY 10029-6574.
q 1997 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
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KEY WORDS: Angiotensinogen gene, ambulatory blood pressure monitoring, hypertension.
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of a smaller sample size.6,7 Furthermore, the effect of a particular gene on BP may depend on time or activity: BP during daytime or activity is under stronger hereditary influence than BP during other intervals.8,9 Hence, ABPM may be more informative than office measurements in a genetic study of hypertension. We determined the relation of the M235T polymorphism of the angiotensinogen ( AGT ) gene with both office BP and ABPM measurements. The M235T polymorphism is the genetic marker that has been most consistently associated with essential hypertension. The M235T variant consists of a thymine to cytosine mutation at nucleotide 704 in exon 2, leading to substitution of threonine for methionine at amino acid 235 position.10 White subjects homozygous for the T235 mutation ( the TT genotype ) have the highest serum AGT levels.10 Although some studies have confirmed these findings, 11,12 lack of association between the M235T polymorphism and hypertension has also been reported in several populations.13 – 15 These conflicting results may at least in part be explained by differences in the design of the studies and the genetic background of the subjects recruited. In addition, studies to date have used office BP measurements ( with its inherent limitations ) to define hypertensive status, whereas there are no published studies using ABPM on this topic.
METHODS Patient Selection We recruited 50 subjects living in the New York City metropolitan area. All subjects were referred to our hypertension clinic for evaluation of office BP’s greater than 140 / 90 mm Hg on at least two separate visits to a physician. Many carried a diagnosis of ‘‘borderline hypertension’’ and only 7 patients were previously treated. In the latter group, all antihypertensive medications were discontinued for at least 2 weeks prior to ambulatory BP measurements and echocardiographic studies. Blacks were excluded because previous studies indicated that the M235T variant is not associated with hypertension in this population.15 None of the subjects were aerobically trained. Daily alcohol consumption was õ 2 drinks / day. Other significant concomitant illnesses ( coronary artery disease, peripheral vascular disease, renal disease, secondary hypertension, etc ) were excluded by a standard history, physical examination, routine laboratory test, and additional diagnostic studies as indicated. The protocol was approved by the Institutional Review Board of the Mount Sinai School of Medicine in New York and informed, signed consent was obtained from all participants. Echocardiography Two-dimensionally guided Mmode echocardiography was performed with an ATL Mark 600 or ATL Ultramark 6 scanner ( Advanced Technology Laboratories, Inc, Bothell, WA ) using a 2.5- or
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3.5-MHz transducer. Echocardiographic tracings were coded and interspersed with other ongoing studies to reduce the chance of reader bias. The left ventricular structures were measured according to the Penn Convention 16 by two observers blinded to the identity and clinical characteristics of the subjects. The left ventricular mass index was calculated by dividing the left ventricular mass ( Penn ) by the body surface area ( grams / meters squared ) .
Blood Pressure Determination Twenty-four hour BP monitoring was performed using the SpaceLabs 90202 and 90207 devices ( SpaceLabs, Redmond, WA ) . Subjects were monitored on a day chosen for typical weekly activity. For calibration, the device was connected to a mercury sphygmomanometer with a Y-tube. The phase V Korotkoff sound was used to determine diastolic BP. After 5 min of rest, three consecutive measurements were taken, 2 to 3 min apart, in a seated position, and calibrated to the monitor readings. The device was used only if measurements differed by °5 mm Hg from the sphygmomanometer. Ambulatory BP recordings were made every 20 min during awake hours and every 60 min during sleep. The data was electronically transferred by software to an IBM-compatible computer and processed for deletion of errors and predefined ‘‘outliers’’ ( systolic õ 70 or ú 260 mm Hg; diastolic õ 40 or ú 150 mm Hg; and HR õ 20 or ú 200 beats /min ) . Hand editing for outliers was not performed to avoid bias. Average 24-h ambulatory BP was used in the correlation of analysis of BP with AGT genotype and left ventricular mass. Awake blood pressure was defined as the average blood pressure during awake hours as reported in a patient diary. The average of the three office BP readings obtained during calibration of the monitor were used to define office BP. Determination of Angiotensinogen Genotypes DNA was extracted from peripheral blood leukocytes using the Qiagen DNA prep kit ( Qiagen, Chatsworth, CA ) . The M235T variants of the angiotensinogen gene were detected by the polymerase chain reaction ( PCR ) using the methods and the one base mismatch primers described by Rutledge et al.17 Statistical Analysis All statistical analyses were performed using SAS software ( SAS Institute Inc., Cary, NC ) on an IBM-compatible personal computer. Subject characteristics by AGT genotype group were analyzed by analysis of variance ( ANOVA ) or x 2 analysis. Office and ambulatory BPs were then analyzed by analysis of covariance ( ANCOVA ) , using AGT genotype, gender, body mass index ( BMI ), and age as independent variables. Left ventricular mass index was analyzed by ANCOVA using angiotensinogen genotype, systolic ambulatory BP, BMI, age, and gender as independent variables. Variables with a P õ .05 were considered significant.
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RESULTS Patient Characteristics We studied 50 subjects ( 19 Whites, 24 Hispanics, 7 Asians ) . There were no significant differences among genotype groups in terms of age, BMI, ethnic and gender distribution, and previous antihypertensive therapy ( Table 1 ) . The distribution of the M235T variants were: MM Å 0.22, MT Å 0.44, and TT Å 0.34. The frequencies for the M and T allele were 0.44 and 0.56, respectively. The genotype distribution did not deviate from the Hardy-Weinberg equilibrium. Office Blood Pressure Office diastolic BP was significantly higher in subjects with the TT genotype compared to the MT genotype ( P õ .05 ) , but this difference was not statistically significant after age, BMI, and gender were taken into account. ANOVA and ANCOVA revealed no other significant differences in office BP between the three genotype groups ( Table 1 ) . Ambulatory Blood Pressure Subjects with the TT genotype had higher systolic, diastolic, and mean 24-h ambulatory BPs, compared with both the MT and MM genotype subjects ( Table 1 ) . Covariate analysis revealed that systolic, diastolic, and mean ambulatory BP were all independently related to AGT genotype and BMI. AGT genotype (P õ .02 ) and BMI (P õ .03 ) were each independently related to systolic 24-h ambulatory BP. Diastolic 24-h ambulatory BP was correlated with BMI ( P õ .03 ) , AGT genotype (P õ .05 ), and age (P õ .05 ) . Mean 24-h ambulatory BP was related to BMI ( P õ .02 ) and AGT genotype (P õ 0.05 ) . Echocardiography The left ventricular mass was not statistically different across genotype groups. On stepwise multiple regression analysis, only systolic 24-h ambulatory BP ( P õ .01 ) and gender (P õ .02 ) were
independent determinants of left ventricular mass ( r 2 Å 0.31 ) . In this relatively nonobese population, there was no independent contribution of BMI or the AGT genotype to left ventricular mass.
DISCUSSION In this group of patients referred because of office hypertension, ABPM revealed an association between the M235T polymorphism of the AGT gene and blood pressure. Our subjects were either untreated or were studied off medications, allowing quantitative comparison of BP level. This advantage combined with the precision afforded by ABPM allowed detection of a genetic association despite the relatively small sample size. A post hoc power analysis revealed that, in our sample size, office measurements had a 29% power to detect a ú10 mm Hg difference in systolic BP between genotype groups, whereas ambulatory measurements had a 72% power to detect the same difference. This finding is consistent with previous reports showing that the use of ABPM can increase the power of a study or reduce the number of subjects required.6,7 The chief novelty of our study is the use of ABPM in a genetic study of hypertension. The ABPM readings are potentially more informative in a genetic study than casual BP values. The large number of readings obtained with ABPM allows analysis of BP as a continuous ( rather than a categorical ) variable. Thus ambulatory measurements more accurately reflect BP load and may be more closely related to morbidity and mortality.4 – 6 Ambulatory BP also identifies the magnitude of the white coat effect, 1 – 3 which may remain unchanged despite successive office visits or institution of antihypertensive therapy.18 In addition,
TABLE 1. SUBJECT CHARACTERISTICS BY AGT GENOTYPE
Number of subjects Age (years) Gender (men/women) BMI (kg/m2) Antihypertensive treatment (%) Ethnicity (Hispanic/white/Asian) Office systolic BP (mm Hg) Office diastolic BP (mm Hg) Office mean BP (mm Hg) Ambulatory systolic BP (mm Hg) Ambulatory diastolic BP (mm Hg) Ambulatory mean BP (mm Hg) Left ventricular mass (g/m2)
MM
MT
TT
11 44 { 9 7/4 27.8 { 2.3 1 (9) 6/4/1 150 { 25 97 { 13 115 { 16 138 { 10 88 { 9 105 { 10 108 { 34
22 45 { 13 13/9 26.1 { 6.9 3 (14) 10/9/3 147 { 23 95 { 13 113 { 16 141 { 15 89 { 11 107 { 12 97 { 23
17 46 { 11 13/4 26.8 { 2.9 3 (18) 8/6/3 161 { 25 104 { 15* 124 { 18 152 { 18*† 97 { 12*† 115 { 14*† 109 { 29
Values are means { standard deviations. BMI, body mass index; BP, blood pressure. * P õ .05 TT v MT; † P õ .03 TT v MM.
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the magnitude of the office pressor response rises with increasing severity of hypertension.2 This phenomenon may therefore interfere with correct classification of subjects in a genetic study because selection criteria are usually based on severity / duration of office hypertension or the number of antihypertensive medications used. Finally, the use of ABPM also allows comparison of BP during activity and different periods of the day. This is important because studies indicate that daytime BP and BP during activity are under stronger genetic control than values during nighttime and inactivity.8,9 In addition, the quantitative contribution of a particular gene to BP load may vary in a time and activity specific manner.8 Hence, the combined advantages of ambulatory monitoring make it an attractive tool for defining phenotype in a genetic study of hypertension. The limitations of our study were small sample size, ethnic heterogeneity, and selection of subjects based on referral for office hypertension. Each of these factors is expected to decrease our analytic power and prevent detection of a genetic association. Despite ethnic heterogeneity, we observed no apparent genotype distortion in our study sample. The M235T genotype distribution did not deviate from the Hardy-Weinberg expectations. The frequency of the T235 allele ( 0.56 ) in our study falls between the values reported for hypertensive White ( 0.44 to 0.51 ) and hypertensive Japanese subjects ( 0.72 to 0.79 ) .10 – 15 The higher frequency of the T235 allele may be due to ethnic admixture and subject selection based on referral for office hypertension. We may have included subjects with high normal BP who may progress to hypertension in the future. However, it is apparent that the precision afforded by ABPM overcame potential limitations of our study. Our study does not resolve the controversy over the true significance of the M235T polymorphism in essential hypertension. It merely demonstrates the potential usefulness of ABPM in genetic study of hypertension. The conflicting results on M235T polymorphism stem largely from study differences in the definition of hypertension, subject recruitment methods, and genetic background of subjects. In addition, the M235T variant itself may only be a marker in linkage disequilibrium with a functional variant. Jeunemaitre et al 10 first reported a significant association of the M235T variant with hypertension, especially in more severe White index cases recruited from Salt Lake City, Utah, and Paris, France. Caufield et al 14 confirmed linkage of the AGT gene with hypertension in a British population but did not find any relationship between the 235T allele and BP. Association of the 235T with hypertension was confirmed in several Japanese populations recruited from hospital clinics.11,12 In Australia, despite selecting subjects with
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severe familial hypertension, Bennet et al 19 found no effect of the M235T variants on BP. Fornage et al 20 conducted a population based study in Rochester, Minnesota, and also did not detect a relationship. These authors emphasized the importance of subject selection criteria and phenotype ascertainment in genetic studies of complex traits. Our study indicates that accurate characterization of BP using ambulatory monitoring may improve the analytic power of a genetic study and allow detection of an association between a gene polymorphism and hypertension with a relatively small sample size. Our report suggests that selection of subjects based on ABPM profile may be a useful complement to other selection strategies for genetic studies of hypertension.
ACKNOWLEDGMENT We would like to thank Mr. Howard Printz for his genuine interest, encouragement and generous support of the Hypertension Section.
REFERENCES 1. Pickering TP, James G, Boddie C, et al: How common is white coat hypertension? JAMA 1988; 259:225 – 228. 2. Verdecchia P, Schillaci G, Borgioni C, et al: White coat hypertension and white coat effect: similarities and differences. Am J Hyperten 1995; 8:790 – 798. 3. Verdecchia P, Porcellati C, Schillaci G, et al: Ambulatory blood pressure: an independent predictor of prognosis. Hypertension 1994; 24:793 – 801. 4. Devereux RB, Pickering TG, Harshfield GA: Left ventricular hypertrophy in patients with hypertension: importance of blood pressure response to regular recurring stress. Circulation 1983; 68:470 – 476. 5. Perloff D, Sokolow M, Cowan R: The prognostic value of ambulatory blood pressure. JAMA 1983; 249:2792 – 2798. 6. Weber M, Cheung D, Graettinger W, et al: Characterization of antihypertensive therapy by whole-day blood pressure monitoring. JAMA 1988; 259:3281 – 3285. 7. Conway J, Coats A, Radaeli A: Ambulatory blood pressure in relation to drug treatment and clinical trials. J Hypertens 1990; 8 ( suppl 6 ) :S83 – S85. 8. Schwartz GL, Turner ST, Sing CF: Association of genetic variation with interindividual variation in ambulatory blood pressure. J Hypertens 1996; 14:251 – 258. 9. Degaute JP, Cauter EV, van de Borne P, et al: Twenty four blood and heart rate profiles in humans: a twin study. Hypertension 1994; 23:244 – 253. 10. Jeunemaitre X, Soubrier F, Kotelevtsev Y, et al: Molecular basis of hypertension: role of angiotensinogen. Cell 1992; 71:169 – 180. 11. Hata A, Namikawa C, Sasaki M, et al: Angiotensinogen as a risk factor for essential hypertension in Japan. J Clin Invest 1994; 93:2185 – 187. 12. Iwai N, Shimoike H, Ohmichi N, et al: Angiotensinogen gene and blood pressure in the Japanese population. Hypertension 1995; 25 ( part 2 ) :688 – 693.
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13. Katsuya T, Koike G, Yee T, et al: Association of the angiotensinogen gene T235 variant with increased risk of coronary heart disease. Lancet 1995; 345:1600 – 1603. 14. Caufield M, Lavende P, Farrall M, et al: Linkage of the angiotensinogen gene to essential hypertension. N Engl J Med 1994; 330:1629 – 1633. 15. Caufield M, Lavender P, Newell-Price J, et al: Linkage of the angiotensinogen gene locus with human essential hypertension in African Caribbeans. J Clin Invest 1995; 96:687 – 692. 16. Devereux RB, Reichek N: Echocardiographic determination of left ventricular mass in man. Circulation 1977; 55:613 – 618.
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17. Rutledge D, Browe C, Kubilis P: Analysis of two variants of the angiotensinogen in essential hypertensive African-Americans. Am J Hypertens 1994; 7:651 – 654. 18. Reeves R: Does this patient have hypertension? JAMA 1995; 273:1211 – 1218. 19. Bennet CL, Scrader AP, Morris BJ: Cross sectional analysis of Met 235rThr variant of the angiotensinogen gene in severe, familial hypertension. Biochem Biophys Res Commun 1993; 197:833 – 839. 20. Fornage M, Turner S, Sing C, et al: Variation at the M235T locus of the angiotensinogen gene and essential hypertension: a population-based case-control study from Rochester, Minnesota. Hum Gen 1995; 96:295 – 300.
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Ambulatory Blood Pressure Monitoring for Detecting the Relation Between Angiotensinogen Gene Polymorphism and Hypertension Ali G. Gharavi, Michael L. Lipkowitz, Joseph A. Diamond, Rima Chamie, and Robert A. Phillips
Compared to office measurements, ambulatory monitoring is a more accurate method of blood pressure ( BP ) characterization and may therefore be useful in a genetics study of hypertension. We studied the relation between the M235T polymorphism of the angiotensinogen gene and hypertension using office and ambulatory ( BP ) measurements. We enrolled untreated subjects ( 33 men and 17 women ) who were referred for evaluation of office BP ú140 / 90 mm Hg on at least two separate occasions. The M235T genotypes of the angiotensinogen gene were determined by polymerase chain reaction ( PCR ) amplification of DNA extracted from peripheral blood leukocytes and digested with BSTU1. The distribution of the genotypes were MM Å 0.22, MT Å 0.44, TT Å 0.34. Based on office measurements, a significant difference in diastolic blood pressure ( BP ) was detected only between the TT and the MT genotype subjects ( office BP:
MM Å 150 { 25 / 97 { 13 mm Hg, MT Å 147 { 23 / 95 { 13 mm Hg, TT Å 161 { 25 / 104 { 15 mm Hg ) . By contrast, with ambulatory BP monitoring, both systolic and diastolic blood pressures were significant higher in TT versus MM and MT ( ambulatory BP, MM Å 138 { 10 / 88 { 9 mm Hg, MT Å 141 { 15 / 89 { 11 mm Hg, TT Å 152 { 18 / 97 { 12 mm Hg ) . Covariate analysis revealed an independent relationship between the M235T genotype and systolic, diastolic, and mean ambulatory BP. Ambulatory monitoring improved the analytic power of our study and allowed detection of a clear and consistent relationship between angiotensinogen polymorphism and hypertension with a relatively small sample size. Am J Hypertens 1997; 10:687 – 691 q 1997 American Journal of Hypertension, Ltd.
enetic studies of essential hypertension usually use office rather than ambulatory blood pressure ( BP ) measurements to characterize phenotype. Office BP measurements are inherently variable and may, therefore, obscure a relationship between a genetic marker and
G
hypertension. Compared to office BP, ambulatory BP monitoring ( ABPM ) accurately defines the level of BP, identifies subjects with white-coat hypertension, 1,2 and may be a more accurate predictor of morbidity and mortality.3 – 5 ABPM measurements may improve the statistical power of a study and allow recruitment
Received October 8, 1996. Accepted January 13, 1997. From the Hypertension Section, Cardiovascular Institute and Division of Nephrology Mount Sinai School of Medicine, New York, New York. This paper was presented in part at the eleventh scientific meeting of the American Society of Hypertension, May 1996, New York,
New York. Address correspondence and reprint requests to Dr. Ali G. Gharavi, MD, Mount Sinai School of Medicine, Hypertension Section, Cardiovascular Institute, Box 1085, One Gustave L. Levy Place, New York, NY 10029-6574.
q 1997 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
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KEY WORDS: Angiotensinogen gene, ambulatory blood pressure monitoring, hypertension.
eajha
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of a smaller sample size.6,7 Furthermore, the effect of a particular gene on BP may depend on time or activity: BP during daytime or activity is under stronger hereditary influence than BP during other intervals.8,9 Hence, ABPM may be more informative than office measurements in a genetic study of hypertension. We determined the relation of the M235T polymorphism of the angiotensinogen ( AGT ) gene with both office BP and ABPM measurements. The M235T polymorphism is the genetic marker that has been most consistently associated with essential hypertension. The M235T variant consists of a thymine to cytosine mutation at nucleotide 704 in exon 2, leading to substitution of threonine for methionine at amino acid 235 position.10 White subjects homozygous for the T235 mutation ( the TT genotype ) have the highest serum AGT levels.10 Although some studies have confirmed these findings, 11,12 lack of association between the M235T polymorphism and hypertension has also been reported in several populations.13 – 15 These conflicting results may at least in part be explained by differences in the design of the studies and the genetic background of the subjects recruited. In addition, studies to date have used office BP measurements ( with its inherent limitations ) to define hypertensive status, whereas there are no published studies using ABPM on this topic.
METHODS Patient Selection We recruited 50 subjects living in the New York City metropolitan area. All subjects were referred to our hypertension clinic for evaluation of office BP’s greater than 140 / 90 mm Hg on at least two separate visits to a physician. Many carried a diagnosis of ‘‘borderline hypertension’’ and only 7 patients were previously treated. In the latter group, all antihypertensive medications were discontinued for at least 2 weeks prior to ambulatory BP measurements and echocardiographic studies. Blacks were excluded because previous studies indicated that the M235T variant is not associated with hypertension in this population.15 None of the subjects were aerobically trained. Daily alcohol consumption was õ 2 drinks / day. Other significant concomitant illnesses ( coronary artery disease, peripheral vascular disease, renal disease, secondary hypertension, etc ) were excluded by a standard history, physical examination, routine laboratory test, and additional diagnostic studies as indicated. The protocol was approved by the Institutional Review Board of the Mount Sinai School of Medicine in New York and informed, signed consent was obtained from all participants. Echocardiography Two-dimensionally guided Mmode echocardiography was performed with an ATL Mark 600 or ATL Ultramark 6 scanner ( Advanced Technology Laboratories, Inc, Bothell, WA ) using a 2.5- or
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3.5-MHz transducer. Echocardiographic tracings were coded and interspersed with other ongoing studies to reduce the chance of reader bias. The left ventricular structures were measured according to the Penn Convention 16 by two observers blinded to the identity and clinical characteristics of the subjects. The left ventricular mass index was calculated by dividing the left ventricular mass ( Penn ) by the body surface area ( grams / meters squared ) .
Blood Pressure Determination Twenty-four hour BP monitoring was performed using the SpaceLabs 90202 and 90207 devices ( SpaceLabs, Redmond, WA ) . Subjects were monitored on a day chosen for typical weekly activity. For calibration, the device was connected to a mercury sphygmomanometer with a Y-tube. The phase V Korotkoff sound was used to determine diastolic BP. After 5 min of rest, three consecutive measurements were taken, 2 to 3 min apart, in a seated position, and calibrated to the monitor readings. The device was used only if measurements differed by °5 mm Hg from the sphygmomanometer. Ambulatory BP recordings were made every 20 min during awake hours and every 60 min during sleep. The data was electronically transferred by software to an IBM-compatible computer and processed for deletion of errors and predefined ‘‘outliers’’ ( systolic õ 70 or ú 260 mm Hg; diastolic õ 40 or ú 150 mm Hg; and HR õ 20 or ú 200 beats /min ) . Hand editing for outliers was not performed to avoid bias. Average 24-h ambulatory BP was used in the correlation of analysis of BP with AGT genotype and left ventricular mass. Awake blood pressure was defined as the average blood pressure during awake hours as reported in a patient diary. The average of the three office BP readings obtained during calibration of the monitor were used to define office BP. Determination of Angiotensinogen Genotypes DNA was extracted from peripheral blood leukocytes using the Qiagen DNA prep kit ( Qiagen, Chatsworth, CA ) . The M235T variants of the angiotensinogen gene were detected by the polymerase chain reaction ( PCR ) using the methods and the one base mismatch primers described by Rutledge et al.17 Statistical Analysis All statistical analyses were performed using SAS software ( SAS Institute Inc., Cary, NC ) on an IBM-compatible personal computer. Subject characteristics by AGT genotype group were analyzed by analysis of variance ( ANOVA ) or x 2 analysis. Office and ambulatory BPs were then analyzed by analysis of covariance ( ANCOVA ) , using AGT genotype, gender, body mass index ( BMI ), and age as independent variables. Left ventricular mass index was analyzed by ANCOVA using angiotensinogen genotype, systolic ambulatory BP, BMI, age, and gender as independent variables. Variables with a P õ .05 were considered significant.
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RESULTS Patient Characteristics We studied 50 subjects ( 19 Whites, 24 Hispanics, 7 Asians ) . There were no significant differences among genotype groups in terms of age, BMI, ethnic and gender distribution, and previous antihypertensive therapy ( Table 1 ) . The distribution of the M235T variants were: MM Å 0.22, MT Å 0.44, and TT Å 0.34. The frequencies for the M and T allele were 0.44 and 0.56, respectively. The genotype distribution did not deviate from the Hardy-Weinberg equilibrium. Office Blood Pressure Office diastolic BP was significantly higher in subjects with the TT genotype compared to the MT genotype ( P õ .05 ) , but this difference was not statistically significant after age, BMI, and gender were taken into account. ANOVA and ANCOVA revealed no other significant differences in office BP between the three genotype groups ( Table 1 ) . Ambulatory Blood Pressure Subjects with the TT genotype had higher systolic, diastolic, and mean 24-h ambulatory BPs, compared with both the MT and MM genotype subjects ( Table 1 ) . Covariate analysis revealed that systolic, diastolic, and mean ambulatory BP were all independently related to AGT genotype and BMI. AGT genotype (P õ .02 ) and BMI (P õ .03 ) were each independently related to systolic 24-h ambulatory BP. Diastolic 24-h ambulatory BP was correlated with BMI ( P õ .03 ) , AGT genotype (P õ .05 ), and age (P õ .05 ) . Mean 24-h ambulatory BP was related to BMI ( P õ .02 ) and AGT genotype (P õ 0.05 ) . Echocardiography The left ventricular mass was not statistically different across genotype groups. On stepwise multiple regression analysis, only systolic 24-h ambulatory BP ( P õ .01 ) and gender (P õ .02 ) were
independent determinants of left ventricular mass ( r 2 Å 0.31 ) . In this relatively nonobese population, there was no independent contribution of BMI or the AGT genotype to left ventricular mass.
DISCUSSION In this group of patients referred because of office hypertension, ABPM revealed an association between the M235T polymorphism of the AGT gene and blood pressure. Our subjects were either untreated or were studied off medications, allowing quantitative comparison of BP level. This advantage combined with the precision afforded by ABPM allowed detection of a genetic association despite the relatively small sample size. A post hoc power analysis revealed that, in our sample size, office measurements had a 29% power to detect a ú10 mm Hg difference in systolic BP between genotype groups, whereas ambulatory measurements had a 72% power to detect the same difference. This finding is consistent with previous reports showing that the use of ABPM can increase the power of a study or reduce the number of subjects required.6,7 The chief novelty of our study is the use of ABPM in a genetic study of hypertension. The ABPM readings are potentially more informative in a genetic study than casual BP values. The large number of readings obtained with ABPM allows analysis of BP as a continuous ( rather than a categorical ) variable. Thus ambulatory measurements more accurately reflect BP load and may be more closely related to morbidity and mortality.4 – 6 Ambulatory BP also identifies the magnitude of the white coat effect, 1 – 3 which may remain unchanged despite successive office visits or institution of antihypertensive therapy.18 In addition,
TABLE 1. SUBJECT CHARACTERISTICS BY AGT GENOTYPE
Number of subjects Age (years) Gender (men/women) BMI (kg/m2) Antihypertensive treatment (%) Ethnicity (Hispanic/white/Asian) Office systolic BP (mm Hg) Office diastolic BP (mm Hg) Office mean BP (mm Hg) Ambulatory systolic BP (mm Hg) Ambulatory diastolic BP (mm Hg) Ambulatory mean BP (mm Hg) Left ventricular mass (g/m2)
MM
MT
TT
11 44 { 9 7/4 27.8 { 2.3 1 (9) 6/4/1 150 { 25 97 { 13 115 { 16 138 { 10 88 { 9 105 { 10 108 { 34
22 45 { 13 13/9 26.1 { 6.9 3 (14) 10/9/3 147 { 23 95 { 13 113 { 16 141 { 15 89 { 11 107 { 12 97 { 23
17 46 { 11 13/4 26.8 { 2.9 3 (18) 8/6/3 161 { 25 104 { 15* 124 { 18 152 { 18*† 97 { 12*† 115 { 14*† 109 { 29
Values are means { standard deviations. BMI, body mass index; BP, blood pressure. * P õ .05 TT v MT; † P õ .03 TT v MM.
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the magnitude of the office pressor response rises with increasing severity of hypertension.2 This phenomenon may therefore interfere with correct classification of subjects in a genetic study because selection criteria are usually based on severity / duration of office hypertension or the number of antihypertensive medications used. Finally, the use of ABPM also allows comparison of BP during activity and different periods of the day. This is important because studies indicate that daytime BP and BP during activity are under stronger genetic control than values during nighttime and inactivity.8,9 In addition, the quantitative contribution of a particular gene to BP load may vary in a time and activity specific manner.8 Hence, the combined advantages of ambulatory monitoring make it an attractive tool for defining phenotype in a genetic study of hypertension. The limitations of our study were small sample size, ethnic heterogeneity, and selection of subjects based on referral for office hypertension. Each of these factors is expected to decrease our analytic power and prevent detection of a genetic association. Despite ethnic heterogeneity, we observed no apparent genotype distortion in our study sample. The M235T genotype distribution did not deviate from the Hardy-Weinberg expectations. The frequency of the T235 allele ( 0.56 ) in our study falls between the values reported for hypertensive White ( 0.44 to 0.51 ) and hypertensive Japanese subjects ( 0.72 to 0.79 ) .10 – 15 The higher frequency of the T235 allele may be due to ethnic admixture and subject selection based on referral for office hypertension. We may have included subjects with high normal BP who may progress to hypertension in the future. However, it is apparent that the precision afforded by ABPM overcame potential limitations of our study. Our study does not resolve the controversy over the true significance of the M235T polymorphism in essential hypertension. It merely demonstrates the potential usefulness of ABPM in genetic study of hypertension. The conflicting results on M235T polymorphism stem largely from study differences in the definition of hypertension, subject recruitment methods, and genetic background of subjects. In addition, the M235T variant itself may only be a marker in linkage disequilibrium with a functional variant. Jeunemaitre et al 10 first reported a significant association of the M235T variant with hypertension, especially in more severe White index cases recruited from Salt Lake City, Utah, and Paris, France. Caufield et al 14 confirmed linkage of the AGT gene with hypertension in a British population but did not find any relationship between the 235T allele and BP. Association of the 235T with hypertension was confirmed in several Japanese populations recruited from hospital clinics.11,12 In Australia, despite selecting subjects with
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severe familial hypertension, Bennet et al 19 found no effect of the M235T variants on BP. Fornage et al 20 conducted a population based study in Rochester, Minnesota, and also did not detect a relationship. These authors emphasized the importance of subject selection criteria and phenotype ascertainment in genetic studies of complex traits. Our study indicates that accurate characterization of BP using ambulatory monitoring may improve the analytic power of a genetic study and allow detection of an association between a gene polymorphism and hypertension with a relatively small sample size. Our report suggests that selection of subjects based on ABPM profile may be a useful complement to other selection strategies for genetic studies of hypertension.
ACKNOWLEDGMENT We would like to thank Mr. Howard Printz for his genuine interest, encouragement and generous support of the Hypertension Section.
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