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Polymorphism of angiotensin I converting enzyme gene in the older Chinese: linked to ambulatory blood pressure levels and circadian blood pressure rhythm
International
Journal
of
cardiology International Journal of Cardiology 55 (1996) 33-40
Polymorphism of angiotensin I converting enzyme gene in the older Chinese: linked to ambulatory blood pressure levels and circadian blood pressure rhythm Jian Ming”‘“, Cao Xinminb, Huang Jianfeng”, Qi Jianhua”, Liu Guozhang”, Wang Jiaruib, Bai Danb, Qiao Deren”, Zhao Jiu Fenc, Feng Shaoru”, Liu Lisheng” “Hypertension
Division, Fu Wai Cardiovascular Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, People’s Republic of China “Xuan Wu Hospital, Capital University of Medical Sciences, Beijing, People’s Republic of China ‘Kai Luan Hospital, Tang San 063000, People’s Republic of China Received 6 December 1995; revised 21 March 1996; accepted 21 March 1996
Abstract This study was undertaken to evaluate the association of polymorphism of angiotensin I converting enzyme (ACE) gene with casual and ambulatory blood pressure in the Chinese population. Genomic DNA was amplified by polymerase chain reaction (PCR) using primers flanking the polymorphic region in intron 16 of the ACE gene. Alleles were detected on agarose gels stained with ethidium bromide. Casual blood pressure was measured in the 133 normotensive and 122 essential hypertensive subjects; of the essential hypertensive subjects, 65 patients underwent non-invasive ambulatory blood pressure. There was no significant difference of ACE genotype and allele frequencies between the hypertensive and normotensive subjects. Non-significant differences were demonstrated in gender, age, casual blood pressure according to the ACE gene genotypes in the 65 patients who underwent non-invasive ambulatory blood pressure; however, effects of the ACE gene genotypes were observed on the derivatives of ambulatory blood pressure, including sleeping blood pressure levels and circadian blood pressure rhythm (P < 0.05). We suggest that insertion/deletion polymorphism of the ACE gene should be associated with blood pressure and ambulatory blood pressure might be superior to casual blood pressure at the molecular level. Angiotensin converting enzyme; Primary hypertension; Insertion-deletion pressure; Circadian rhythm
Keywords:
1. Introduction Angiotensin I converting enzyme (ACE) is a zinc metallopeptidase, the primary known functions of *Corresponding author, Tel.: +86 1 831 4466 ext. 498; fax: +86 1 831 3012.
polymorphism;
Ambulatory
blood
which are to convert angiotensin I to angiotensin II and to inactivate bradykinin [ 11. Regulation of the ACE expression might play a role in hypertension. In humans, the plasma level of ACE is determined genetically. The ACE gene has insertion/deletion (I/D) polymorphism in intron 16. The ACE genotype has been reported to be associated with plasma
0167-5273/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PZZ SO167-5273(96)02629-O
34
J. Ming et al. I International
Journal of Cardiology 55 (1996) 33-40
ACE activity [2]. The DD genotype might increase the risk of myocardial infarction [3]. Therefore, determining whether there is an I/D polymorphism of ACE gene associated with hypertension could be useful in developing the screening strategies. However, the contradictory communications have been published, some reporting the association [4,5] and the others no association [6-8,231. Hypertension and blood pressure level in general have methodological problems in measurement. Blood pressure is influenced by the instrument and the individual taking the measure, the subject’s physical activity and behaviour, and the location and setting in which the blood pressure is obtained [9111. Ambulatory blood pressure monitoring offers an alternative measurement to casual blood pressure. Ambulatory blood pressure has been reported to be superior to casual blood pressure at the organic level in predicting the target organ damages associated with hypertension [ 12-141 and reveal the characteristic of circadian blood pressure rhythm which is essentially important in the pathophysiology of hypertension [15-171. To date, no data are available, describing the association of ACE gene polymorphism with ambulatory blood pressure, including ambulatory blood pressure levels and circadian blood pressure rhythm, especially in the Chinese population and proving whether ambulatory blood pressure is still superior to casual blood pressure at the gene molecular level. Therefore, the present study was designed to examine the relationships of ACE gene polymorphism with casual blood pressure and ambulatory blood pressure in the Chinese population.
stroke; seated casual blood pressure > 140190 mmHg [4]. The protocol and procedures were approved by the Fu Wai Hospital and Kai Luan Hospital Ethics Committee. Normotensive subjects were healthy people referred to our laboratory for complete physical examinations and showing casual blood pressure 5 140/90 mmHg, who did not have specific symptoms.
2. Materials
2.3. DNA studies
and methods
2.1. Subjects To be included in the study, subjects had to be more than 60 years of age. Those with essential hypertension were the outpatients. Of 205 outpatients, 122 met all the following criteria: no antihypertensive drugs for > 4 weeks; absence of clinical electrocardiographic and echo cardiographic evidence of coronary or valvular ventricular disease, renal disease, transient cerebral ischemic attacks or
2.2. Blood pressure measurements and calculation of cumulative sum (cusum)-derived statistics Clinical blood pressure was measured after 15 min of seated rest by using a mercury sphygmomanometer (Phase 1 and 5), with deflation rate fixed at 2 mmHg/beat. Ambulatory blood pressure was recorded using the fully automatic unit Accutracker II monitor (Suntech Instruments, Raleigh, NY, USA), that had been validated [ 181. All patients underwent monitoring in the ambulatory circumstances and were instructed to act and rest as usual. A total of 48 blood-pressures and 48 heart-rate values/24 h one recording per 30 min were obtained and fed into an IBM PB/XT 386 (IBM Corp., Purchase, NY). The accuracy of the Accutracker II was tested against mercury sphygmomanometer readings both before each patient left the hospital and when he returned. The 48 blood-pressures and 48 heart-rate values were analyzed further only when the readings obtained in the preceding two instances were found to differ by no more than 5 mmHg from the corresponding manometric readings. Cusum method and calculation of cusum-derived statistics were outlined precisely by both Stanton et al. [19] and us [16] (Appendix 1).
Genomic DNA was isolated from peripheral leukocytes as previously reported [20]. The genotype of the ACE gene was determined by the polymerase chain reaction (PCR) according to Rigat et al. [21]. The sense oligonucleotide primer was 5’-CTG GAG ACC ACT CCC ATC CTT TCT-3’, and the antisense primer was 5’-GAT GTG CCC ATC ACA TTC GTC AGAT-3’. These primers allowed detection of a genomic DNA segment with 490 bp corresponding to the insertion allele (I) as well as a
.I. Ming et al. / International
Journal of Cardiology
segment with 190 bp corresponding to the deletion allele (D). Reactions were performed in a final volume of 25 ~1, containing 10 pmol of each primer, 2.5 mmol/l MgCl,, 50 mmol/l KCl, 10 mmol/l Tris-HCl (pH 8.4), 0.1 mg/ml gelatin, 0.2 mmol/l of each dNTP, and 0.5 Taq DNA polymerase. The amplification profile included a denaturation at 94°C for 60 s and 35 cycles of denaturation at 94°C for 60 s, annealing at 58°C for 60 s, and extension at 74°C for 120 s. The PCR products were resolved in 1.5% agarose gels and visualized with ethidium bromide staining. 2.4. Statistical analysis The Statistical Analysis System was used to analyse the data [22]. Data are expressed as mean + S.D. Differences in the distribution of ACE genotype and sex were analyzed using x2-tests. One way ANOVA was carried out to evaluate the relationships between genotype and both casual blood pressure and the derivatives of ambulatory blood pressure. The derivatives of ambulatory blood pressure included both cusum-derived statistics and ambulatory blood pressure levels. Ambulatory blood pressure levels were defined as mean blood pressure values during 24-h, waking (06:00-22:00) and sleeping (22:00-06:OO) periods respectively. The size of a two-sided test equals to 0.05.
3. Results 3.1. Study population The characteristics of the 133 normotensive subjects and 122 hypertensive patients who met the criteria are summarized in Table 1, and the attributes of the 65 hypertensive patients who had the good quality ambulatory blood pressure monitories epitomized in Table 2 and Table 3. 3.2. Genotype frequencies in the hypertensive and normotensive subjects The polymorphism was readily detected by this method as a 490 bp PCR product in the presence of the insertion and/or as a 190 bp fragment in the
55 (1996) 33-40
35
Table 1 Characteristics and frequencies of the insertion/deletion polymorphism of ACE gene in the normotensive subjects and essential hypertensive patients based upon casual blodd pressure
n Sex (male/female) Age (year) Genotypes II ID DD Alleles I D
Normotensive
Hypertensive
P
133 lll/ll 66.3 t 6.3
122 97125 66.8 i 6.6
n.s. n.s.
61 (45.9%) 50 (37.6%) 22 (16.5%)
50 (41.0%) 54 (44.3%) 18 (14.8%)
ILS.
0.65 0.35
0.63 0.37
n.s.
n.s., non-significant.
presence of the deletion. Genotype and allele frequencies for the Chinese are shown in Table 1, indicating that there was no significant difference between the hypertensive and normotensive subjects. 3.3. Phenotype analysis with ACE gene genotypes in the hypertensive subjects x*-test and one way ANOVA demonstrated no differences in gender, age, casual blood pressure according to the ACE gene genotypes (Table 2). Significant effects of the ACE gene genotypes were observed on the derivatives of ambulatory blood pressure, including sleeping systolic and diastolic blood pressure levels and circadian blood pressure rhythm (Table 2 and Table 3). Fig. 1 depicts the systolic and diastolic blood pressure profile respectively according to the ACE gene genotypes.
4. Discussion The major findings in the present study are that in the Chinese, there is not a significant association of an I/D polymorphism of the ACE gene with essential hypertension based upon casual blood pressure, and that no significant differences in casual blood pressure are found according to the ACE gene genotypes; however, we have shown a relation of the ACE gene genotypes with the derivatives of ambulatory blood pressure, including sleeping systolic
36
.I. Ming et al. I International
Journal
of Cardiology 55 (1996) 33-40
Table 2 Characteristics and casual and ambulatory blood pressure levels from 65 essential hypertensive subjects
n Sex (male/female) Age (w-) sCBP** dCBP** sWBP** dWBP** sSBP** dSBP** s24BP** d24BP**
II
ID
DD
25 2312 69 ” 170 + 85 i 150 + 80 2 135 I! 71 -c 144 2 77 2
26 26/O 65 t165 i 87 i154 i 83 2 148 i 79 i 151 r 82 2
14 13/l 68 164 83 152 83 142 76 148 80
6.7 17.1 11.6 13.1 8.2 14.2* 9.9* 12.2 8.1
6.9 16.0 10.9 14.8 8.2 20.0 11.4 15.5 8.4
F
P
n.s.
2 tt -+ t i I! i t
6.3 15.9 10.4 12.8 7.9 10.7 8.3 11.9 7.3
n.s. n.s. ll.S.
3.30 3.88
ns. ns. 0.0442 0.0266 n.s. n.s.
*P < 0.05, II VS. ID. **, mmHg; sCBP, sWBP, sSBP, s24BP, dCBP, dWBP, dSBP, d24BP, = systolic and diastolic clinic, waking, sleeping, 24-h, respectively.
and diastolic blood pressure levels and circadian blood pressure rhythm. Our results are consistent with those of the reports, describing that there is an no association of I/D polymorphism of ACE gene with hypertension based upon casual blood pressure, [6-8,231 and that the genotype of the ACE gene apparently had no significant effects on (clinical) blood pressure [24]. Nevertheless, other studies show that the I/D polymorphism of ACE gene is associated with hypertension based upon casual blood pressure [4,5]. Linkage analysis has demonstrated that polymorphisms in the ACE gene are linked to the regulation of blood pressure in the rat [25,26]. Therefore, these findings seem to support an association between ACE gene
and the genesis of hypertension. Whether the controversy is due to ethnic or inclusion criteria variances is unknown and merits further investigation. However, one substantial reason for the controversy, we consider, could be due to the methodological problems in blood pressure measurement. It has been proved that casual blood pressure on which the diagnosis and management of hypertension has been based is not always a true reflection of a patient’s blood pressure profile [27]. Ambulatory blood pressure monitoring, as an alternative measurement to casual blood pressure, has been reported to be superior to casual blood pressure in predicting the target organ damages associated with hypertension [ 12-141 and reveal the characteristic of circa-
Table 3 Circadian blood pressure rhythm from 65 essential hypertensive subjects II n sCDC** sCDT** sCDCAM** sCPH*** dCDC** dCDT** dCDCAM** dCPH***
25 139 2 156 2 - 17.0 329 2 73 r 83 i -9.8 219 -t
DD
ID 13.4* 15.5 I! 13.2* 91.0* 10.0* 9.0 i 9.4 62.5
26 150 2 157 -t -7.3 261 2 81 ? 86 2 -4.5 199 ?
18.0 15.2 + 15.7 68.7 9.9 9.2 t 9.7 63.6
14 145 k 154 2 -8.9 287 2 78 +84 t -5.8 198 L
12.5 12.0 ? 9.8 50.7 9.4 8.9 ? 6.7 26.8
F
P
3.21
0.0478 n.s. 0.0448 0.0084 0.0228 ns. n.s. n.s.
3.28 5.21 4.05
*P < 0.05, II vs. ID. **, mmHg; ***, mmHg.h; sCDT, sCDC, sCDCAM, sCPH, dCDT, dCDC, dCDCAM, dCPH = systolic and diastolic cusum-detived crest, and cusum-derived trough blood pressures and cusum-derived circadian alteration magnitude, cusum plot height, respectively.
.I. Ming et al. I Intermational Journal of Cardiology
-601
II
ID
12
I6
-
16
II
20 hour
22
II
+
ID
-DD
6
*
II
6
II
37
55 (1996) 33-40
\
16
I?.
/
1 ” ” / * 1 5 ” 16 16 a* 16 2.2 6 hour -
II
-ID
c j 2 ,
”
6
I
-cDD
Fig. 1. (A) 24-h ambulatory systolic blood pressure (SBP) profile and (a) corresponding cumulative sums (cusum) plot according to the ACE gene genotypes. (B) 24-h ambulatory diastolic blood pressure (DBP) profile and (b) corresponding cumulative sums (cusum) plot according to the ACE gene genotypes.
dian blood pressure rhythm which is not available only using casual blood pressure measurement. It is more important that in the derivatives of ambulatory blood pressure, cusum-derived statistics reflecting circadian blood pressure rhythm, have the different relevances from 24-h blood pressure levels. Chau et al. [15] proposed that cusum-derived active blood pressure span is associated with salt intake in normal and borderline blood pressure groups and no relationships are detected between salt output and both casual blood pressure and ambulatory blood pressure levels. Our previous study has shown that left ventricular structure in isolated systolic hypertension appears to be related to ambulatory blood pressure levels, whereas left ventricular function appears to be related to circadian blood pressure rhythm [16]. Hence, ambulatory blood pressure is not only more accurate in reflecting blood pressure level but also supplies more information about blood pressure profile than casual blood pressure. The results in the present study agrees with those of the previous studies, suggesting that ambulatory
blood pressure should be superior to casual blood pressure at the organic level, [ 12-161 and extend their validity as this was the case with association of an I/D polymorphism of the ACE gene with ambulatory blood pressure at the molecular level. Meanwhile, the present study could provide the interesting evidence for an association between I/D polymorphism of the ACE gene with blood pressure in humans; and we suggest that the methodological problems in blood pressure measurement should be one of the most essential reasons for the controversy. Four caveats should be kept in mind. Firstly is that positive histories of hypertension which other studies [4-81 had was not one of the inclusion criteria in the present study. We considered that it was difficult to confirm unquestionably the positive and negative histories of hypertension due to the methodological problems in blood pressure measurement. The frequencies of the insertion/deletion polymorphism of ACE gene in the present study are similar with those of the Japanese population which had the positive histories of hypertension [8]. Secondly, in the present
38
J. Ming et al. / International
Journal
study, the hypertensives and normotensive controls are rather old (66 years). The reason why we chose the older subjects is that a normotensive control of 40-years-old might be a hypertensive at his 60 years. Therefore, it is advantageous to analyze the relation of blood pressure with gene involved in the hypertensive etiology in the older population. Thirdly, one disturbing features of the present study is that the DD genotype always has nonsignificantly lower blood pressure than the ID genotypes. ACE alleles are a co-dominant system on ACE levels [2] and would be expected to produce a graduated increase in blood pressure with one D allele and again with 2 D alleles even if nonsignificantly. The answers may be in the exclusion criteria, where we excluded persons with coronary heart disease. Since ACE DD genotype is associated with coronary heart disease [3], excluding these subjects might remove those persons who might have elevated blood pressures leading to coronary heart disease. Therefore, the remaining DD subjects might have lower blood pressure. Hence, the further studies including the subjects with coronary heart disease need conforming this hypothesis. However, in the present study, the fact that the ID subjects have significantly higher sleeping blood pressure levels than the II ones supports in part the expectation of the production of a graduated increase in blood pressure with D allele. Finally, only sleeping but not waking blood pressure was significant in the present study. It is possible that the more variable blood pressure was occurring during daily activities while at home and the waking blood pressure was disturbed by the doctor while at the hospital, [28] obscuring the effect of the genotype on the waking blood pressure. Therefore, our findings of the significant increases in the sleeping blood pressure, but not waking blood pressure, may be interpreted as the consequence of the fact that the ACE gene directly influence basal blood pressure, whereas waking blood pressure are additionally influenced by both activity and psychosocial factors. Although four caveats are mentioned, at the very least, two important conclusions emerge from our findings that there would be an association of I/D polymorphism of the ACE gene with blood pressure and ambulatory blood pressure might be superior to casual blood pressure at the molecular level.
of Cardiology 5.5 (1996) 33-40 Appendix
1
Let’s briefly recall the cumulative sum method. Each interval blood pressure is taken as the mean of the blood pressure readings at the start and finish of the interval. Mean 24-h blood pressure, the reference value for construction of the plot, is calculated from these interval pressures, weighted by the duration of the interval; intended interval duration was 60 min: mean 24 - h BP = $$(BPi)(di) 1-l
where BP is blood pressure, D total duration of ambulatory recording, n total number of intervals, BPi blood pressure (systolic or diastolic of the ith interval), and di is ith interval duration. Similarly, mean pressures of all other periods are calculated from interval pressures weighted for time. To construct the cusum plot, each interval pressure value in succession is subtracted from mean 24-h pressure; and remainder (mmHg) is multiplied by the duration of the interval (h) and then the resultant pressuretime product (mmHg.h) is added to the previous sum. This cusum, plotted against time, is the cusum plot. The cusum plot slope (CPS) for any given time period is defined as the change in the cusum over a given period of time divided by the change in time for that period: (a period must be composed of whole numbers of intervals.) CPS = (CSE - CSB)Idp where CSE is the cumulative sum at the end of the period, CSB is the cumulative sum at the beginning of the period, and dp is the duration of the period. The steepest positive slope (trough CPS) was defined as trough CPS = (CSO4 - CS22)/6 where CS04 and CS22 are cusums at 4 and 22 o’clock respectively. It was stipulated that crest (the most negative slope) CPS = (CS18 - CSOS)/ 10 where CS18 and CS8 are cusums at 18 and 8 o’clock respectively. Since the cusum plot slope over a given time period equals the difference between the mean blood
J. Ming et al. / International
Journal of Cardiology
pressure for that period and mean 24-h blood pressure, cusum-derived crest blood pressure may be calculated as cusum-derived
crest BP
= crest CPS + mean 24-h BP
and cusum-derived trough blood pressure is defined as cusum-derived
trough BP
= trough CPS + mean 24-h BP
Cusum plot height (CPH) was calculated as CPH
= 2 I(BPi -
mean 24 - h BP)(di)l
i=l
where n is number of intervals between maximum and minimum cumulative sums (or between minimum and maximum cumulative sum values where the minimum value precedes the maximum), BPi is blood pressure of the ifh interval, and di is ith interval duration. Cusum-derived circadian alteration magnitude (CDCAM) is defined as the difference between cusum-derived crest and trough blood pressures and is calculated from the difference between the cusum plot slopes (CPS) of the two periods: CDCAM
= crest CPS - trough CPS
References [l] Ehlers MRW, Riordan JF. Angiotensin converting enzyme: new concepts concerning its biological role. Biochemistry 1989; 28: 5311-531s. [2] Rigat B, Hubert C, Alhegc-Gelas F, Corvol P, Sorbrier F. An insertion/deletion polymorphism in the angiotensin I converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest 1990; 86: 1343-1346. [3] Tiret L, Kee F, Porier 0, NIcaud V, Lecerf L, Cambou J, Luc G, Amouyel P, Cambien F. Deletion polymorphism in angiotensin converting enzyme gene associated with parental history of myocardial infarction. Lancet 1993; 341: 991-999. [4] Zee RYL, Lou YK, Griffiths LR, Morris BJ, Association of a polymorphism of the angiotensin 1 converting enzyme gene with essential hypertension. Biochem Biophys Res Commun 1992; 184: 9-15.
55 (1996) 33-40
39
[5] Rutledge DR, Kubilis P, Browe CS, Ross EA. Polymorphism of the angiotensin I converting enzyme gene in essential hypertensive patients. Biochem Mol Biol Inter 1995; 35: 661-668. %I Summers KM, West JA, Huggard PR, West MJ, Angiotensin converting enzyme and regulation of blood pressure in a large Australian family. Clin Exp Pharmacol Physiol 1993; 20: 320-323. [71 Jeunemattre X, Lifton RP, Hunt SC, Williams RR, Lalouel JM. Absence of linkage between the angiotensin converting enzyme locus and human essential hypertension. Nature Genet 1992; 1: 72-75. K31Higashimori K, Zhao Y, Higaki J, Kamitani A, Katysuya T, Nakura J, Miki T, Mikami H, Ogihara T. Association analysis of a polymorphism of the angiotensin converting enzyme gene with essential hypertension in the Japanese population. Biochem Biophys Res Commun 1993; 191: 399404. 191 Gordon T, Sorlie P, Kamrel WB. Problems in the assessment of blood pressure: The Framingham Study. Int J Epidemiol 1976; 5: 327-334. UOI Carey RM, Reid RA, Ayers CR, Lunch SS, McLain WL, Vaughan ED. The Charlottesville blood pressure survey: value of repeated blood pressure measurements. J Am Med Assoc 1976; 236: 847-851. t111Picking TG, James GD. Some implications of the differences between home, clinic and ambulatory blood pressure in normotensive and hypertensive patients. J Hypertens 1989; (suppl 3): S65-S72. WI Shimada K, Kawamoto A, Matsubayashi K, Ozawa T. Silent cerebrovascular disease in the elderly: correlation with ambulatory pressure. Hypertension 1990; 16: 692-699. 1131 Verdecchia P, Schillaci G, Guerrieri M, Gatteschi C, Benemio G, Boldrini G, Porcellati C. Circadian blood pressure changes and left ventricular hypertrophy in essential hypertension. Circulation 1990; 81: 528-536. u41 Schulte KL, Meryer-Sabellek W, Liederwald K, Germmeren DV, Lenz T. Relation of regression of left ventricular hypertrophy to changes in ambulatory blood pressure after long-term therapy with perindopril versus nifedipine. Am J Cardiol 1992; 70: 468-473. WI Chau NP, Chanudet X, Bauduceau B, Gautier D, Larroque P. Active ambulatory blood pressures and urinary electrolytes in young male subjects with normal blood pressure or borderline hypertension. J Hypertens 1993; 11: 203-209. U61 Ming J, Guozhang L, Deren Q, Xiaoqing L, Fen ZJ, Honglian N, Shaoru F, Aili W, Lisheng L. Effect of 24-h blood pressure levels and circadian blood pressure rhythm on left ventricular structure and function in isolated systolic hypertension. Int J Cardiol 1994; 47: 51-58. Cl71 Calhoun DA, Zhu S, Wyss JM, Oparil S. Diurnal blood pressure variation and dietary salt in spontaneously hypertensive rats. Hypertension 1994; 24: l-7. 1181White WB, Schulman P, ,McCabe EJ. Clinical evaluation on the Accutracker ambulatory blood pressure monitor: assessment of performance in two countries and comparison with sphygmomanometry and intraarterial blood pressure at rest and during exercise. J Hypertens 1989; 7: 967-978.
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[19] Stanton A, Cox .I, Atkins N, O’Malley K, O’Brien E. Cumulative sums in quantifying circadian blood pressure patterns. Hypertension 1992; 19: 93-101. [20] Bell GF, Karam JH, Rutter WJ. Polymorphic DNA region adjacent to the 5’-end of the human insulin gene. Proc Nat1 Acad Sci USA 1981; 78: 5759-5763. [21] Rigat B, Hubert C, Corvol P, Soubrier F. PCR detection of the insertion/deletion polymorphism of the human angiotensin I-converting enzyme gene (DCPI) (dipeptidyl carboxypeptidase I). Nucl Acids Res 1992; 20: 1433. [22] SAS Institute: SASlSTAT guide for personal computers, version 6. Cary, North Carolina: SAS Institute 1985; 229235. [23] Schmidt S, Hooft IMS van, Grobbee DE, GAnten D, Ritz E. Polymorphism of the angiotensin 1 converting enzyme gene is apparently not related to high blood pressure: Dutch hypertension and offspring study. J Hypertens 1993; 11: 345-348. [24] Iwai N, Ohmichi N, Nakamura Y, Kinoshita M. DD genotype of the angiotensin converting enzyme gene is a risk factor for left ventricular hypertrophy. Circulation 1994; 90: 26222628.
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[25] Jacob Hj, Lindpainter K, Lincoln SE, Kusumi K, Bunker RK, Mao YP, Ganten D, Dzau VJ, Lander ES. Genetic mapping of a gene causing hypertension in the stroke-prone spontaneously hypertensive rat. Cell 1991; 67: 213-224. 1261 Hilbert P, Lindpainter K, Beckmann JS, Serikawa T, Soubtier F, Dubay C, Cartwright P, DeGouyon B, Julier C, Takahasi S,Vincent M, Ganten D, Georges M, Latjhrop GM. Chromosomal mapping of two genetic loci associated with blood pressure regulation in hereditary hypertensive rats. Nature 1991; 353: 521-529. [27] Zachariah K, Krier JD. Clinical uses of ambulatory blood pressure monitoring. J Hypertens 1991; 9(suppl 1): s7-~12. [28] Mancia G. Ambulatory blood pressure monitoring: research and clinical applications. J Hypertens 1990; 8(suppl 7): Sl-s13.
Journal
of
cardiology International Journal of Cardiology 55 (1996) 33-40
Polymorphism of angiotensin I converting enzyme gene in the older Chinese: linked to ambulatory blood pressure levels and circadian blood pressure rhythm Jian Ming”‘“, Cao Xinminb, Huang Jianfeng”, Qi Jianhua”, Liu Guozhang”, Wang Jiaruib, Bai Danb, Qiao Deren”, Zhao Jiu Fenc, Feng Shaoru”, Liu Lisheng” “Hypertension
Division, Fu Wai Cardiovascular Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, People’s Republic of China “Xuan Wu Hospital, Capital University of Medical Sciences, Beijing, People’s Republic of China ‘Kai Luan Hospital, Tang San 063000, People’s Republic of China Received 6 December 1995; revised 21 March 1996; accepted 21 March 1996
Abstract This study was undertaken to evaluate the association of polymorphism of angiotensin I converting enzyme (ACE) gene with casual and ambulatory blood pressure in the Chinese population. Genomic DNA was amplified by polymerase chain reaction (PCR) using primers flanking the polymorphic region in intron 16 of the ACE gene. Alleles were detected on agarose gels stained with ethidium bromide. Casual blood pressure was measured in the 133 normotensive and 122 essential hypertensive subjects; of the essential hypertensive subjects, 65 patients underwent non-invasive ambulatory blood pressure. There was no significant difference of ACE genotype and allele frequencies between the hypertensive and normotensive subjects. Non-significant differences were demonstrated in gender, age, casual blood pressure according to the ACE gene genotypes in the 65 patients who underwent non-invasive ambulatory blood pressure; however, effects of the ACE gene genotypes were observed on the derivatives of ambulatory blood pressure, including sleeping blood pressure levels and circadian blood pressure rhythm (P < 0.05). We suggest that insertion/deletion polymorphism of the ACE gene should be associated with blood pressure and ambulatory blood pressure might be superior to casual blood pressure at the molecular level. Angiotensin converting enzyme; Primary hypertension; Insertion-deletion pressure; Circadian rhythm
Keywords:
1. Introduction Angiotensin I converting enzyme (ACE) is a zinc metallopeptidase, the primary known functions of *Corresponding author, Tel.: +86 1 831 4466 ext. 498; fax: +86 1 831 3012.
polymorphism;
Ambulatory
blood
which are to convert angiotensin I to angiotensin II and to inactivate bradykinin [ 11. Regulation of the ACE expression might play a role in hypertension. In humans, the plasma level of ACE is determined genetically. The ACE gene has insertion/deletion (I/D) polymorphism in intron 16. The ACE genotype has been reported to be associated with plasma
0167-5273/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PZZ SO167-5273(96)02629-O
34
J. Ming et al. I International
Journal of Cardiology 55 (1996) 33-40
ACE activity [2]. The DD genotype might increase the risk of myocardial infarction [3]. Therefore, determining whether there is an I/D polymorphism of ACE gene associated with hypertension could be useful in developing the screening strategies. However, the contradictory communications have been published, some reporting the association [4,5] and the others no association [6-8,231. Hypertension and blood pressure level in general have methodological problems in measurement. Blood pressure is influenced by the instrument and the individual taking the measure, the subject’s physical activity and behaviour, and the location and setting in which the blood pressure is obtained [9111. Ambulatory blood pressure monitoring offers an alternative measurement to casual blood pressure. Ambulatory blood pressure has been reported to be superior to casual blood pressure at the organic level in predicting the target organ damages associated with hypertension [ 12-141 and reveal the characteristic of circadian blood pressure rhythm which is essentially important in the pathophysiology of hypertension [15-171. To date, no data are available, describing the association of ACE gene polymorphism with ambulatory blood pressure, including ambulatory blood pressure levels and circadian blood pressure rhythm, especially in the Chinese population and proving whether ambulatory blood pressure is still superior to casual blood pressure at the gene molecular level. Therefore, the present study was designed to examine the relationships of ACE gene polymorphism with casual blood pressure and ambulatory blood pressure in the Chinese population.
stroke; seated casual blood pressure > 140190 mmHg [4]. The protocol and procedures were approved by the Fu Wai Hospital and Kai Luan Hospital Ethics Committee. Normotensive subjects were healthy people referred to our laboratory for complete physical examinations and showing casual blood pressure 5 140/90 mmHg, who did not have specific symptoms.
2. Materials
2.3. DNA studies
and methods
2.1. Subjects To be included in the study, subjects had to be more than 60 years of age. Those with essential hypertension were the outpatients. Of 205 outpatients, 122 met all the following criteria: no antihypertensive drugs for > 4 weeks; absence of clinical electrocardiographic and echo cardiographic evidence of coronary or valvular ventricular disease, renal disease, transient cerebral ischemic attacks or
2.2. Blood pressure measurements and calculation of cumulative sum (cusum)-derived statistics Clinical blood pressure was measured after 15 min of seated rest by using a mercury sphygmomanometer (Phase 1 and 5), with deflation rate fixed at 2 mmHg/beat. Ambulatory blood pressure was recorded using the fully automatic unit Accutracker II monitor (Suntech Instruments, Raleigh, NY, USA), that had been validated [ 181. All patients underwent monitoring in the ambulatory circumstances and were instructed to act and rest as usual. A total of 48 blood-pressures and 48 heart-rate values/24 h one recording per 30 min were obtained and fed into an IBM PB/XT 386 (IBM Corp., Purchase, NY). The accuracy of the Accutracker II was tested against mercury sphygmomanometer readings both before each patient left the hospital and when he returned. The 48 blood-pressures and 48 heart-rate values were analyzed further only when the readings obtained in the preceding two instances were found to differ by no more than 5 mmHg from the corresponding manometric readings. Cusum method and calculation of cusum-derived statistics were outlined precisely by both Stanton et al. [19] and us [16] (Appendix 1).
Genomic DNA was isolated from peripheral leukocytes as previously reported [20]. The genotype of the ACE gene was determined by the polymerase chain reaction (PCR) according to Rigat et al. [21]. The sense oligonucleotide primer was 5’-CTG GAG ACC ACT CCC ATC CTT TCT-3’, and the antisense primer was 5’-GAT GTG CCC ATC ACA TTC GTC AGAT-3’. These primers allowed detection of a genomic DNA segment with 490 bp corresponding to the insertion allele (I) as well as a
.I. Ming et al. / International
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segment with 190 bp corresponding to the deletion allele (D). Reactions were performed in a final volume of 25 ~1, containing 10 pmol of each primer, 2.5 mmol/l MgCl,, 50 mmol/l KCl, 10 mmol/l Tris-HCl (pH 8.4), 0.1 mg/ml gelatin, 0.2 mmol/l of each dNTP, and 0.5 Taq DNA polymerase. The amplification profile included a denaturation at 94°C for 60 s and 35 cycles of denaturation at 94°C for 60 s, annealing at 58°C for 60 s, and extension at 74°C for 120 s. The PCR products were resolved in 1.5% agarose gels and visualized with ethidium bromide staining. 2.4. Statistical analysis The Statistical Analysis System was used to analyse the data [22]. Data are expressed as mean + S.D. Differences in the distribution of ACE genotype and sex were analyzed using x2-tests. One way ANOVA was carried out to evaluate the relationships between genotype and both casual blood pressure and the derivatives of ambulatory blood pressure. The derivatives of ambulatory blood pressure included both cusum-derived statistics and ambulatory blood pressure levels. Ambulatory blood pressure levels were defined as mean blood pressure values during 24-h, waking (06:00-22:00) and sleeping (22:00-06:OO) periods respectively. The size of a two-sided test equals to 0.05.
3. Results 3.1. Study population The characteristics of the 133 normotensive subjects and 122 hypertensive patients who met the criteria are summarized in Table 1, and the attributes of the 65 hypertensive patients who had the good quality ambulatory blood pressure monitories epitomized in Table 2 and Table 3. 3.2. Genotype frequencies in the hypertensive and normotensive subjects The polymorphism was readily detected by this method as a 490 bp PCR product in the presence of the insertion and/or as a 190 bp fragment in the
55 (1996) 33-40
35
Table 1 Characteristics and frequencies of the insertion/deletion polymorphism of ACE gene in the normotensive subjects and essential hypertensive patients based upon casual blodd pressure
n Sex (male/female) Age (year) Genotypes II ID DD Alleles I D
Normotensive
Hypertensive
P
133 lll/ll 66.3 t 6.3
122 97125 66.8 i 6.6
n.s. n.s.
61 (45.9%) 50 (37.6%) 22 (16.5%)
50 (41.0%) 54 (44.3%) 18 (14.8%)
ILS.
0.65 0.35
0.63 0.37
n.s.
n.s., non-significant.
presence of the deletion. Genotype and allele frequencies for the Chinese are shown in Table 1, indicating that there was no significant difference between the hypertensive and normotensive subjects. 3.3. Phenotype analysis with ACE gene genotypes in the hypertensive subjects x*-test and one way ANOVA demonstrated no differences in gender, age, casual blood pressure according to the ACE gene genotypes (Table 2). Significant effects of the ACE gene genotypes were observed on the derivatives of ambulatory blood pressure, including sleeping systolic and diastolic blood pressure levels and circadian blood pressure rhythm (Table 2 and Table 3). Fig. 1 depicts the systolic and diastolic blood pressure profile respectively according to the ACE gene genotypes.
4. Discussion The major findings in the present study are that in the Chinese, there is not a significant association of an I/D polymorphism of the ACE gene with essential hypertension based upon casual blood pressure, and that no significant differences in casual blood pressure are found according to the ACE gene genotypes; however, we have shown a relation of the ACE gene genotypes with the derivatives of ambulatory blood pressure, including sleeping systolic
36
.I. Ming et al. I International
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of Cardiology 55 (1996) 33-40
Table 2 Characteristics and casual and ambulatory blood pressure levels from 65 essential hypertensive subjects
n Sex (male/female) Age (w-) sCBP** dCBP** sWBP** dWBP** sSBP** dSBP** s24BP** d24BP**
II
ID
DD
25 2312 69 ” 170 + 85 i 150 + 80 2 135 I! 71 -c 144 2 77 2
26 26/O 65 t165 i 87 i154 i 83 2 148 i 79 i 151 r 82 2
14 13/l 68 164 83 152 83 142 76 148 80
6.7 17.1 11.6 13.1 8.2 14.2* 9.9* 12.2 8.1
6.9 16.0 10.9 14.8 8.2 20.0 11.4 15.5 8.4
F
P
n.s.
2 tt -+ t i I! i t
6.3 15.9 10.4 12.8 7.9 10.7 8.3 11.9 7.3
n.s. n.s. ll.S.
3.30 3.88
ns. ns. 0.0442 0.0266 n.s. n.s.
*P < 0.05, II VS. ID. **, mmHg; sCBP, sWBP, sSBP, s24BP, dCBP, dWBP, dSBP, d24BP, = systolic and diastolic clinic, waking, sleeping, 24-h, respectively.
and diastolic blood pressure levels and circadian blood pressure rhythm. Our results are consistent with those of the reports, describing that there is an no association of I/D polymorphism of ACE gene with hypertension based upon casual blood pressure, [6-8,231 and that the genotype of the ACE gene apparently had no significant effects on (clinical) blood pressure [24]. Nevertheless, other studies show that the I/D polymorphism of ACE gene is associated with hypertension based upon casual blood pressure [4,5]. Linkage analysis has demonstrated that polymorphisms in the ACE gene are linked to the regulation of blood pressure in the rat [25,26]. Therefore, these findings seem to support an association between ACE gene
and the genesis of hypertension. Whether the controversy is due to ethnic or inclusion criteria variances is unknown and merits further investigation. However, one substantial reason for the controversy, we consider, could be due to the methodological problems in blood pressure measurement. It has been proved that casual blood pressure on which the diagnosis and management of hypertension has been based is not always a true reflection of a patient’s blood pressure profile [27]. Ambulatory blood pressure monitoring, as an alternative measurement to casual blood pressure, has been reported to be superior to casual blood pressure in predicting the target organ damages associated with hypertension [ 12-141 and reveal the characteristic of circa-
Table 3 Circadian blood pressure rhythm from 65 essential hypertensive subjects II n sCDC** sCDT** sCDCAM** sCPH*** dCDC** dCDT** dCDCAM** dCPH***
25 139 2 156 2 - 17.0 329 2 73 r 83 i -9.8 219 -t
DD
ID 13.4* 15.5 I! 13.2* 91.0* 10.0* 9.0 i 9.4 62.5
26 150 2 157 -t -7.3 261 2 81 ? 86 2 -4.5 199 ?
18.0 15.2 + 15.7 68.7 9.9 9.2 t 9.7 63.6
14 145 k 154 2 -8.9 287 2 78 +84 t -5.8 198 L
12.5 12.0 ? 9.8 50.7 9.4 8.9 ? 6.7 26.8
F
P
3.21
0.0478 n.s. 0.0448 0.0084 0.0228 ns. n.s. n.s.
3.28 5.21 4.05
*P < 0.05, II vs. ID. **, mmHg; ***, mmHg.h; sCDT, sCDC, sCDCAM, sCPH, dCDT, dCDC, dCDCAM, dCPH = systolic and diastolic cusum-detived crest, and cusum-derived trough blood pressures and cusum-derived circadian alteration magnitude, cusum plot height, respectively.
.I. Ming et al. I Intermational Journal of Cardiology
-601
II
ID
12
I6
-
16
II
20 hour
22
II
+
ID
-DD
6
*
II
6
II
37
55 (1996) 33-40
\
16
I?.
/
1 ” ” / * 1 5 ” 16 16 a* 16 2.2 6 hour -
II
-ID
c j 2 ,
”
6
I
-cDD
Fig. 1. (A) 24-h ambulatory systolic blood pressure (SBP) profile and (a) corresponding cumulative sums (cusum) plot according to the ACE gene genotypes. (B) 24-h ambulatory diastolic blood pressure (DBP) profile and (b) corresponding cumulative sums (cusum) plot according to the ACE gene genotypes.
dian blood pressure rhythm which is not available only using casual blood pressure measurement. It is more important that in the derivatives of ambulatory blood pressure, cusum-derived statistics reflecting circadian blood pressure rhythm, have the different relevances from 24-h blood pressure levels. Chau et al. [15] proposed that cusum-derived active blood pressure span is associated with salt intake in normal and borderline blood pressure groups and no relationships are detected between salt output and both casual blood pressure and ambulatory blood pressure levels. Our previous study has shown that left ventricular structure in isolated systolic hypertension appears to be related to ambulatory blood pressure levels, whereas left ventricular function appears to be related to circadian blood pressure rhythm [16]. Hence, ambulatory blood pressure is not only more accurate in reflecting blood pressure level but also supplies more information about blood pressure profile than casual blood pressure. The results in the present study agrees with those of the previous studies, suggesting that ambulatory
blood pressure should be superior to casual blood pressure at the organic level, [ 12-161 and extend their validity as this was the case with association of an I/D polymorphism of the ACE gene with ambulatory blood pressure at the molecular level. Meanwhile, the present study could provide the interesting evidence for an association between I/D polymorphism of the ACE gene with blood pressure in humans; and we suggest that the methodological problems in blood pressure measurement should be one of the most essential reasons for the controversy. Four caveats should be kept in mind. Firstly is that positive histories of hypertension which other studies [4-81 had was not one of the inclusion criteria in the present study. We considered that it was difficult to confirm unquestionably the positive and negative histories of hypertension due to the methodological problems in blood pressure measurement. The frequencies of the insertion/deletion polymorphism of ACE gene in the present study are similar with those of the Japanese population which had the positive histories of hypertension [8]. Secondly, in the present
38
J. Ming et al. / International
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study, the hypertensives and normotensive controls are rather old (66 years). The reason why we chose the older subjects is that a normotensive control of 40-years-old might be a hypertensive at his 60 years. Therefore, it is advantageous to analyze the relation of blood pressure with gene involved in the hypertensive etiology in the older population. Thirdly, one disturbing features of the present study is that the DD genotype always has nonsignificantly lower blood pressure than the ID genotypes. ACE alleles are a co-dominant system on ACE levels [2] and would be expected to produce a graduated increase in blood pressure with one D allele and again with 2 D alleles even if nonsignificantly. The answers may be in the exclusion criteria, where we excluded persons with coronary heart disease. Since ACE DD genotype is associated with coronary heart disease [3], excluding these subjects might remove those persons who might have elevated blood pressures leading to coronary heart disease. Therefore, the remaining DD subjects might have lower blood pressure. Hence, the further studies including the subjects with coronary heart disease need conforming this hypothesis. However, in the present study, the fact that the ID subjects have significantly higher sleeping blood pressure levels than the II ones supports in part the expectation of the production of a graduated increase in blood pressure with D allele. Finally, only sleeping but not waking blood pressure was significant in the present study. It is possible that the more variable blood pressure was occurring during daily activities while at home and the waking blood pressure was disturbed by the doctor while at the hospital, [28] obscuring the effect of the genotype on the waking blood pressure. Therefore, our findings of the significant increases in the sleeping blood pressure, but not waking blood pressure, may be interpreted as the consequence of the fact that the ACE gene directly influence basal blood pressure, whereas waking blood pressure are additionally influenced by both activity and psychosocial factors. Although four caveats are mentioned, at the very least, two important conclusions emerge from our findings that there would be an association of I/D polymorphism of the ACE gene with blood pressure and ambulatory blood pressure might be superior to casual blood pressure at the molecular level.
of Cardiology 5.5 (1996) 33-40 Appendix
1
Let’s briefly recall the cumulative sum method. Each interval blood pressure is taken as the mean of the blood pressure readings at the start and finish of the interval. Mean 24-h blood pressure, the reference value for construction of the plot, is calculated from these interval pressures, weighted by the duration of the interval; intended interval duration was 60 min: mean 24 - h BP = $$(BPi)(di) 1-l
where BP is blood pressure, D total duration of ambulatory recording, n total number of intervals, BPi blood pressure (systolic or diastolic of the ith interval), and di is ith interval duration. Similarly, mean pressures of all other periods are calculated from interval pressures weighted for time. To construct the cusum plot, each interval pressure value in succession is subtracted from mean 24-h pressure; and remainder (mmHg) is multiplied by the duration of the interval (h) and then the resultant pressuretime product (mmHg.h) is added to the previous sum. This cusum, plotted against time, is the cusum plot. The cusum plot slope (CPS) for any given time period is defined as the change in the cusum over a given period of time divided by the change in time for that period: (a period must be composed of whole numbers of intervals.) CPS = (CSE - CSB)Idp where CSE is the cumulative sum at the end of the period, CSB is the cumulative sum at the beginning of the period, and dp is the duration of the period. The steepest positive slope (trough CPS) was defined as trough CPS = (CSO4 - CS22)/6 where CS04 and CS22 are cusums at 4 and 22 o’clock respectively. It was stipulated that crest (the most negative slope) CPS = (CS18 - CSOS)/ 10 where CS18 and CS8 are cusums at 18 and 8 o’clock respectively. Since the cusum plot slope over a given time period equals the difference between the mean blood
J. Ming et al. / International
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pressure for that period and mean 24-h blood pressure, cusum-derived crest blood pressure may be calculated as cusum-derived
crest BP
= crest CPS + mean 24-h BP
and cusum-derived trough blood pressure is defined as cusum-derived
trough BP
= trough CPS + mean 24-h BP
Cusum plot height (CPH) was calculated as CPH
= 2 I(BPi -
mean 24 - h BP)(di)l
i=l
where n is number of intervals between maximum and minimum cumulative sums (or between minimum and maximum cumulative sum values where the minimum value precedes the maximum), BPi is blood pressure of the ifh interval, and di is ith interval duration. Cusum-derived circadian alteration magnitude (CDCAM) is defined as the difference between cusum-derived crest and trough blood pressures and is calculated from the difference between the cusum plot slopes (CPS) of the two periods: CDCAM
= crest CPS - trough CPS
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[5] Rutledge DR, Kubilis P, Browe CS, Ross EA. Polymorphism of the angiotensin I converting enzyme gene in essential hypertensive patients. Biochem Mol Biol Inter 1995; 35: 661-668. %I Summers KM, West JA, Huggard PR, West MJ, Angiotensin converting enzyme and regulation of blood pressure in a large Australian family. Clin Exp Pharmacol Physiol 1993; 20: 320-323. [71 Jeunemattre X, Lifton RP, Hunt SC, Williams RR, Lalouel JM. Absence of linkage between the angiotensin converting enzyme locus and human essential hypertension. Nature Genet 1992; 1: 72-75. K31Higashimori K, Zhao Y, Higaki J, Kamitani A, Katysuya T, Nakura J, Miki T, Mikami H, Ogihara T. Association analysis of a polymorphism of the angiotensin converting enzyme gene with essential hypertension in the Japanese population. Biochem Biophys Res Commun 1993; 191: 399404. 191 Gordon T, Sorlie P, Kamrel WB. Problems in the assessment of blood pressure: The Framingham Study. Int J Epidemiol 1976; 5: 327-334. UOI Carey RM, Reid RA, Ayers CR, Lunch SS, McLain WL, Vaughan ED. The Charlottesville blood pressure survey: value of repeated blood pressure measurements. J Am Med Assoc 1976; 236: 847-851. t111Picking TG, James GD. Some implications of the differences between home, clinic and ambulatory blood pressure in normotensive and hypertensive patients. J Hypertens 1989; (suppl 3): S65-S72. WI Shimada K, Kawamoto A, Matsubayashi K, Ozawa T. Silent cerebrovascular disease in the elderly: correlation with ambulatory pressure. Hypertension 1990; 16: 692-699. 1131 Verdecchia P, Schillaci G, Guerrieri M, Gatteschi C, Benemio G, Boldrini G, Porcellati C. Circadian blood pressure changes and left ventricular hypertrophy in essential hypertension. Circulation 1990; 81: 528-536. u41 Schulte KL, Meryer-Sabellek W, Liederwald K, Germmeren DV, Lenz T. Relation of regression of left ventricular hypertrophy to changes in ambulatory blood pressure after long-term therapy with perindopril versus nifedipine. Am J Cardiol 1992; 70: 468-473. WI Chau NP, Chanudet X, Bauduceau B, Gautier D, Larroque P. Active ambulatory blood pressures and urinary electrolytes in young male subjects with normal blood pressure or borderline hypertension. J Hypertens 1993; 11: 203-209. U61 Ming J, Guozhang L, Deren Q, Xiaoqing L, Fen ZJ, Honglian N, Shaoru F, Aili W, Lisheng L. Effect of 24-h blood pressure levels and circadian blood pressure rhythm on left ventricular structure and function in isolated systolic hypertension. Int J Cardiol 1994; 47: 51-58. Cl71 Calhoun DA, Zhu S, Wyss JM, Oparil S. Diurnal blood pressure variation and dietary salt in spontaneously hypertensive rats. Hypertension 1994; 24: l-7. 1181White WB, Schulman P, ,McCabe EJ. Clinical evaluation on the Accutracker ambulatory blood pressure monitor: assessment of performance in two countries and comparison with sphygmomanometry and intraarterial blood pressure at rest and during exercise. J Hypertens 1989; 7: 967-978.
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[19] Stanton A, Cox .I, Atkins N, O’Malley K, O’Brien E. Cumulative sums in quantifying circadian blood pressure patterns. Hypertension 1992; 19: 93-101. [20] Bell GF, Karam JH, Rutter WJ. Polymorphic DNA region adjacent to the 5’-end of the human insulin gene. Proc Nat1 Acad Sci USA 1981; 78: 5759-5763. [21] Rigat B, Hubert C, Corvol P, Soubrier F. PCR detection of the insertion/deletion polymorphism of the human angiotensin I-converting enzyme gene (DCPI) (dipeptidyl carboxypeptidase I). Nucl Acids Res 1992; 20: 1433. [22] SAS Institute: SASlSTAT guide for personal computers, version 6. Cary, North Carolina: SAS Institute 1985; 229235. [23] Schmidt S, Hooft IMS van, Grobbee DE, GAnten D, Ritz E. Polymorphism of the angiotensin 1 converting enzyme gene is apparently not related to high blood pressure: Dutch hypertension and offspring study. J Hypertens 1993; 11: 345-348. [24] Iwai N, Ohmichi N, Nakamura Y, Kinoshita M. DD genotype of the angiotensin converting enzyme gene is a risk factor for left ventricular hypertrophy. Circulation 1994; 90: 26222628.
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[25] Jacob Hj, Lindpainter K, Lincoln SE, Kusumi K, Bunker RK, Mao YP, Ganten D, Dzau VJ, Lander ES. Genetic mapping of a gene causing hypertension in the stroke-prone spontaneously hypertensive rat. Cell 1991; 67: 213-224. 1261 Hilbert P, Lindpainter K, Beckmann JS, Serikawa T, Soubtier F, Dubay C, Cartwright P, DeGouyon B, Julier C, Takahasi S,Vincent M, Ganten D, Georges M, Latjhrop GM. Chromosomal mapping of two genetic loci associated with blood pressure regulation in hereditary hypertensive rats. Nature 1991; 353: 521-529. [27] Zachariah K, Krier JD. Clinical uses of ambulatory blood pressure monitoring. J Hypertens 1991; 9(suppl 1): s7-~12. [28] Mancia G. Ambulatory blood pressure monitoring: research and clinical applications. J Hypertens 1990; 8(suppl 7): Sl-s13.