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Angiotensin I converting enzyme (ACE) gene polymorphism and essential hypertension in Japan
AJH 1995; 8:95-97
Angiotensin I Converting Enzyme (ACE) Gene Polymorphism and Essential Hypertension in Japan Ethnic Difference of ACE Genotype Tomoaki Ishigami, Tamio Iwamoto, Kouichi Tamura, Satoshi Yamaguchi, Kan Iwasawa, Kazuaki Uchino, Satoshi Umemura, and Masao Ishii
A polymorphism of the angiotensin I converting enzyme (ACE) gene has recently been reported and analysis of this polymorphism has indicated that it is associated with several cardiovascular diseases. However, the results are still controversial and such association has not yet been established conclusively. To determine whether the ACE gene may be responsible for essential hypertension in a Japanese population, we also compared the distribution of genotypes and the allele frequency of this polymorphism in our findings of a Japanese population with these features in other countries. Eighty-seven hypertensive patients with a family history of essential hypertension and 95 normotensive patients whose parents had no such history were enrolled in the study. Polymorphism of the ACE gene was determined by using the polymerase chain reaction. Homozygotes for this polymorphism had either a 490-bp band (I/) or a 190-bp band (DD) and het-
KEY WORDS: Angiotensin I converting enzyme, gene polymorphism, essential hypertension, ethnicity.
Received April 12, 1994. Accepted October 11, 1994. From the Second Department of Internal Medicine, Yokohama City University, Yokohama, Japan. This study was supported in part by Grant-in-Aid 05670956 for Scientific Research from the Ministry of Education, Science and Culture, Japan and by a grant from the Kihara Memorial Foundation, Yokohama, Japan. Address correspondence and reprint requests to Satoshi Umemura, Second Department of Internal Medicine, Yokohama City University, Fukuura 3-9, Kanazawa-ku, Yokohama, 236, Japan.
Polymorphism of the angiotensin I converting enzyme (ACE) gene has recently been reported 1 and analysis of this polymorphism has indicated that it is associated with several cardiovascular diseases. 2'3 However, the results are still controversial and such association has not yet been established conclusively, especially in essential hypertension. 4-1° To determine whether the ACE gene may be responsible for essen-
© 1995 by the American Journal of Hypertension, Ltd.
erozygotes had both bands (ID). In hypertensive subjects, the numbers and frequency of the ACE genotypes were: II, 44 (0.51); ID, 26 (0.30); DD, 17 (0.19). In normotensive subjects these were: II, 35 (0.37); ID, 43 (0.45); DD, 17 (0.18). There were no significant differences between the two groups in derived allele frequencies (X2 = 1.41). The difference between the overall allelic frequency in Japan and that reported in several other countries was significant. We did not find any association between ACE gene polymorphism and essential hypertension in Japan. However, there were significant differences in derived allele frequencies b e t w e e n our findings in a Japanese population and those reported from Europe and Australia. Am J Hypertens 1995;8:95-97
A
0895-7061/95/$9.50 0895-7061(94)00184-D
96
ISHIGAMI ET AL
AJH-JANUARY 1995-VOL. 8, NO. 1
tial hypertension, we examined the association of this polymorphism with essential hypertension in a Japanese population. Since there are genetic heterogeneities among different ethnic groups, n'12 we also compared the distribution of genotypes and the allele frequency of this polymorphism in our findings of a Japanese population with these features in other ethnic populations.
TABLE 2. FREQUENCY OF INSERTION/DELETION POLYMORPHISM OF THE ACE GENE IN ESSENTIAL HYPERTENSIVE A N D NORMOTENSIVE SUBJECTS
II ID DD
HT (n = 87)
NT (n = 95)
44 (0.51) 26 (0.30) 17 (0.19)
35 (0.37) 43 (0.45) 17 (0.18) Allele
SUBJECTS AND METHODS
Eighty-seven hypertensive patients with a family history of essential hypertension (HT) who had at least one hypertensive parent, and 95 normotensive patients whose parents had no such history (NT) were enrolled in the study. We extracted genomic DNA from these patients' peripheral blood leukocytes by standard methods, 13 using proteinase K digestion of nuclei. Phenol extraction was followed by ethanol precipitation of the DNA. We amplified the insertion/ deletion portion in intron 16 of the ACE gene by the polymerase chain reaction (PCR) method. 1 The PCR product was electrophoresed in 1.6% agarose gels and visualized by ethidium bromide staining. The insertion/deletion polymorphism of the ACE gene was detected as follows: homozygotes for this polymorphism have either a 490-bp band, named II or a 190bp band, named D D , while heterozygotes have both bands, named ID. RESULTS
Table 1 shows the several characteristics in patients with hypertension (HT) and normotensives (NT). There was no significant difference between the two groups. Table 2 shows the genotype distribution and derived allele frequency in the two groups. There were no significant differences in genotype distribution between the HT and NT groups and there was also no significant differences in allele frequency between the groups. The allele frequency reported by Rigat et al in TABLE 1. C O M P A R I S O N OF CHARACTERISTICS IN PATIENTS WITH ESSENTIAL HYPERTENSION (HT) A N D NORMOTENSIVE SUBJECTS (NT) HT
N Age Sex Male Female Onset of hypertension Height (cm) Weight (kg) BMI (kg/m -2)
NT
87 59.3 + 1.3
95 57.4 -!--1.8
43 44 48.7 -+ 2.0 158.8 -+ 1.1 59.5 -+ 1.4 23.6 +- 0.5
51 44 158.1 + 1.5 55.7 + 1.8 22.3 +-- 0.5
I D
0.66 0.60 0.34 0.40 X2 = 1.41 (P > .05)
France (I = 0.41, D = 0.59, X 2 = 22.63), 13 by Zee et al in Australia (I = 0.48, D = 0.52, X2 = 15.64, 4 and by Schmidt et al in the Netherlands (I = 0.37, D = 0.63, X2 = 54.56) 5 were significantly different (all P < .05) from the allele frequency in our study in Japan (I = 0.63, D = 0.37). DISCUSSION
In this study of a Japanese population, we found no association between the ACE gene polymorphism and essential hypertension. However, we did find that the allele frequency in Japanese differed from those reported in French, 13 Australian, 4 and Dutch populations. ~ This ACE gene polymorphism is reported to be associated with cardiovascular disease such as myocardial infarction2 and ischemic or idiopathic dilated cardiomyopathy.3 However, the results of an association analysis of this polymorphism with essential hypertension are still controversial, 4-9'13 although this polymorphism is thought to account for half of the variance of serum enzyme levels. 13 Further pathophysiologic implications of this polymorphism have not yet been established. In this study, we found no association between essential hypertension and this polymorphism. This negative result is consistent with results previously reported in Dutch s and US populations, 6 in young Caucasian adults, and in an Osaka-based study, 8 while the result is not consistent with an Australian report 4 and that in a Kanazawa-based study in Japan. 9 The reasons for these different results are not clear. However, possible reasons for this difference are reported in the recent 15th ISH meeting by the Australian group. They reported that this difference was due to a higher death rate in patients having a D D genotype: no difference in I/D was seen in young hypertensives v normotensives. In addition they thought that older hypertensives were reported to show marked depletion of the D D genotype. 1° It is generally accepted that there are genetic het-
ACE GENOTYPE AND EH IN JAPAN 97
AJH-JANUARY 1995-VOL. 8, NO. 1
erogeneities a m o n g different ethnic groups. This hereditary h e t e r o g e n e i t y m a y account for the ethnic variety of m a n y p h e n o m e n a . In this s t u d y we confirmed that there w e r e significant differences in the allele f r e q u e n c y b e t w e e n previously r e p o r t e d findings in Western E u r o p e a n countries and in Japan. Such differences have also b e e n r e p o r t e d b e t w e e n blacks a n d whites in the US. 11 The implications for these various allele differences in ACE gene p o l y m o r p h i s m s are not k n o w n . Epidemiologic studies have s h o w n that cardiovascular disease is more prevalent in E u r o p e a n - W e s t e r n countries than in Japan. 15'16 The DD g e n o t y p e of this polym o r p h i s m has recently b e e n r e p o r t e d to be associated with m a n y cardiovascular diseases. Interestingly, we n o t e d here that this D allele was more frequent in E u r o p e a n - W e s t e r n c o u n t r i e s t h a n in Japan. It is therefore possible that this ethnic difference in ACE p o l y m o r p h i s m m a y account, in part, for these epidemiologic findings. In a Chinese p o p u l a t i o n similar differences w e r e seen and r e p o r t e d recently by E.J.D. Lee.12 H o w e v e r , further studies are required to elucidate the implications of these ethnic differences in the ACE gene p o l y m o r p h i s m . REFERENCES
1. Rigat B, Hubert C, Corvol P, Soubrier F: PCR detection of the insertion/deletion polymorphism of the human angiotensin converting enzyme gene. Nucl Acid Res 1992;20:1433. 2. Cambien F, Poirier O, Lecerf L, et al: Deletion polymorphism in the gene for angiotensin-converting enzyme is a potent risk factor for myocardial infarction. Nature 1992;359:641-644. 3. Raynolds MV, Bristow MR, Bush EW, et al: Angiotensin-converting enzyme DD genotype in patients with ischemic or idiopathic dilated cardiomyopathy. Lancet 1993;342:1073-1075. 4. Zee RYZ, Lo Y, Griffiths LR, Morris BJ: Association of a polymorphism of the angiotensin I converting enzyme gene with essential hypertension. Biochem Biophys Res Commun 1992;184:9-15. 5. Schrnidt S, Van Hooft IMS, Grobbee DG, et al: Polymorphism of the angiotensin I converting enzyme
6.
7.
8.
9.
10.
11.
12.
13.
gene is apparently not related to high blood pressure: Dutch Hypertension and Offspring Study. J Hypertens 1993;11:345-348. Jeunemaitre X, Lifton RP, Hunt SC, et al: Absence of linkage between the angiotensin converting enzyme locus and human essential hypertension. Neture Genetics 1992;1:72-75. Harrap SB, Davidson H, Conor JM, et al: The Angiotensin I Converting Enzyme Gene and Predisposition to High Blood Pressure. Hypertension 1993;21: 455--460. Higashimori K, Zhao Y, Higaki J, et al: Association analysis of a polymorphism of the angiotensin converting enzyme gene with essential hypertension in the Japanese population. Biochem Biophys Res Commun 1993;191:399-404. Morise T, Takeuti Y, Takeda R: Angiotensin-converting enzyme polymorphism and essential hypertension (abst). Lancet 1994;343:125. Morris BJ, Zee RYL, Schrader AP, Bennet CL: Identification of genes for hypertension (HT), obesity and accelerated death rate in patients with severe, familial HT (abst). ] Hypertens 1994;12(suppl 3):128. Bloem LJ, Manatunga AK, Boatright E, Pratt JH: Relation of race and a polymorphism in the angiotensin converting enzyme gene to enzyme levels (abst). Hypertension 1993;22:407. Lee EJD: Population genetics of the angiotensinconverting enzyme in Chinese. Br J Clin Pharmac 1994;37:212-214.
Rigat B, Hubert C, Cambie F, et al: An insertion/ deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half of the variance of serum enzyme levels. J Clin Invest 1990;86:1343-1346. 14. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press, 1989, pp. 9.16-9.19. 15. Dawber TR: The Framingham study. The epidemiology of atherosclerotic disease. Cambridge, Harvard University Press, 1980. 16. Ueda K, Omae T, Hasuo Y, et al: Prevalence and longterm prognosis of mild hypertensives and hypertensives in a Japanese community, Hisayama. J Hypertens 1988;6:981-989.
Angiotensin I Converting Enzyme (ACE) Gene Polymorphism and Essential Hypertension in Japan Ethnic Difference of ACE Genotype Tomoaki Ishigami, Tamio Iwamoto, Kouichi Tamura, Satoshi Yamaguchi, Kan Iwasawa, Kazuaki Uchino, Satoshi Umemura, and Masao Ishii
A polymorphism of the angiotensin I converting enzyme (ACE) gene has recently been reported and analysis of this polymorphism has indicated that it is associated with several cardiovascular diseases. However, the results are still controversial and such association has not yet been established conclusively. To determine whether the ACE gene may be responsible for essential hypertension in a Japanese population, we also compared the distribution of genotypes and the allele frequency of this polymorphism in our findings of a Japanese population with these features in other countries. Eighty-seven hypertensive patients with a family history of essential hypertension and 95 normotensive patients whose parents had no such history were enrolled in the study. Polymorphism of the ACE gene was determined by using the polymerase chain reaction. Homozygotes for this polymorphism had either a 490-bp band (I/) or a 190-bp band (DD) and het-
KEY WORDS: Angiotensin I converting enzyme, gene polymorphism, essential hypertension, ethnicity.
Received April 12, 1994. Accepted October 11, 1994. From the Second Department of Internal Medicine, Yokohama City University, Yokohama, Japan. This study was supported in part by Grant-in-Aid 05670956 for Scientific Research from the Ministry of Education, Science and Culture, Japan and by a grant from the Kihara Memorial Foundation, Yokohama, Japan. Address correspondence and reprint requests to Satoshi Umemura, Second Department of Internal Medicine, Yokohama City University, Fukuura 3-9, Kanazawa-ku, Yokohama, 236, Japan.
Polymorphism of the angiotensin I converting enzyme (ACE) gene has recently been reported 1 and analysis of this polymorphism has indicated that it is associated with several cardiovascular diseases. 2'3 However, the results are still controversial and such association has not yet been established conclusively, especially in essential hypertension. 4-1° To determine whether the ACE gene may be responsible for essen-
© 1995 by the American Journal of Hypertension, Ltd.
erozygotes had both bands (ID). In hypertensive subjects, the numbers and frequency of the ACE genotypes were: II, 44 (0.51); ID, 26 (0.30); DD, 17 (0.19). In normotensive subjects these were: II, 35 (0.37); ID, 43 (0.45); DD, 17 (0.18). There were no significant differences between the two groups in derived allele frequencies (X2 = 1.41). The difference between the overall allelic frequency in Japan and that reported in several other countries was significant. We did not find any association between ACE gene polymorphism and essential hypertension in Japan. However, there were significant differences in derived allele frequencies b e t w e e n our findings in a Japanese population and those reported from Europe and Australia. Am J Hypertens 1995;8:95-97
A
0895-7061/95/$9.50 0895-7061(94)00184-D
96
ISHIGAMI ET AL
AJH-JANUARY 1995-VOL. 8, NO. 1
tial hypertension, we examined the association of this polymorphism with essential hypertension in a Japanese population. Since there are genetic heterogeneities among different ethnic groups, n'12 we also compared the distribution of genotypes and the allele frequency of this polymorphism in our findings of a Japanese population with these features in other ethnic populations.
TABLE 2. FREQUENCY OF INSERTION/DELETION POLYMORPHISM OF THE ACE GENE IN ESSENTIAL HYPERTENSIVE A N D NORMOTENSIVE SUBJECTS
II ID DD
HT (n = 87)
NT (n = 95)
44 (0.51) 26 (0.30) 17 (0.19)
35 (0.37) 43 (0.45) 17 (0.18) Allele
SUBJECTS AND METHODS
Eighty-seven hypertensive patients with a family history of essential hypertension (HT) who had at least one hypertensive parent, and 95 normotensive patients whose parents had no such history (NT) were enrolled in the study. We extracted genomic DNA from these patients' peripheral blood leukocytes by standard methods, 13 using proteinase K digestion of nuclei. Phenol extraction was followed by ethanol precipitation of the DNA. We amplified the insertion/ deletion portion in intron 16 of the ACE gene by the polymerase chain reaction (PCR) method. 1 The PCR product was electrophoresed in 1.6% agarose gels and visualized by ethidium bromide staining. The insertion/deletion polymorphism of the ACE gene was detected as follows: homozygotes for this polymorphism have either a 490-bp band, named II or a 190bp band, named D D , while heterozygotes have both bands, named ID. RESULTS
Table 1 shows the several characteristics in patients with hypertension (HT) and normotensives (NT). There was no significant difference between the two groups. Table 2 shows the genotype distribution and derived allele frequency in the two groups. There were no significant differences in genotype distribution between the HT and NT groups and there was also no significant differences in allele frequency between the groups. The allele frequency reported by Rigat et al in TABLE 1. C O M P A R I S O N OF CHARACTERISTICS IN PATIENTS WITH ESSENTIAL HYPERTENSION (HT) A N D NORMOTENSIVE SUBJECTS (NT) HT
N Age Sex Male Female Onset of hypertension Height (cm) Weight (kg) BMI (kg/m -2)
NT
87 59.3 + 1.3
95 57.4 -!--1.8
43 44 48.7 -+ 2.0 158.8 -+ 1.1 59.5 -+ 1.4 23.6 +- 0.5
51 44 158.1 + 1.5 55.7 + 1.8 22.3 +-- 0.5
I D
0.66 0.60 0.34 0.40 X2 = 1.41 (P > .05)
France (I = 0.41, D = 0.59, X 2 = 22.63), 13 by Zee et al in Australia (I = 0.48, D = 0.52, X2 = 15.64, 4 and by Schmidt et al in the Netherlands (I = 0.37, D = 0.63, X2 = 54.56) 5 were significantly different (all P < .05) from the allele frequency in our study in Japan (I = 0.63, D = 0.37). DISCUSSION
In this study of a Japanese population, we found no association between the ACE gene polymorphism and essential hypertension. However, we did find that the allele frequency in Japanese differed from those reported in French, 13 Australian, 4 and Dutch populations. ~ This ACE gene polymorphism is reported to be associated with cardiovascular disease such as myocardial infarction2 and ischemic or idiopathic dilated cardiomyopathy.3 However, the results of an association analysis of this polymorphism with essential hypertension are still controversial, 4-9'13 although this polymorphism is thought to account for half of the variance of serum enzyme levels. 13 Further pathophysiologic implications of this polymorphism have not yet been established. In this study, we found no association between essential hypertension and this polymorphism. This negative result is consistent with results previously reported in Dutch s and US populations, 6 in young Caucasian adults, and in an Osaka-based study, 8 while the result is not consistent with an Australian report 4 and that in a Kanazawa-based study in Japan. 9 The reasons for these different results are not clear. However, possible reasons for this difference are reported in the recent 15th ISH meeting by the Australian group. They reported that this difference was due to a higher death rate in patients having a D D genotype: no difference in I/D was seen in young hypertensives v normotensives. In addition they thought that older hypertensives were reported to show marked depletion of the D D genotype. 1° It is generally accepted that there are genetic het-
ACE GENOTYPE AND EH IN JAPAN 97
AJH-JANUARY 1995-VOL. 8, NO. 1
erogeneities a m o n g different ethnic groups. This hereditary h e t e r o g e n e i t y m a y account for the ethnic variety of m a n y p h e n o m e n a . In this s t u d y we confirmed that there w e r e significant differences in the allele f r e q u e n c y b e t w e e n previously r e p o r t e d findings in Western E u r o p e a n countries and in Japan. Such differences have also b e e n r e p o r t e d b e t w e e n blacks a n d whites in the US. 11 The implications for these various allele differences in ACE gene p o l y m o r p h i s m s are not k n o w n . Epidemiologic studies have s h o w n that cardiovascular disease is more prevalent in E u r o p e a n - W e s t e r n countries than in Japan. 15'16 The DD g e n o t y p e of this polym o r p h i s m has recently b e e n r e p o r t e d to be associated with m a n y cardiovascular diseases. Interestingly, we n o t e d here that this D allele was more frequent in E u r o p e a n - W e s t e r n c o u n t r i e s t h a n in Japan. It is therefore possible that this ethnic difference in ACE p o l y m o r p h i s m m a y account, in part, for these epidemiologic findings. In a Chinese p o p u l a t i o n similar differences w e r e seen and r e p o r t e d recently by E.J.D. Lee.12 H o w e v e r , further studies are required to elucidate the implications of these ethnic differences in the ACE gene p o l y m o r p h i s m . REFERENCES
1. Rigat B, Hubert C, Corvol P, Soubrier F: PCR detection of the insertion/deletion polymorphism of the human angiotensin converting enzyme gene. Nucl Acid Res 1992;20:1433. 2. Cambien F, Poirier O, Lecerf L, et al: Deletion polymorphism in the gene for angiotensin-converting enzyme is a potent risk factor for myocardial infarction. Nature 1992;359:641-644. 3. Raynolds MV, Bristow MR, Bush EW, et al: Angiotensin-converting enzyme DD genotype in patients with ischemic or idiopathic dilated cardiomyopathy. Lancet 1993;342:1073-1075. 4. Zee RYZ, Lo Y, Griffiths LR, Morris BJ: Association of a polymorphism of the angiotensin I converting enzyme gene with essential hypertension. Biochem Biophys Res Commun 1992;184:9-15. 5. Schrnidt S, Van Hooft IMS, Grobbee DG, et al: Polymorphism of the angiotensin I converting enzyme
6.
7.
8.
9.
10.
11.
12.
13.
gene is apparently not related to high blood pressure: Dutch Hypertension and Offspring Study. J Hypertens 1993;11:345-348. Jeunemaitre X, Lifton RP, Hunt SC, et al: Absence of linkage between the angiotensin converting enzyme locus and human essential hypertension. Neture Genetics 1992;1:72-75. Harrap SB, Davidson H, Conor JM, et al: The Angiotensin I Converting Enzyme Gene and Predisposition to High Blood Pressure. Hypertension 1993;21: 455--460. Higashimori K, Zhao Y, Higaki J, et al: Association analysis of a polymorphism of the angiotensin converting enzyme gene with essential hypertension in the Japanese population. Biochem Biophys Res Commun 1993;191:399-404. Morise T, Takeuti Y, Takeda R: Angiotensin-converting enzyme polymorphism and essential hypertension (abst). Lancet 1994;343:125. Morris BJ, Zee RYL, Schrader AP, Bennet CL: Identification of genes for hypertension (HT), obesity and accelerated death rate in patients with severe, familial HT (abst). ] Hypertens 1994;12(suppl 3):128. Bloem LJ, Manatunga AK, Boatright E, Pratt JH: Relation of race and a polymorphism in the angiotensin converting enzyme gene to enzyme levels (abst). Hypertension 1993;22:407. Lee EJD: Population genetics of the angiotensinconverting enzyme in Chinese. Br J Clin Pharmac 1994;37:212-214.
Rigat B, Hubert C, Cambie F, et al: An insertion/ deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half of the variance of serum enzyme levels. J Clin Invest 1990;86:1343-1346. 14. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press, 1989, pp. 9.16-9.19. 15. Dawber TR: The Framingham study. The epidemiology of atherosclerotic disease. Cambridge, Harvard University Press, 1980. 16. Ueda K, Omae T, Hasuo Y, et al: Prevalence and longterm prognosis of mild hypertensives and hypertensives in a Japanese community, Hisayama. J Hypertens 1988;6:981-989.