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(Circulation. 1997;95:2348-2350.)
© 1997 American Heart Association, Inc.

Articles

Hypertension, Serum Angiotensinogen, and Molecular Variants of the Angiotensinogen Gene Among Nigerians

Charles Rotimi, PhD; Richard Cooper, MD; Olufemi Ogunbiyi, MD; Linda Morrison, MS; Modupe Ladipo, MD; Duane Tewksbury, PhD; Ryk Ward, PhD

the Department of Preventive Medicine and Epidemiology (C.R., R.C.), Loyola University, Chicago, Ill; University College Hospital (O.O., M.L.), Ibadan, Nigeria; Department of Human Genetics (L.M., R.W.), University of Utah (Salt Lake City); and Marshfield Medical Research Foundation (D.T.), Marshfield, Wisc.

	Abstract

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Background We evaluated the association among the M235T and T174M variants of the angiotensinogen (AGT) gene, plasma AGT, and hypertension status in a sample of Nigerians.

Methods and Results Participants were selected from the extremes of the blood pressure distribution obtained from the population survey of 2509 men and women aged 25 to 74 years. Cases (hypertensive subjects) were individuals who had high blood pressure or were taking antihypertensive medication, and control subjects were individuals with low blood pressure who had never taken antihypertensive medication. We found a significant association between the M235T variant and plasma AGT level. Hypertensive subjects had higher plasma AGT levels compared with control subjects. The allele frequencies of the two variants were similar in the hypertensive patients and the control subjects.

Conclusions The consistent relationships observed between the M235T variant and the protein product and between plasma level of the protein and hypertension status in different ethnic groups provide some evidence for a biochemical mechanism linking DNA variation in the renin-angiotensin system with the hypertension phenotype.

Key Words: genes • hypertension

	Introduction

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The role of the RAS in BP regulation is well established,^{1 2} and molecular variants (M235Tand T174M) of the gene encoding one of its primary components—AGT—have been shown to be associated with essential hypertension in several human populations.^{3 4 5 6} Furthermore, a correlation between plasma AGT concentration and BP has been reported by several investigators.³ Curiously, however, the association among variants of the AGT gene, plasma AGT concentration, and hypertension status among black populations has been, at best, inconsistent.^{7 8 9 10 11}

Our group^{8 11} and others^{7 9 10} have reported that among the two important molecular variants of AGT that have been characterized, the 235T allele is the predominant allele in blacks (ranging from 80% to 93%). Evidence of the linkage of this allele to hypertension and the observed higher allelic frequency in African Americans have led some investigators to suggest that this locus may help explain, at least in part, the higher prevalence of hypertension observed among blacks in North America.⁹ To date, no direct evidence for this hypothesis has been obtained.This analysis was undertaken to evaluate the relationship among AGT polymorphisms, AGT plasma level, and hypertension status in a black population with little or no European admixture. Although currently living in different environmental settings, the Nigerian population shares considerable genetic similarity with blacks in North America.¹² In addition, because of the low prevalence of hypertension in Nigeria and the low level of awareness, treatment, and control, antihypertensive therapy does not intrude on the effort to define the relationship among the AGT gene, its product (AGT level), and the physiological end point (BP) as forcefully as occurs in many epidemiological settings.

	Methods

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Participant Recruitment
Subjects were recruited from an ongoing population survey of hypertension in the Ibadan area of the state of Oyo, Nigeria. To enroll persons with maximal contrasts with respect to genetic predisposition to hypertension, an algorithm was used to select persons with the lowest and highest BPs from the main database of the population survey of 2509 men and women aged 25 to 74 years.¹³ Cases (hypertensive subjects) were individuals with the highest BP (mean systolic BP, 162.0±28.7 mm Hg; mean diastolic BP, 96.6±15.1 mm Hg), and control subjects were individuals with the lowest BP who had never taken antihypertensive medication (mean systolic BP, 118.0±13.1 mm Hg; mean diastolic BP, 70.6±10.2 mm Hg). No women were taking estrogens.

BP, height, and weight were measured according to a standardized protocol.^{13 14} All BPs were measured from the right arm (with an appropriate-size cuff) with the subjects in the sitting position. Systolic and diastolic BPs were recorded to the nearest 2 mm Hg as the first and fifth Korotkoff phases with the use of a mercury manometer. BMI was calculated as current measured weight in kilograms divided by height in meters squared.

AGT Measurement and Identification of Molecular Variants of AGT Gene
Venous blood was obtained from each participant. Plasma and buffy coat were separated through centrifugation of EDTA blood at 800g to 900g for 10 minutes and stored at -70°C until shipped to Marshfield Medical Research Foundation for the determination of AGT concentration and to the Department of Human Genetics at the University of Utah for detection of molecular alleles as previously described.^{8 11} AGT concentration was measured through a two-step procedure involving the conversion of AGT to angiotensin I by incubation of an aliquot of plasma with human renin at pH 6 until the release of angiotensin I was complete. The released angiotensin I was determined with radioimmunoassay.¹⁵ The intra-assay CV was 5.8%, and the interassay CV was 12%.⁹

Statistical Analysis
Group mean values were compared with the use of the t test procedure (SAS Institute). Differences in the distribution of genotypes between groups were determined with the ² procedure. The possible relationship between AGT concentration and the molecular variants was tested separately for men and women with the use of ANOVA. Multiple regression analysis was used to evaluate the relationship between hypertension status and AGT concentration while controlling for other covariates, including BMI, age, and sex. Statistical significance was attributed for computed two-tailed P<.05. The study protocol was approved by the institutional review boards at Loyola University, Chicago, and University College Hospital, Ibadan, and each subject gave informed consent to participate in this study.

	Results

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A total of 257 persons (129 men and 128 women) with a mean age of 54.2 years were recruited into this study from a larger community survey. As a result of the selection criteria (ie, sampling from the tails of the BP distribution), the mean systolic and diastolic BPs (137.4 and 82.3 mm Hg) for these participants were significantly higher than those for all participants in the population survey (n=2509; systolic BP, 120.3 mm Hg; diastolic BP, 72.7 mm Hg).¹³ Consequently, the mean BMI among the hypertensive subjects was also higher (23.0 kg/m²) than in the survey as a whole (22.2 kg/m²). Although AGT concentration was significantly higher for women compared with men (1598.2±389.1 versus 1466.7±408; P=.01), the difference was attributable in large measure to the effect of age. Women were significantly younger than the men, and age was correlated with AGT level (r=-.22, P<.01). In a multivariate model, therefore, no effect was observed (P=.13 for sex from the regression model with age as a covariate; ß[age]=-5.97, P<.01).

The presence of the 235T allele was significantly associated with a higher mean AGT concentration in the combined sample of men and women (Table 1; P<.01). There was no association between the T174M locus and AGT level for all participants combined (P=.17) or for men or women separately.

View this table: [in this window] [in a new window]   Table 1.

In the analysis combining men and women, mean AGT concentration was significantly related to hypertension status (P=.01; Table 2), and the association remained significant after accounting for the variation in hypertension status due to BMI. This finding was, however, inconsistent by sex; although a significant difference was observed between AGT level and hypertension status for men (P=.0016), this was not the case for women (P=.99). Both treated and untreated hypertensive men had AGT levels (1573 and 1644, respectively) that were higher than those of the control subjects. There were no significant differences in the allele frequencies of either the M235T or T174M variant in hypertensive patients compared with the control subjects.

View this table: [in this window] [in a new window]   Table 2.

	Discussion

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As reported previously, the 235T allele is the predominant AGT allele in black populations, ranging from 80% to 93%.^{7 8 9 10 11} In this sample of Nigerian adults, individuals who were homozygous for the 235T allele had a significantly higher plasma AGT level. Furthermore, plasma AGT level was significantly higher among hypertensives, and this was true for both men and women. Although the 235T allele was slightly more common among hypertensives, the difference did not approach statistical significance (P=.42). Although Caulfield and colleagues⁷ found no association between the M235T variant and hypertension, they reported evidence of linkage of the AGT polymorphism with hypertension in a black population using an AGT dinucleotide repeat sequence. On the one hand, this lack of a consistent association and replication of the only reported evidence of linkage⁷ might be taken to suggest that the M235T polymorphism may not be a useful index of susceptibility to hypertension in black populations. On the other hand, the clear association between AGT genotype and plasma AGT level demonstrates the physiological relevance of the AGT gene in the RAS in different ethnic groups. It is also important that the sample size and the high population frequency (0.90) of the 235T allele may have resulted in sufficient power to detect a significant increase in the 235T allele frequency among the hypertensives relative to the control subjects in this study.

We observed a relationship between AGT level and BMI that was sex specific. In addition to the strong association observed with BMI, AGT level was significantly associated with all body measures, including weight; arm, waist, and hip circumferences; and waist-to-hip ratio in men. In sharp contrast, none of these variables were significantly associated with AGT level in women. Sex hormones could modulate the expression of AGT, and sex differences in the association between AGT and hypertension status have been observed,^{3 6} leading some investigators to suggest that AGT may indirectly contribute to hypertension in women.⁶

Although the available data do not support the contention that the higher frequency of the 235T allele in blacks explains in part the higher prevalence of hypertension, the association between the 235T allele and increased plasma AGT concentration in this study warrants further evaluation. Clearly, this finding suggests that the AGT variants are of pathophysiological relevance in black populations. This finding may open new opportunities to study the role of salt sensitivity among North American blacks. Increased sodium sensitivity could be due to the susceptibility allele for the AGT gene, and higher plasma AGT concentration may result in increased production of angiotensin II, leading to increased vascular tone and sodium reabsorption. Furthermore, increased AGT level may result in low renin levels and subsequent downregulation of the RAS. However, because the AGT gene is not regulated by renin levels, the production of plasma AGT will continue despite low plasma renin levels; because the AGT concentration is at the Michaelis-Menton constant for its interaction with renin,^{9 17} higher AGT level could lead to increased production of angiotensin II. The interaction of these components under normal physiological conditions is not well understood at the present time. The identification of molecular variants with predictable life-long effects on circulating levels should help to elucidate these mechanisms.

Our findings provide direct evidence linking the 235T allele of the AGT gene with higher plasma AGT levels. In addition, plasma AGT level was associated with hypertension status among men. The lack of a direct association, however, between the molecular variant and hypertension status detracted from a causal interpretation. Those making attempts at further clarification of the involvement of the AGT variants in the pathophysiology of hypertension should consider defining haplotypes of the 235T allele that are more directly associated with risk in black populations.

	Selected Abbreviations and Acronyms

 

AGT = angiotensinogen BMI = body mass index BP = blood pressure CV = coefficient of variation RAS = renin-angiotensin system

	Acknowledgments

 
This work was supported by grants from the National Institutes of Health (HL-45508, HL-52075, and HL-53353), the Veterans Administration, and American Heart Association, Wisconsin Affiliate (Dr Tewksbury).

	Footnotes

 
Reprint requests to Dr C. Rotimi, Department of Preventive Medicine, Loyola University Stritch School of Medicine, Maywood, IL 60153.

Dr Ward's present address: Institute of Biological Anthropology, Oxford University, Oxford, UK.

Received January 16, 1997; revision received March 13, 1997; accepted March 20, 1997.

	References

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