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(Hypertension. 2000;35:1297.)
© 2000 American Heart Association, Inc.

Scientific Contributions

Lack of Association of 3 Functional Gene Variants With Hypertension in African Americans

Nicole Larson; Richard Hutchinson; Eric Boerwinkle

From the Human Genetics Center and Institute of Molecular Medicine, University of Texas–Houston Health Science Center, Houston, Tex (N.L., E.B.); and the Department of Medicine, University of Mississippi Medical Center, Jackson, Miss (R.H.).

Correspondence to Eric Boerwinkle, PhD, Human Genetics Center and Institute of Molecular Medicine, University of Texas–Houston Health Science Center, 6901 Bertner, Houston, TX 77030.

	Abstract

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Abstract—African Americans are a critical population in which to study the impact of physiologically important candidate gene mutations on the occurrence of hypertension. African Americans not only have a higher prevalence of hypertension, but the disease strikes earlier, with greater severity, and often results in death at an earlier age compared with whites in the United States. In this study, 3 physiologically important candidate gene mutations (angiotensinogen A[-6], -Adducin Gly460Trp, and G-Protein ß₃-subunit C825T) were examined for their association with hypertension status in a sample of 904 African Americans from Jackson, Mississippi. Tests of simple association and multivariate logistic regression analyses revealed no association between hypertension status and any of the studied polymorphisms. This lack of association persisted after stratification of the sample by gender and body size. These data indicate that these polymorphisms do not contribute in a significant way to interindividual variation in the risk of hypertension in this sample of African Americans, and further genome-wide studies should be performed to identify genes that may influence blood pressure levels in this population.

Key Words: hypertension, genetic • genes • blacks • blood pressure

	Introduction

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Hypertension is a disease of significant public health importance because it is a major risk factor for the development of cardiovascular, cerebrovascular, and renal diseases.¹ Numerous studies have shown that blacks have a higher prevalence of hypertension than whites in the United States. When the hypertension cut point is defined as 140/90, the disease is 50% more common in African Americans than whites; when the cut point is 160/90, hypertension is 100% more common.² African Americans not only have a higher prevalence of hypertension, but it strikes earlier and with greater severity and often results in death at an earlier age for blacks than for whites in the United States.³

There is strong evidence that interindividual variation in the risk of hypertension has a genetic component. It has been estimated that 20% to 60% of the interindividual variability in blood pressure levels is due to genetic differences.⁴ Various twin studies, adoption studies, pedigree analyses, and association studies reveal a genetic component to blood pressure levels and hypertension status in numerous populations, including African Americans.⁵ Identification and characterization of the individual genes underlying interindividual variation in blood pressure levels or hypertension status has largely relied on the study of candidate genes, although entire genomic approaches are emerging.⁶

One likely pathway for blood pressure regulation is the renin-angiotensinogen system, which influences both salt and water homeostasis and local blood vessel characteristics. Inoue et al⁷ reported a guanine-for-alanine substitution at position -6 in the promoter of the human angiotensinogen gene, which affects interactions with various transcription factors and the basal transcription rate of the angiotensinogen gene. Therefore, this angiotensinogen (AGT) A(-6) polymorphism is an obvious candidate gene polymorphism for interindividual variation in the occurrence of hypertension in the African American population. Another promising candidate gene is ADDA, which codes for the cytoskeleton protein -Adducin. This protein is found in renal tubules and is thought to regulate ion transport through changes in the actin cytoskeleton. A polymorphism, Gly460Trp, found in ADDA, was shown to be associated with hypertension in humans.⁸ A third candidate gene, GNB3, codes for the ß₃-subunit of the heterotrimeric G protein. A polymorphism in this gene (C825T) has been associated with hypertension.^{9 10} The mutation causes a splice variant in the ß₃-subunit that causes the transcript to be aberrantly spliced and results in the loss of a 41–amino acid segment of a propeller-like structure in this protein. Removal of this "propeller blade" causes an apparent dominant gain of function.¹¹

Despite the high prevalence of hypertension in the African American community and the presumed importance of these variants on the regulation of blood pressure levels, there is a paucity of data relating gene information to the occurrence of hypertension in African Americans. Results from this study include genotype and allele frequencies for the physiologically important candidate gene mutations of AGT A(-6), ADDA, and GNB3 and tests for association between these polymorphisms and hypertension status in a sample of African Americans from the Atherosclerosis Risk in Communities (ARIC) Study.

	Methods

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The study sample is a subset of the cohort collected from the large multicenter ARIC Study. The ARIC Study is a prospective investigation of the pathogenesis and natural history of atherosclerosis and the pathogenesis of clinical atherosclerotic diseases in 4 communities in the United States. The study also measures variation in cardiovascular risk factors, medical care, and disease by race, gender, place, and time.¹² The sample used for this ancillary substudy consists of 904 (472 hypertension cases and 432 control subjects) African Americans from the community of Jackson, Mississippi.

Blood pressure was measured with the use of a random zero sphygmomanometer. Three readings were taken on the right arm, each 2 minutes apart after the subject had been sitting quietly for 5 minutes. Subjects were classified as hypertensive cases if (a) they had a systolic blood pressure 160 mm Hg, (b) they had a diastolic blood pressure 95 mm Hg, or (c) they had taken any antihypertensive medication in the past 2 weeks. Patients were classified as control subjects if (a) they had a systolic blood pressure <140 mm Hg, (b) they had a diastolic blood pressure <90 mm Hg, and (c) they were not taking antihypertensive medication. The sample was designed to include approximately equal distributions of age and gender between case and control groups. Risk factors were assessed by interview, examination, measurement of blood lipids and hemostatic factors, and other methods.¹² Risk factors evaluated in this study included body size, serum ion levels, fasting serum glucose levels, fasting serum insulin levels, various lipid measures, and smoking and drinking status. All participants signed an informed consent form that included information about genetic studies.

Genotyping
Genomic DNA was isolated by phenol-chloroform extraction of frozen buffy coats. Each polymorphism was typed after polymerase chain reaction amplification of 10 ng/µL proteinase K–treated DNA with the use of standard methods. For AGT A(-6), the forward primer sequence was 5'CTG TGA GTG CGA CCC TGG AGA3' and the reverse primer sequence was 5'CTT ACC TTC TGC TGT AGT ACC3'. For ADDA Gly460Trp, the forward primer sequence was 5'CTC CTT TGC TAG TGA CGG TGA TTC3' and the reverse primer sequence was 5'GAC TTG GGA CTG CTT CCA TCC GGC C3'. For GNB3 C825T, the forward primer sequence was 5'TGA CCC ACT TGC CAC CCG TGC3' and the reverse primer sequence was 5'GCA GCA GCC AGG GCT GGC3'. For typing the AGT A(-6), ADDA Gly460Trp, and GNB3 C825T polymorphisms, the amplified products were digested with Bst01, Sau96I, and BseDI, respectively. Digested DNA was electrophoresed on polyacrylamide gels for the AGT and ADDA polymorphisms and on agarose gels for the GNB3 polymorphism. Bands were visualized and typed after ethidium bromide staining. All gels were scored independently by 2 laboratory personnel who were blind to the case-control status of the study participants.

Statistical Analyses
Routine descriptive statistics were used throughout. Allele frequencies were computed by gene counting. A ² test of goodness-of-fit was used to test for agreement to Hardy-Weinberg equilibrium expectations. Multivariate logistic regression analysis was used to assess the relation between each of the polymorphisms and the probability of having hypertension while simultaneously considering the effects of other predictor variables. A probability value of 0.05 was used to assess statistical significance for each of the primary hypotheses of interest.

	Results

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Basic descriptive statistics for this sample of African American hypertensive cases and control subjects are shown in Table 1. All effects were as expected, for example, hypertensive cases had, on average, a larger body size and higher fasting serum glucose levels. Three quarters of the hypertension cases were taking blood pressure–lowering medications.

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

Genotype and allele frequencies of the AGT A(-6), ADDA Gly460Trp, and GNB3 C825T polymorphisms in the African American hypertensive cases and control subjects are reported in Table 2. Observed genotype frequencies were found to be in Hardy-Weinberg equilibrium for each polymorphism. These analyses did not reveal any significant association between genotype and hypertension status for any of the polymorphisms studied, either when the full sample was evaluated, when the sample was stratified by gender or body size, or when individuals with diabetes were excluded (data not shown).

View this table: [in this window] [in a new window]   Table 2. Genotypes and Allele Frequencies

To determine which risk factor variables were significant predictors of hypertension status in this study, logistic regression analyses were performed, first on the full sample, then on men and women separately. None of the polymorphisms studied were observed to be significant predictors of hypertension status after considering the effects of other predictor variables (eg, age, body size, serum calcium, and potassium; data not shown).

	Discussion

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The polymorphisms investigated for this study (AGT A (-6), ADDA Gly460Trp, and GNB3 C825T) were selected because they are the loci to date for which functional differences between allele products indicate a potential biological role in the development of high blood pressure. This evidence combined with results from previously published association studies makes these variants likely candidates for association with hypertension status in African Americans. However, no other published reports have evaluated the importance of these polymorphisms in a sample of African Americans.

Studies by Inoue et al⁷ showed a potential mechanistic relation between the AGT A(-6) polymorphism and hypertension status. Tests of promoter function in cultured cells and studies of binding between AGT oligonucleotides and nuclear proteins strongly suggested that the substitution at nucleotide -6 affected the interaction between at least 1 trans-acting nuclear factor and the AGT promoter, thereby influencing the basal rate of transcription of the AGT gene. Jeunemaitre et al¹³ previously reported a significant association between the AGT A(-6) polymorphism and hypertension status in both whites and Japanese in which the A allele was associated with hypertension. Hegele et al¹⁴ reported a significant association between the AGT A(-6) polymorphism and elevated systolic blood pressure in a sample of young Canadian aborigines. Evidence from this study, however, does not extend these findings to African Americans. No association was found between AGT A(-6) genotype and hypertension status in this sample of African Americans from Jackson, Mississippi. For this sample, the allele frequencies obtained for the AGT A(-6) polymorphism (HT, A=0.82; NT, A=0.83) were similar to those reported for African Caribbeans (A=0.82), aboriginal Canadians (A=0.85), and a Japanese population (HT, A=0.90). A white population, however, showed a considerably lower frequency of the A allele (HT, A=0.47) (Table 3). The extent, if any, to which the observed allele frequency differences among populations are contributing to differences in the prevalence of hypertension cannot be easily determined. It is important to keep in mind that the factors that are contributing to interindividual differences within a population are not necessarily the same as those that are contributing to differences among populations.

View this table: [in this window] [in a new window]   Table 3. Summary of Allele Frequencies Across Populations

The Gly460Trp polymorphism of ADDA modulates the overall capacity of tubular epithelial cells to transport ions through variations in sodium-potassium ATPase activity and through modifications in the assembly of the actin cytoskeleton.¹⁵ Further findings indicated a reduced pressure-natriuresis slope in individuals with 1 T allele after sodium depletion or sodium load.¹⁶ Cusi et al¹⁷ reported a positive association between the occurrence of hypertension and the ADDA polymorphism in both Italian and French populations, with hypertension being associated with the presence of the T allele. An association between the ADDA polymorphism and hypertension status was also found in 2 Japanese studies.^{18 19} Other studies, however, reported no association between this polymorphism and hypertension status in Japanese.^{20 21} Results of this study indicate a lack of association between ADDA and hypertension status in African Americans. For the ADDA polymorphism, the frequency of the T allele was considerably lower for this sample of African Americans (HT, T=0.07; NT, T=0.07) than the T allele frequency reported for Italian, French, or Japanese populations. Of all the populations studied, the frequency of the T allele was highest in the sample of Japanese and lowest in the sample of African Americans (Table 3).

Siffert et al⁹ demonstrated that the T allele of the C825T polymorphism resulted in the formation of a truncated protein with increased G-protein reactivity and increased odds of being hypertensive. Enhanced G-protein–mediated signals may elevate blood pressure by increased sodium-hydrogen exchanger activity, increased cytoplasmic calcium accumulation, and increased contractility or proliferation of vascular smooth muscle cells.¹¹ Hegele et al²² also reported an association between the T allele and elevated systolic blood pressure levels in a sample of Canadian aborigines. Schunkert et al¹⁰ reported a mild association between this polymorphism and diastolic blood pressure level but no association for systolic blood pressure level. Finally, a study of whites with a strong family history of hypertension reported an association between hypertension status and GNB3 genotype.²³ In African Americans in this study, however, there was no association found between this polymorphism and hypertension status. For the GNB3 polymorphism, the frequency of the T allele in this sample of African Americans (NT, T=0.75; HT T=0.74) was considerably greater than the allele frequencies reported for whites (NT, C=0.25; HT C=0.31) or an aboriginal Canadian population (T=0.50) (Table 3).

Despite the presumed functional significance of the AGT A(-6), ADDA Gly460Trp, and GNB3 C825T polymorphisms and their previously reported association with hypertension status in other populations, no significant association was found between hypertension status and any of these polymorphisms in a sample of African Americans from Jackson, Mississippi. Taken together, this evidence indicates that these polymorphisms do not contribute in a significant way to interindividual variation in the risk of hypertension in African Americans. These data, however, do not preclude the possibility that other variants in and around these same genes may influence blood pressure levels and the occurrence of hypertension in the African American population. Further genome-wide studies should be done to localize and identify genes that may influence blood pressure levels and hypertension status in this important population.

	Acknowledgments

 
This study was supported in part by National Institutes of Health grant HL-51021. The ARIC study was funded by National Heart, Lung, and Blood Institute contracts N01-HC-55015, N01-HC-55016, N01-HC-55018, N01-HC-55019, N01-HC-55020, N01-HC-55021, and N01-HC-55022. We would like to thank Cathy Britt of the University of Mississippi Medical Center for her valuable assistance.

Received June 7, 1999; first decision July 7, 1999; accepted January 11, 2000.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Larson, N. Articles by Boerwinkle, E. Search for Related Content PubMed PubMed Citation Articles by Larson, N. Articles by Boerwinkle, E. Related Collections Epidemiology Genomics