Vol. 43, No. 1, September 1997 BIOCHEMISTRY ond MOLECULAR BIOLOGY INTERNATIONAL Pages

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1 Vol. 43, No. 1, September 1997 BIOCHEMISTRY ond MOLECULAR BIOLOGY INTERNATIONAL Pages ANGIOTENSIN II TYPE I RECEPTOR POLYMORPHISM IN AFRICAN AMERICANS LOWER FREQUENCY OF THE C n66 VARIANT Short title: ANGIOTENSIN RECEPTOR GENE IN AFRICAN AMERICANS James V. Gainer I, Tracy E. Hunley 2, Valentina Kon 2, John H. Nadeau I, James A.S. Muldowney, III ~, Nancy J. Brown 1 Vanderbilt University Medical Center Divisions of Clinical Pharmacology 1 and Pediatric Nephrology 2, Nashville, TN Received July 8, 1997 SUMMARY The C 1166 variant, an A to C substitution polymorphism at the 1166 position of the angiotensin II type I (AT 0 receptor, has been previously associated with hypertension in Caucasians. This study determines the frequency of the C 1166 variant in an African American population. Normotensive African American (n = 99) and Caucasian (n = 100) subjects were genotyped to determine the frequency of the C 1166 variant. This study establishes the frequency of the C t166 variant in African Americans ( ) and demonstrates a significantly lower frequency in African Americans compared with Caucasians (0.05 vs. 0.25, respectively, )(=30.7, p < < 0.001, ld:). Keywords: blacks, angiotensin receptors, renin-angiotensin system, polymorphism (genetics), essential hypertension, blood pressure INTRODUCTION A polymorphism of the angiotensin II type I (AT1) receptor, an A to C substitution at the 1166 position in the 3' untranslated region, has recently been described by Bonnardeaux et al. The C 1166 variant of this polymorphism has been associated with hypertension (1) and aortic stiffness (2), a correlate of left ventricular hypertrophy, in Caucasian populations. African Americans have a higher prevalence of hypertension and associated target organ damage (3,4). Racial differences have Corresponding author and address for reprints: James V. Gainer, M.D. 556 MRB-1, 23rd Avenue S. at Pierce Ave. Vanderbilt University Nashville, TN Phone: (615) Fax: (615) j ay.gainer@mcmail.vanderbilt.edu /97/ /0 Copyright by Academic Press Australia. ~AII rights of reproduction in any form reserved.

2 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL been described for gene polymorphisms in the renin angiotensin system which may account for differences in the expression of hypertension (5-14). To determine the feasiblity of finding such an association in Afi'ican Americans, we determined the frequency of the AT1-C ''66 variant in an African American population. METHODS Patient Pop.ulation The study population was comprised of 199 normotensive subjects (99 African American and 100 Caucasian). All subjects were unpaid volunteers and method of ascertainment was the same for both African Americans and Caucasians. Each subject was interviewed regarding known history of hypertension, medication use, and comorbid disease. The subject's height, weight, and seated blood pressure were recorded. Subjects were considered normotensive with a systolic blood pressure below 140 mmhg and a diastolic blood pressure below 90 mmhg and the absence of a current or past use of antihypertensive medication. Patients were not excluded oil the basis of comorbid disease. The study was approved by the Vanderbilt University Institutional Review Board. Informed consent was obtained and blood was drawn for DNA analysis. Laborato~ Analysis The AT, receptor polymorphism was detected by a PCR-RFLP method in which an 897 base pair (bp) DNA fragment was PCR-amplified from genomic DNA samples extracted from white blood cells. The C I~66 allele contains an additional recognition site for the restriction endonuclease DdeI that is absent in the A I166 allele (15). Therefore, the amplification products were then subjected to the restriction enzyme DdeI for 12 hours. All resultant products then contained a 609 bp fragment. The presence of a 288 bp fragment corresponded to the A ~ 166 allele and separation of this fragment into 145 and 143 bp sections marked the presence of the C ~66 allele. Statistical Anal~ The genotype frequencies for each polymorphism by race were tested for deviation from Hardy-Weinberg equilibrium by chi-square goodness-of-fit analysis with 1 degree of freedom. The frequencies of the AT,-1166 alleles by race were compared by chi-square analysis with 1 degree of freedom. P values <0.05 were considered significant. RESULTS The characteristics of the normotensive subjects in this study are shown in Table 1. The African American and Caucasian groups did not significantly differ by age, gender, or mean arterial pressure (MAP). African American subjects, however, had a slightly increased body mass index (BMI) compared to Caucasians. Table 2 shows the number of observed ATl-1166 genotypes which conform to Hardy- Weinberg equilibrium within racial groups and the frequency of the AT,'-1166 alleles by race. The frequency of the C 'z66 allele in Ati'ican Americans was 0.05 _4-0.01, and the C H~~ allele in Caucasians was (X 2 = 30.7, p < <0.001, 1 djo. 228

3 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL DISCUSSION The ATI-A n66 to C 1166 substitution has been associated with hypertension (1) and aortic stiffness (2), a correlate of left ventricular hypertrophy, in small studies of Caucasians. Hypertension and left ventricular hypertrophy are more prevalent among African Americans than Caucasians (3,4). To determine the feasibility for a disease-gene association in African Americans, we determined the degree ofpolymorphism at the AT locus. We report here that the frequency of the C I166 allele is dramatically lower in normotensive African Americans compared to Caucasians. Racial differences in allele frequencies have been correlated with differences in the prevalence of hypertension and the expression of the hypertension phenotype. The 235T variant of the angiotensinogen gene has been associated with hypertension in Caucasian (16) and Japanese (5) Table 1. Subject characteristics. African American (n = 99) Caucasian (n = 100) n (men) 99 (46) 100 (51) age, years 35.4 :~ MAP, mm Hg BMI, kg/m 2 * MAP = mean arterial pressure (DBP+I/3(SBP-DBP)); BMI = body mass index. Values are mean + SEM. *p < 0.05 vs. Caucasian BMI. Table 2. Comparison of AT genotypes and allele frequencies by race. Genotypes African American Caucasian (99) (100) 'AA' 'AC' 7 34 'CC' 1 7 Alleles 'A' 'C' Overall, African American vs. Caucasian 'C' allele (%z =30.7, p<<0.001, 1 dj). 229

4 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL populations but not in a sample of African Americans (6). Duru et al. reported an increased frequency of the angiotensin converting enzyme (ACE) deletion allele among hypertensive compared to normotensive African Americans (7). In a small study of hypertensive subjects, Benetos et al. recently reported a greater antihypertensive effect of ACE inhibitors in subjects who were either heterozygous or homozygous for the AT~-C H66 allele, Whereas subjects who were homozygous for the A ~ ~6~ allele were more likely to respond to calcium channel blockers (8). Linkage disequilibrium or the degree of a disease-gene association can be expected to be affected by racial admixture (17). The identification of racial differences in the frequency of polymorphisms in an admixed population could aid in the detection of significant disease-gene associations (17,18), and ultimately the genetic determinants of a disease. Further a polymorphism that can act as a marker for racial admixture, such as AT~-C J 166 which may represent a unique Caucasian allele, could augment the detection of significant associations. This study effectively establishes the lower frequency of the ATj-C H66 allele in African Americans. Even with this low frequency of the AT~-C ]166 allele in African Americans, the detection of a significant association with hypertension would require a large sample size. For example, if hypertensive African Americans had a 4% increase in the ATI-C n66 allele frequency, 500 subjects with and 500 without hypertension would be needed to demonstrate a significant association. Thus, the AT~-1166 polymorphism remains a potential tool to connect the AT~ receptor gene with cardiovascular disease, and in addition, may serve as a useful marker to improve detection of disease-causing genes. ACKNOWLEDGEMENTS This work was supported by grants from the National Institutes of Health (NIH GM-07569, DK 42159, DK 44757). N. J. Brown is a Clinical Associate Physician, General Clinical Research Center (MO1-RR ) and a recipient of a Grant-in-Aid from the American Heart Association, Tennessee Affiliate. V. Kon is a recipient of the Established Investigator Award for the American Heart Association. REFERENCES t. Bonnardeaux, A., Davies, E., Jeunemaitre, X., Fdry, I., Charm, A., Clauser, E., Tiret, L., Cambien, F., Corvol, P., and Soubrier, F. (1994) Hypertension 24, Benetos, A., Topouchian, J., Ricard, S., Gautier, S., Bonnardeaux, A., Asmar, R., Poirier, O., Soubrier, F., Safar, M., and Cambien, F. (1995) Hypertension 26, Dtmn, F.G., Oigman, W., Sungarrd-Riise, K,, Messerli, F.H., Ventura, H., Reisin, E., and Frohlich E.D. (1983) J. Am. Coll. Cardiol. 1,

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