- Email: [email protected]
α2-Adrenergic receptor gene polymorphism and hypertension in blacks
AJH 1995; 8:390-394
(lz-Adrenergic receptors are found on presynaptic neurons of the central and peripheral nervous systems, on blood vessels, on platelets, on adipocytes, and in the kidney and pancreas. Activation of these ubiquitous adrenoreceptors results in decreased neuronal norepinephrine release, vasodilation, a fall in blood pressure, platelet aggregation, increased sodium excretion, and decreased insulin release. We hypothesized that defects in (lz-adrenerglc receptors, or postreceptor defects, could explain the increased prevalence of hypertension in blacks. To test our hypothesis, we first determined whether or not a polymorphism of the (lz-adrenergic receptor gene was associated with pathologic elevations in blood pressure in American blacks. Dra-I identified a restriction fragment-length polymorphism (RFLP) of 6.3 and 6.7 kb of the (lz-adrenergic receptor gene on chromosome 10 in humans. Of 227 patients studied, 13/107 hypertensive
A
bnormalities of genetic regulation and environmental factors contribute to pathologic elevations of blood pressure in humans.l'? Although specific genetic differ-
Received December 23, 1993. Accepted December 27, 1994. From the Department of Medicine, Wayne State University School ofMedicine, Detroit; Veterans Administration Medical Center, Aflen Park; Department of Physiology, University of Michigan Medical School, and Department of Biostatistics, University of Michigan School of Public Health, Ann Arbor, MI. Warren Lockette, MD, is an Established Investigator of the American Heart Association. These studies were supported by a grantfrom the American Diabetes Association and a Veterans Administration Merit Award. Address correspondence and reprint requests to Warren Lockette, MO, Division of Endocrinology, Department ofMedicine, 4H Universily HealthCenter, Wayne StateUniversity School of Medicine, 4201 S1. Antoine, Detroit, MI, 48201.
subjects were homozygous for the 6.3-kb allele, whereas only 3/120 normotensive volunteers were homozygotes (P = .008). When analyzed by race, 13/8~.black hypertensive subjects were homozygous for the 6.3-kb allele, whereas only 2/59 normotensive blacks were homozygous for the 6.3-kb alleles (P = .02). However, only 1/61 white normotensive and 0/25 white hypertensive subjects were homozygous for the 6.3-kb allele (P = 1.00). Ethnic variation among blacks may explain our findings. Alternatively, a genetic polymorphism in, or near, the az-adrenergic receptor on chromosome 10 caa contribute to the development of hypertension in blacks. Am J Hypertens 1995;8:390394
KEY WORDS: Adrenergic receptors, hypertension, geaetic polymorphism, RFLP, humans.
ences have been linked with the development of hypertension in laboratory animals.v" only genetic polymorphisms of the angiotensinogen gene have been conclusively linked with essential hypertension in humans.7- 14 The analysis of the genetics of high blood pressure is difficult because this disease has multiple etiologies, and the contribution of Mendelian traits to the developmentof hypertension can be obscured by the rarity, late onset, or incomplete penetrance of its heritable features. Alternatively, different genetic defects may result in similar phenotypic presentation, le, pathologic elevations in blood pressure. It should be possible to identify discrete genes which contribute to pathologic elevations in blood pressure in humans. The contribution of a single 0895-7061195/$0.00 0895-7061(95)00024~
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gene to a polygenic disease such as hypertension can be measured by case-control studies designed to seek an association between the disease and a marker ge· notype at the "candidate" gene locus; these investigations are known as population studies. Candidate genesare firstidentified and selected for study by the high likelihood that alterations in the peptide they encode will raise or lower blood pressure. Next, to determine the contribution of a particular candidate gene in the development of hypertension, quantitative or qualitative differences in the gene product between hypertensives and norrnotensives, or polymorphisms, are sought.P Mendelian traits are polymorphic when more than two phenotyres exist at a significant rate within the population.' Once a significant association between a particular polymorphism and the development of hypertensionis found among individuals of the population, the role of the candidate gene in the development of hypertension can be confirmed using linkage analysis and other strategies involving family pedigrees. A key step in identifying a genetic component to essential hypertension is the selection of the appropriate candidate genes. We and others17,18 have been impressed by the high frequency with which hypertension coexists with hyperinsulinemia, tendency towards thrombotic stroke, hyperlipidemia, and salt sensitivity in many black patients. We have recently reported decreased vascular responsiveness to an (X2adrenergic antagonist, as well as abnormalities in a2~ adrenergic receptor-mediated insulin release in subjects with diabetes mellitus. 19,20 Genetic abnormalities of a2-adrenergic receptors could sxplaln tile elevated peripheral vascular resistance, increased sensitivity to salt, and hyperinsulinemia found concomitantly in human and experimental models of genetichypertension.21- 26 However, to demonstrate the feasibility of this hypothesis, we must firstdetermine whether or not a defect in a gene coding for the a 2adrenergic receptor is associated with the development of hypertensionin American blacks. Thegoal of this study was to determine whether or not hypertension in blacks was associated with a polymorphism of the alA gene, located on chromosome 10, which encodes the adrenoreceptor found in the brain and em platelets. METHODS Selection of Subjects Two hundred twenty-sever. men and women who readily ider.tified themselves as either black or white were recruited from the Endocrinology, Metabolism, and 1. !ypertension Clinics of the Wayne State University School of Medicinel Veterans Administration Medical Center. These studies were approved by our Institutional Committee for the Use of Humans in Research, ana. all participants
gave informed consent. The volunteers were examined, and their m-edical records were reviewed by at least tWO investigators. Subjects were classified as normotensive if they had no history of hypertension, no clinical evidence of underlying occult hypertension, were taking no antihypertensive medication, were not receiving vasodilators or other drug therapies that could affect blood pressure (eg, treatment for diseases such as angina pectoris), and had sitting systolic blood pressure less than 140 mm Hg and diastolic blood pressure less than 90 mm Hg on their three most recent clinic visits. Patients with hypertension had significant and sustained elevations of blood pressure (greater than 160 mm Hg systolic and 95 mm Hg diastolic) on at least three separate occasions. The hypertensive subjects were at least 21 years old; to obviate the problems inherent in the late onset of essential hypertension in some individuals, all normotensive subjects were at least 45 years of age. Self-description was used to identify individuals as either black or white. Genomic Analysis The designations of hypertensive and nonhypertensive were made prospectively, and genomic ana~sis using standard tech....iques of Southern blotting" was performer' by in'; 'i .dgators blinded to the clinical designation of the volunteers. Genomic DNA was digested with the restriction enzyme Dra-I the restriction digests were size fractionated using electrophoretic techniques, and the DNA was then transferred to nylon membranes. A 5.5-kb Bam HI fragment (provided IfY R. I efkowitz and R. Regan, Duke University) eno-ding the chromosome 10 a2-adrenergic receptor v....s digested out of a pUC 18 plasmid vector,2B labeled nonisotopically using a commercially available kit (Boehringer Mannheim), and hybridized to the restriction digests. Following stringent washes, the restdction fragment-length polymorphisms were visualized, and individuals were classified as either 6.7 homozygotes (6.716.7), heterozygotes (6.7/6.3), or 6.3 homozygotes (6.3/6.3). Statistical comparisons of the frequencies of the 6.7 and 6.3 alleles in the population and subject subgroups were analyzed using Fisher's exact test. I,
RESULTS Werecruited 120 normotensive and 107 hypertensive subjects from our clinic population; there was no significant racial difference in the rate of inclusion/ refusal for participation in this study. Subjects with secondary forms of hypertension were identified using standard clinical and laboratory assessments, and they were excluded from our studies. Digestion of genomic DNA with Dra I and subsequent hybridization with a Bam HI probe complementary to the a2-adrenergic receptorgene found on
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LOCKETI'E ET At
human chromosome 10(ClO) resulted in either a 6.3or 6.7-kb aHele29 in homozygotes, and both 6.3- and 6.7-kb alleles in subjects heterozygous for this polymorphism (Figure 1). The frequency of the 6.7-kb (81%) and 6.3-kb (19%) alleles in our normotensive white group matched the geue frequency for these alleles reported by others.29 Therewar a greater prevalence of the 6.3-kb allele in normotensive blacks (30%), compared to all whites (19%) (P == .03) or to normotensive whites (19%) (P = .07). Most striking was the increased prevalence of 6.3-kb homozygotes among the hypertensive blacks compared to the normotensive whites (P = .004), to the normotensive blacks (P == .02), and to all normotensive subjects combined (ie, hypertensive blacks v nonhypertensive blacks, nonhypertensive whites, and hypertensive whites) (P = .0002). The frequency of
FIGURE 1. An example of the genetic polymorphism ofthe
homozygote,<:; 2,3,4,6,8 Lane 7 :;:: 6.3-kb homllzygote. =
=
6.3/6.7-kb heterozygotes;
sential hypertension in American blacks. Assuming Hardy-Weinberg equilibrium, and a demonstrated frequency of 19% for the 6.3-kb allele, 1/25 whites should be homozygous forthe 6.3-kb allele. Lessthan the predicted number of 6.3-kb homozygotes were found in whites (1/61 normotensive whites and 0/25 hypertensive whites); however, these observed differences were not statistically.' different from those expected. Given an allele frequency of 30%, we found a significantly increasednumber of 6.3-kb homozygotes, 13/62, compared to the expected frequency of 7/D'1 among American blacks with moderate to severe essential hypertension. Other investigators have attempted to identify C'P. complex quantitative traits associated with hypertension in laboratory animals and humans. Polymorphisms for the renin and kallikreinv" genes have been linked to the developmentof genetic hypertension in laboratory rats. In humans, MN blood group antigens," isozymes of the haptoglobin protein," the rate of kallikrein excretion," certainhistocompatibility antigens.l" and genes controlling lipid metabolism" are associated with the developmentof hypertension. Although polymorphisms of the genes for the sodium-hydrogen antiporterand atrial natriuretic factor have been studied, no associations with essential hypertension have been found.13,30 Although no populationstudy has found an association ofhypertension and polymorphism of the renin gene in the American1 or general European population.P a genetic polymorphism of the renin gene has recently. Lcen reported in hypertensive blacks of Caribbean descent." Also, a chimeric gene defect has been reported to causehypertensionin humans." VIle report a possible association between a candidate gene and hypertension in American blacks. However, these data must be interpreted with caution; we recognize that ethnic variation within the black population can also contribute to our findings. The mechanism(s) by which a genetic polymorphism of the
GENETICS OF HYPERTENSION 393
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TABLE 1. FREQUENCY OF GENOTYPE OF THE C10 (l2-ADRENERGIC RECEPTOR IN NORMOTENSIVE AND HYPERTENSIVE BLACKS
Black
White NT (n
6.7/6.7-kb homozygote 6.7/6.3-kb heterozygote 6.3/6.3-kb homozygote
= 61)
39 21 1
HT (n
= 25)
16 9
o
NT (n
=59)
26 31 2
HT (n
=82)
46 23 13""
Frequency ofalleles and genotypes identified with a probe complementary to tile C10 Cl2-adrenergic receptor in 227 subjects prospectively identified as nonnotensive or hypertensive. Normotensive blacks had an increased frequency ofthe 6.3-kb allele (30%) compared to normotensive whites (19%, P
= .07).
*Analysis using Fisher's exact test for comparison of 6.3-kb 110mozygotes to tIle remainder of the subjects showed significant difference for all hypertensive subjects (white and black) v all normotensive subjects (white and black) (P = .008); and significatlt differences for hypertensive black subjects v hypertensive whites (P = .036) and v normotensive blacks (P = .024).
quence ATTTA encodes a rapid degradation signal in complementary messenger RNA. These observations are compatible with numerous reports of abnormal modulation ofthe number of(X2-adrenergic receptors, but differing reports of changes in li~and binding, in genetically hypert.ensive animals.P" 5 It is suggested from our study that a polymorphism of the C-lO (X2-adrenergic receptor may be associated with the development of severe hypertension. The frequency of the 6.3-kb allele was the same in the black normotensive and hypertensive group, but there was a fourfold increase in the prevalence of 6.3-kb homozygotes in hypertensive blacks compared to normotensive blacks. We attempted to study the phenotypic expression (salt excretion, glucose tolerance, and vascular reactivity) in our 6.3-kb homozygct.es, but nearly all of these blacks had diastolic blood pressures greater than 105 mm Hg as their antihypertensive medicationswere tapered: this precluded safecompletion of these studies. Accordingly, we are now attempting to identify and study 6.3-kb homozygotes who are younger and have milder hypertension to determine whether presence of the 6.3-kb homozygosity is a marker forthe subsequentdevelopment ofsevere hypertension. It is possible that homozygosity of the 6.3-kb allele may identify silent, intermediate phenotypes such as abnormal salt excretion, which subsequently contributes to pathologic elevations in blood pressure. We have recently sequenced the 3' portion of this gene (unpublished observarior.s), and we are now using the polymerase chain reaction to identify homozygotes more rapidly. Although these studies are by no means conclusive, they suggest the need for linkage studies of the Clf)
platelet aggregation, or salt sensitivity with genetic polymorphisms of (X2-adrenergic receptors encoded by chromosomes 2 and 4, as well as genetic defects in postreceptor mechanisms such as the gene encoding the GTP binding protein associated with the (X2-adrenergic receptor. REFERENCES 1. Wilson AF, Elston RC, Tran LO, Siervogel RM: Useof the robust sib-pair method to SCT!:en for single locus, multiple locus, and pleitropic effects: application of traits related to hypertension. Am J Hum Gen 1991; 48:862-872. 2. Jeunemaitre X, Soubrier F, Kotelevtsev YV, et al: Molecular basis ofhuman hypertension: roleof angiotensinogen. Cell 1992;71:169-180. 3. Williams R..~, Hunt SC, Hasstedt SJ, et al: Multigenic human hypertension: evidence forsubtypesand hope for haplotypes. J Hypertens 1990;8(suppl 7):539-546. 4. Kurtz TW, Simonet L, Kabra PM, et al: Cosegregation of the renin allele of the spontaneously hypertensive rat with an increase in blood pressure. J Clin Invest 1990;85:1328-1332. 5. Rapp JP, Wang S-M, Dene H: A genetic polymorphismin the renin geneof Dahlrats cosegregaies with blood pressure.Science 1989;243:542-544. 6. Pravenec M, Kren V, Kunes J, et al: Cosegregation of blood pressure with a kallikrein gene family polymorphism. Hypertension 1991;17:1-5. 7. Heise ER, Moore MA, Roeid QB, Goodman HO: Possible association of MN locus haplotypes with essential hypertension. Hypertension 1987;9:634-640. 8. Weinberger MH, Miller J2, Fineberg NS, et al: Association of haptoglobin with sodium sensitivity and resistance of blood pressure. Hypertension 1987;10:443446. 9. Berry TO, Hasstedt SJ, Hunt SC, et al:A gene for high urinarykallikrein may protectagainsthypertension in Utah kindreds. Hypertension 1989;13:3-8. 10. Gerbase-OeLima M, Delima JJG, Persoli LB, et al: Essential hypertension and histocompatibility antigens: a linkage study. Hypertension 1989;14:604-609. 11. Naftilan AI, Williams R, Burt 0, el al:A lack of genetic linkage ofrenin generestriction fragment length poly-
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14. 15. 16. 17. 18. 19. 20. 21.
22.
23.
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morphisms with human hypertension. Hypertension 1989;14:614-618. Soubrier F, Ieunemaitre X, Rigat B, et al: Similar frequencies of renin gene restriction fragment length polymorphisms in hypertensive subjects. Hypertension 1990;16:712-717. Lifton RP, Hunt SC, Williams RR, et al: Exclusion of the sodium-hydrogen antiporter as a candidate gene in human essential hypertension. Hypertension 1991; 17:S-14. Caulfield M, Lavender P, Farral M, et al: Linkage of the angiotensinogen gene to essential hypertension. N Engl J Med 1994;330:1629-1633. Kurtz TW, Spence MA: Genetics of essential hypertension. Am J Med 1993;94:77-84. Cooper ON, Clayton JF: DNA polymorphism and the study of disease associations. Human G~p\et 1988;/'8: 299-312. Fuh MM-T, Shieh S, Wu OA, et al. Abnormalities of carbohydrate and lipid metabolism in patients with hypertension. Arch Int Med 1987;147:1037-1038. Douglas JG: Hypertension and diabetes in Blacks. Diabetes Care 1990;13(suppl 4):1191-1195. Farrow 5, Mers A, Banta G, et al: Effect of the alpha-Z adrenergic antagonist yohimbine on orthostatic tolerance, Hypertension 1990;15:877-880. Farrow 5, Lockette W: Decreased vascular responsiveness to thealpha-2 adrenergic antagonist yohimbine. J Assoc Acad Minority Physicians 1991;2:182A. Smyth DD, Umemura 5, Pettinger WA: Alpha-2 adrenoreceplor antagonism of vasopressin-induced changes in sodium excretion. Am J Physiol 1985;248 (Renal Fluid Electrolyte Physiol1?): F767-772. Kline RL, Mercer PF: Contribution of renal nerves to the natriuretic and diuretic effect of alpha-2 adrenoceptor activation. J Pharmacol Exp Ther 1990;253:266271. Graham RM, Pettinger WA, Sagalowsky, et al: Renal alpha-adrenergic receptor abnormality in the spontaneously hypertensive rat. Hypertension 1982;4:881887.
24. Diop L, Parini A, Dausse JP, et al: Cerebral and renal alpha-adrenoreceptors in Sabra hypertensive and normotensive rats: effects of high sodiumdiets. J Cardiovase PharmacoI1984;6(suppI5):S742-S747. 25. Parini A, Diop L, Dause [-P, et al: Sabra rats as a model to differentiate between Na+ and GTP regulation of alpha-2 adrenoceptor densities. Eur J Pharmacol 1985;112:97-104. 26. Meldrum MJ, Singleteary NM, Dawson R: Upregulation of renalalpha-Z adrenergic receptors is not lndicative of salt-related increases in blood pressure in spontaneously hypertensive rats. Hypertension 1990; 16:49-54. 27. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold SpringHarbor Laboratory Press, 1989. 28. Kobilka DK, Matsui H, Kobilka TS, et al: Cloning, seouencing, and expression of the gene coding for the human platelet alpha-2 adrenergic receptor. Science 1988;238:65~56.
29. Hoehe MR, Berrettini WH, Lentes KU: Ora I identifies a twoallele DNA polymorphism in the human alpha-2 adrenergic receptor gene (ADRAR), using a 5.5 kb probe. Nucleic Acids Res 1988;16:9070. 30. Barley J, Carter NO, Cruickshank JK, et al: Renin and atrial natriuretic peptide restriction fragment length polymorphisms: association with ethnicity and blood pressure. J Hypertens 1991;9:993-996. 31. Lifton RP, Dluhy RG, Powers M, et al: A chimaeric ll-beta-hydroxylaselaldosterone synthase. gene causes glucocorticoid-remediable aldosteronism and human hypertension. Nature 1992;355:262-265. 32. Jackson RJ, Standart N: Dothe poly(a) and 3' untranslated region control mRNA translation? Cell 1990;62: 15--24. 33. Gaugitsch HW, Prieschl EE, Kalthoff F, et al: A novel transiently expressed integral membrane protein linked to cell activation: molecular cloning via the rapid degradation signal AUUUA. J BioI Chern 1992; 16:11267-11273.
(lz-Adrenergic receptors are found on presynaptic neurons of the central and peripheral nervous systems, on blood vessels, on platelets, on adipocytes, and in the kidney and pancreas. Activation of these ubiquitous adrenoreceptors results in decreased neuronal norepinephrine release, vasodilation, a fall in blood pressure, platelet aggregation, increased sodium excretion, and decreased insulin release. We hypothesized that defects in (lz-adrenerglc receptors, or postreceptor defects, could explain the increased prevalence of hypertension in blacks. To test our hypothesis, we first determined whether or not a polymorphism of the (lz-adrenergic receptor gene was associated with pathologic elevations in blood pressure in American blacks. Dra-I identified a restriction fragment-length polymorphism (RFLP) of 6.3 and 6.7 kb of the (lz-adrenergic receptor gene on chromosome 10 in humans. Of 227 patients studied, 13/107 hypertensive
A
bnormalities of genetic regulation and environmental factors contribute to pathologic elevations of blood pressure in humans.l'? Although specific genetic differ-
Received December 23, 1993. Accepted December 27, 1994. From the Department of Medicine, Wayne State University School ofMedicine, Detroit; Veterans Administration Medical Center, Aflen Park; Department of Physiology, University of Michigan Medical School, and Department of Biostatistics, University of Michigan School of Public Health, Ann Arbor, MI. Warren Lockette, MD, is an Established Investigator of the American Heart Association. These studies were supported by a grantfrom the American Diabetes Association and a Veterans Administration Merit Award. Address correspondence and reprint requests to Warren Lockette, MO, Division of Endocrinology, Department ofMedicine, 4H Universily HealthCenter, Wayne StateUniversity School of Medicine, 4201 S1. Antoine, Detroit, MI, 48201.
subjects were homozygous for the 6.3-kb allele, whereas only 3/120 normotensive volunteers were homozygotes (P = .008). When analyzed by race, 13/8~.black hypertensive subjects were homozygous for the 6.3-kb allele, whereas only 2/59 normotensive blacks were homozygous for the 6.3-kb alleles (P = .02). However, only 1/61 white normotensive and 0/25 white hypertensive subjects were homozygous for the 6.3-kb allele (P = 1.00). Ethnic variation among blacks may explain our findings. Alternatively, a genetic polymorphism in, or near, the az-adrenergic receptor on chromosome 10 caa contribute to the development of hypertension in blacks. Am J Hypertens 1995;8:390394
KEY WORDS: Adrenergic receptors, hypertension, geaetic polymorphism, RFLP, humans.
ences have been linked with the development of hypertension in laboratory animals.v" only genetic polymorphisms of the angiotensinogen gene have been conclusively linked with essential hypertension in humans.7- 14 The analysis of the genetics of high blood pressure is difficult because this disease has multiple etiologies, and the contribution of Mendelian traits to the developmentof hypertension can be obscured by the rarity, late onset, or incomplete penetrance of its heritable features. Alternatively, different genetic defects may result in similar phenotypic presentation, le, pathologic elevations in blood pressure. It should be possible to identify discrete genes which contribute to pathologic elevations in blood pressure in humans. The contribution of a single 0895-7061195/$0.00 0895-7061(95)00024~
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gene to a polygenic disease such as hypertension can be measured by case-control studies designed to seek an association between the disease and a marker ge· notype at the "candidate" gene locus; these investigations are known as population studies. Candidate genesare firstidentified and selected for study by the high likelihood that alterations in the peptide they encode will raise or lower blood pressure. Next, to determine the contribution of a particular candidate gene in the development of hypertension, quantitative or qualitative differences in the gene product between hypertensives and norrnotensives, or polymorphisms, are sought.P Mendelian traits are polymorphic when more than two phenotyres exist at a significant rate within the population.' Once a significant association between a particular polymorphism and the development of hypertensionis found among individuals of the population, the role of the candidate gene in the development of hypertension can be confirmed using linkage analysis and other strategies involving family pedigrees. A key step in identifying a genetic component to essential hypertension is the selection of the appropriate candidate genes. We and others17,18 have been impressed by the high frequency with which hypertension coexists with hyperinsulinemia, tendency towards thrombotic stroke, hyperlipidemia, and salt sensitivity in many black patients. We have recently reported decreased vascular responsiveness to an (X2adrenergic antagonist, as well as abnormalities in a2~ adrenergic receptor-mediated insulin release in subjects with diabetes mellitus. 19,20 Genetic abnormalities of a2-adrenergic receptors could sxplaln tile elevated peripheral vascular resistance, increased sensitivity to salt, and hyperinsulinemia found concomitantly in human and experimental models of genetichypertension.21- 26 However, to demonstrate the feasibility of this hypothesis, we must firstdetermine whether or not a defect in a gene coding for the a 2adrenergic receptor is associated with the development of hypertensionin American blacks. Thegoal of this study was to determine whether or not hypertension in blacks was associated with a polymorphism of the alA gene, located on chromosome 10, which encodes the adrenoreceptor found in the brain and em platelets. METHODS Selection of Subjects Two hundred twenty-sever. men and women who readily ider.tified themselves as either black or white were recruited from the Endocrinology, Metabolism, and 1. !ypertension Clinics of the Wayne State University School of Medicinel Veterans Administration Medical Center. These studies were approved by our Institutional Committee for the Use of Humans in Research, ana. all participants
gave informed consent. The volunteers were examined, and their m-edical records were reviewed by at least tWO investigators. Subjects were classified as normotensive if they had no history of hypertension, no clinical evidence of underlying occult hypertension, were taking no antihypertensive medication, were not receiving vasodilators or other drug therapies that could affect blood pressure (eg, treatment for diseases such as angina pectoris), and had sitting systolic blood pressure less than 140 mm Hg and diastolic blood pressure less than 90 mm Hg on their three most recent clinic visits. Patients with hypertension had significant and sustained elevations of blood pressure (greater than 160 mm Hg systolic and 95 mm Hg diastolic) on at least three separate occasions. The hypertensive subjects were at least 21 years old; to obviate the problems inherent in the late onset of essential hypertension in some individuals, all normotensive subjects were at least 45 years of age. Self-description was used to identify individuals as either black or white. Genomic Analysis The designations of hypertensive and nonhypertensive were made prospectively, and genomic ana~sis using standard tech....iques of Southern blotting" was performer' by in'; 'i .dgators blinded to the clinical designation of the volunteers. Genomic DNA was digested with the restriction enzyme Dra-I the restriction digests were size fractionated using electrophoretic techniques, and the DNA was then transferred to nylon membranes. A 5.5-kb Bam HI fragment (provided IfY R. I efkowitz and R. Regan, Duke University) eno-ding the chromosome 10 a2-adrenergic receptor v....s digested out of a pUC 18 plasmid vector,2B labeled nonisotopically using a commercially available kit (Boehringer Mannheim), and hybridized to the restriction digests. Following stringent washes, the restdction fragment-length polymorphisms were visualized, and individuals were classified as either 6.7 homozygotes (6.716.7), heterozygotes (6.7/6.3), or 6.3 homozygotes (6.3/6.3). Statistical comparisons of the frequencies of the 6.7 and 6.3 alleles in the population and subject subgroups were analyzed using Fisher's exact test. I,
RESULTS Werecruited 120 normotensive and 107 hypertensive subjects from our clinic population; there was no significant racial difference in the rate of inclusion/ refusal for participation in this study. Subjects with secondary forms of hypertension were identified using standard clinical and laboratory assessments, and they were excluded from our studies. Digestion of genomic DNA with Dra I and subsequent hybridization with a Bam HI probe complementary to the a2-adrenergic receptorgene found on
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LOCKETI'E ET At
human chromosome 10(ClO) resulted in either a 6.3or 6.7-kb aHele29 in homozygotes, and both 6.3- and 6.7-kb alleles in subjects heterozygous for this polymorphism (Figure 1). The frequency of the 6.7-kb (81%) and 6.3-kb (19%) alleles in our normotensive white group matched the geue frequency for these alleles reported by others.29 Therewar a greater prevalence of the 6.3-kb allele in normotensive blacks (30%), compared to all whites (19%) (P == .03) or to normotensive whites (19%) (P = .07). Most striking was the increased prevalence of 6.3-kb homozygotes among the hypertensive blacks compared to the normotensive whites (P = .004), to the normotensive blacks (P == .02), and to all normotensive subjects combined (ie, hypertensive blacks v nonhypertensive blacks, nonhypertensive whites, and hypertensive whites) (P = .0002). The frequency of
FIGURE 1. An example of the genetic polymorphism ofthe
homozygote,<:; 2,3,4,6,8 Lane 7 :;:: 6.3-kb homllzygote. =
=
6.3/6.7-kb heterozygotes;
sential hypertension in American blacks. Assuming Hardy-Weinberg equilibrium, and a demonstrated frequency of 19% for the 6.3-kb allele, 1/25 whites should be homozygous forthe 6.3-kb allele. Lessthan the predicted number of 6.3-kb homozygotes were found in whites (1/61 normotensive whites and 0/25 hypertensive whites); however, these observed differences were not statistically.' different from those expected. Given an allele frequency of 30%, we found a significantly increasednumber of 6.3-kb homozygotes, 13/62, compared to the expected frequency of 7/D'1 among American blacks with moderate to severe essential hypertension. Other investigators have attempted to identify C'P. complex quantitative traits associated with hypertension in laboratory animals and humans. Polymorphisms for the renin and kallikreinv" genes have been linked to the developmentof genetic hypertension in laboratory rats. In humans, MN blood group antigens," isozymes of the haptoglobin protein," the rate of kallikrein excretion," certainhistocompatibility antigens.l" and genes controlling lipid metabolism" are associated with the developmentof hypertension. Although polymorphisms of the genes for the sodium-hydrogen antiporterand atrial natriuretic factor have been studied, no associations with essential hypertension have been found.13,30 Although no populationstudy has found an association ofhypertension and polymorphism of the renin gene in the American1 or general European population.P a genetic polymorphism of the renin gene has recently. Lcen reported in hypertensive blacks of Caribbean descent." Also, a chimeric gene defect has been reported to causehypertensionin humans." VIle report a possible association between a candidate gene and hypertension in American blacks. However, these data must be interpreted with caution; we recognize that ethnic variation within the black population can also contribute to our findings. The mechanism(s) by which a genetic polymorphism of the
GENETICS OF HYPERTENSION 393
-APRIL 1995-VOL. 8, NO.4, PART 1
TABLE 1. FREQUENCY OF GENOTYPE OF THE C10 (l2-ADRENERGIC RECEPTOR IN NORMOTENSIVE AND HYPERTENSIVE BLACKS
Black
White NT (n
6.7/6.7-kb homozygote 6.7/6.3-kb heterozygote 6.3/6.3-kb homozygote
= 61)
39 21 1
HT (n
= 25)
16 9
o
NT (n
=59)
26 31 2
HT (n
=82)
46 23 13""
Frequency ofalleles and genotypes identified with a probe complementary to tile C10 Cl2-adrenergic receptor in 227 subjects prospectively identified as nonnotensive or hypertensive. Normotensive blacks had an increased frequency ofthe 6.3-kb allele (30%) compared to normotensive whites (19%, P
= .07).
*Analysis using Fisher's exact test for comparison of 6.3-kb 110mozygotes to tIle remainder of the subjects showed significant difference for all hypertensive subjects (white and black) v all normotensive subjects (white and black) (P = .008); and significatlt differences for hypertensive black subjects v hypertensive whites (P = .036) and v normotensive blacks (P = .024).
quence ATTTA encodes a rapid degradation signal in complementary messenger RNA. These observations are compatible with numerous reports of abnormal modulation ofthe number of(X2-adrenergic receptors, but differing reports of changes in li~and binding, in genetically hypert.ensive animals.P" 5 It is suggested from our study that a polymorphism of the C-lO (X2-adrenergic receptor may be associated with the development of severe hypertension. The frequency of the 6.3-kb allele was the same in the black normotensive and hypertensive group, but there was a fourfold increase in the prevalence of 6.3-kb homozygotes in hypertensive blacks compared to normotensive blacks. We attempted to study the phenotypic expression (salt excretion, glucose tolerance, and vascular reactivity) in our 6.3-kb homozygct.es, but nearly all of these blacks had diastolic blood pressures greater than 105 mm Hg as their antihypertensive medicationswere tapered: this precluded safecompletion of these studies. Accordingly, we are now attempting to identify and study 6.3-kb homozygotes who are younger and have milder hypertension to determine whether presence of the 6.3-kb homozygosity is a marker forthe subsequentdevelopment ofsevere hypertension. It is possible that homozygosity of the 6.3-kb allele may identify silent, intermediate phenotypes such as abnormal salt excretion, which subsequently contributes to pathologic elevations in blood pressure. We have recently sequenced the 3' portion of this gene (unpublished observarior.s), and we are now using the polymerase chain reaction to identify homozygotes more rapidly. Although these studies are by no means conclusive, they suggest the need for linkage studies of the Clf)
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