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(Stroke. 2001;32:822.)
© 2001 American Heart Association, Inc.

Original Contributions

G-Protein ß3 Subunit and -Adducin Polymorphisms and Risk of Subclinical and Clinical Stroke

Alanna C. Morrison, BS; Peter A. Doris, PhD; Aaron R. Folsom, MD; F. Javier Nieto, MD, PhD; Eric Boerwinkle, PhD for the Atherosclerosis Risk in Communities Study

From the Human Genetics Center, University of Texas–Houston Health Science Center, (A.C.M, E.B.); Institute of Molecular Medicine, Houston, Tex (P.A.D., E.B.); University of Minnesota, School of Public Health, Division of Epidemiology, Minneapolis (A.R.F.); and Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Md (F.J.N.).

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. E-mail eboerwin{at}gsbs.gs.uth.tmc.edu

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References Introduction  References

 
Background and Purpose—Essential hypertension is a significant risk factor for stroke. Genes contributing to interindividual variation in blood pressure levels and essential hypertension status may play a role in the etiology of stroke either through their effects on blood pressure levels or through separate pathways. For this reason, we sought to examine the association between the -adducin (ADD1) G/W460 and G-protein ß3 subunit (GNß3) 825C/T polymorphisms and subclinical and clinical stroke in the Atherosclerosis Risk in Communities (ARIC) Study.

Methods—Subclinical stroke was determined by cerebral MRI. Subclinical cerebral infarct cases (n=202) were compared with a stratified random sample (MRI-CRS) identified from individuals participating in the MRI examination (n=211). Incidence of clinical ischemic stroke was determined by following the ARIC cohort for an average of 7.2 years for potential cerebrovascular events; 231 validated clinical ischemic strokes were identified. A stratified random sample of the ARIC cohort (CRS) (n=984) was used as the comparison group for the clinical cases.

Results—The frequency of the ADD1 W460 allele was determined for the subclinical cases (0.12), MRI-CRS (0.16), clinical cases (0.14), and CRS (0.17). The frequency of the GNß3 825T allele was determined in whites and blacks, respectively, for the subclinical cases (0.26, 0.73), MRI-CRS (0.31, 0.75), clinical cases (0.36, 0.72), and CRS (0.30, 0.72). The ADD1 W460 and GNß3 825T alleles were not significantly associated with subclinical stroke. The ADD1 W460 allele was also not a significant predictor of clinical stroke. The GNß3 825T allele was significantly associated with clinical stroke in whites after adjustment for age and sex (hazard rate ratio, 1.45; 95% CI, 1.05 to 2.00) and after further adjustment for multiple stroke risk factors (hazard rate ratio, 1.68; 95% CI, 1.18 to 2.41). The GNß3 825T allele was not significantly associated with clinical stroke in blacks for either adjustment model.

Conclusions—The GNß3 gene 825C/T polymorphism is significantly associated with incident clinical ischemic stroke in a white middle-aged American population, but not in blacks. This association does not appear to be mediated by established stroke risk factors, specifically blood pressure levels or hypertension status.

Key Words: cerebral infarction • genetics • risk factors • stroke, ischemic

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References Introduction  References

 
One third of the deaths each year in the United States due to the complications of untreated essential hypertension are stroke deaths,¹ and hypertensive individuals are reported to have a greater incidence of stroke than normotensive individuals.² The established relationship between hypertension and stroke, as well as the fact that both conditions demonstrate marked familial aggregation,^{3 4} indicates that they may share at least some genes in common. While numerous studies have investigated the effect of specific genes on blood pressure levels and essential hypertension status, there is a paucity of information related to the role of specific genes on the onset and outcome of stroke. Polymorphisms of the -adducin (ADD1) and G-protein ß3 subunit (GNß3) genes have received considerable attention as candidate genes for essential hypertension. These genes may play a role in the etiology of stroke through their effects on blood pressure levels or through separate pathways.

Adducin is a heteromeric cytoskeleton protein composed of - and ß-subunits. Variation in the -adducin protein may affect ion transport through modification of actin cytoskeleton assembly and modulation of sodium pump activity^{5 6} and may play a role in both human and rat hypertension.^{7 8 9} In humans, significant linkage was detected in hypertensive sib pairs for markers located near the ADD1 gene.¹⁰ Many investigators have reported a significant association between the ADD1 W460 allele and hypertension.^{10 11 12 13} Other studies, however, report that the ADD1 W460 allele does not significantly contribute to variation in blood pressure levels¹⁴ or hypertension status.^{13 15 16 17 18 19 20}

Studies in lymphoblasts and fibroblasts from patients with essential hypertension demonstrated enhanced signal transduction involving pertussis toxin–sensitive G-proteins.²¹ Most membrane receptors rely on heterotrimeric G-proteins to activate or inhibit intracellular signaling cascades.²² Siffert et al²³ detected a novel polymorphism (825C/T) in exon 10 of the gene encoding the ß3 subunit of heterotrimeric G-proteins (GNß3). An association was observed between the 825T allele and generation of a splice variant, in which the nucleotides of exon 9 are deleted. The splice variant is believed to result in a dominant gain of function.²⁴ Many, but not all, studies have reported a significant association between the GNß3 825T allele and variation in blood pressure levels,^{22 25} hypertension status,^{15 23 26 27 28} and related phenotypes, such as measures of body weight and obesity.^{29 30 31 32}

The effect of the ADD1 and GNß3 polymorphisms on cellular ion transport and their hypothesized importance in predisposing certain individuals to hypertension prompted us to evaluate their association with stroke in the large prospective Atherosclerosis Risk in Communities (ARIC) Study. Our objective was to consider both subclinical and clinical disease and to gain insight into whether any observed association was independent of established stroke risk factors, specifically blood pressure levels or hypertension status.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References Introduction  References

 
The ARIC Study
Study participants were selected from the ARIC Study, a prospective investigation of atherosclerosis and its clinical sequelae involving 15 792 individuals aged 45 to 64 years at recruitment (1986–1989). Subjects were selected by probability sampling from 4 communities: Forsyth County, North Carolina; Jackson, Mississippi (blacks only); northwestern suburbs of Minneapolis, Minnesota; and Washington County, Maryland. The initial clinical examinations included a home interview to ascertain cardiovascular risk factors, socioeconomic factors, and family medical history; clinical examination; and blood drawing for laboratory determinations. Medical events were identified by an annual questionnaire, 3-year cycles of examinations, and hospital and death certificate surveillance. A detailed description of the ARIC Study design and methods is published elsewhere.³³

Prevalent Subclinical Cerebral Infarction
During 1993 and 1994, all cohort members aged 56 years from 2 of the 4 ARIC field centers (Jackson, Mississippi, and Forsyth County, North Carolina) were screened for eligibility to participate in a cerebral MRI examination (n=2877). For participants’ safety, specific criteria were used to exclude individuals as ineligible for the MRI examination.³⁴ Subclinical cerebral infarct cases (n=202) and an age- and sex-stratified random sample (MRI-CRS; n=211) were identified from a total sample of white and black MRI examination participants aged 56 years who did not have a positive or unknown history of prevalent stroke or coronary heart disease or history of transient ischemic attack/stroke symptoms at the initial clinic visit (n=1719). Subclinical cerebral infarct cases were defined by the presence of cerebral infarcts >3 mm. MRI scanning and image interpretation were based on previously published protocols.^{35 36} Before analyses involving the ADD1 G/W460 polymorphism, an additional 32 individuals were excluded because of insufficient DNA. A total of 195 subclinical cerebral infarct cases and 186 MRI-CRS individuals were included in analyses of the ADD1 G/W460 polymorphism. Similarly, 19 individuals were excluded before analyses involving the GNß3 825C/T polymorphism because of insufficient DNA. A total of 196 subclinical cerebral infarct cases and 198 MRI-CRS individuals were included in analyses of the GNß3 825C/T polymorphism.

Incident Clinical Ischemic Stroke
Clinical ischemic stroke was determined by contacting participants annually to identify hospitalizations during the previous year and by surveying discharge lists from local hospitals and death certificates from state vital statistics offices for potential cerebrovascular events.^{33 37} Hospital records were obtained, abstracted, and classified by computer algorithm and physician review. Details on quality assurance for ascertainment and classification of ischemic stroke events have been published elsewhere.³⁷ Ischemic strokes were defined as validated definite or probable hospitalized embolic or thrombotic brain infarctions. Participants were excluded for this analysis if they had a positive or unknown history of prevalent stroke or coronary heart disease or history of transient ischemic attack/stroke symptoms at the initial clinic visit or if they had ethnic background other than white or black (n=1499). The remaining 14 293 participants were followed for incident clinical ischemic stroke for an average of 7.2 years, and 231 incident ischemic stroke cases were identified. A comparison group for the clinical cases was selected from the cohort of 14 293 participants. Selection of this cohort random sample (CRS) was stratified on the basis of ultrasound examination of carotid arteries, age, and sex. The resulting CRS included 984 individuals. A nested case-control design was used to account for individuals selected as part of the CRS who were also identified as incident ischemic stroke cases. Before analyses involving the ADD1 G/W460 polymorphism, 55 individuals were excluded because of insufficient genotype information, leaving 210 incident clinical cases and 950 CRS individuals for ADD1 G/W460 polymorphism analyses. Similarly, 89 individuals were excluded before analyses involving the GNß3 825C/T polymorphism because of insufficient genotype information, leaving 212 clinical cases and 914 CRS individuals.

Examination and Laboratory Measures
Seated blood pressure was measured 3 times with a random-zero sphygmomanometer, and the last 2 measurements were averaged. Questionnaires were used to assess use of antihypertensive medication. The ratio of waist (umbilical level) and hip (maximum buttocks) circumference was calculated as a measure of fat distribution. Diabetes was defined by a fasting glucose level 126 mg/dL, a nonfasting glucose level 200 mg/dL, and/or a history of or treatment for diabetes. Cigarette smoking status was analyzed by comparing current smokers with individuals who had formerly or never smoked. Von Willebrand factor antigen levels were measured by enzyme immunoassay as previously described.³⁸

Genotype Determination
A 147-bp fragment surrounding the ADD1 G/W460 variant was amplified with the use of the forward primer 5'-CTCCTTTGCTAGTGACGGTGATTC-3' and the reverse primer 5'-GACTTGGGACTGCTTCCATTCGGCC-3'. A mismatch, which introduces a Sau96I restriction site, was placed in the 3' region of the reverse primer to enable genotyping via restriction digest (the mismatched nucleotide is underlined). Polymerase chain reaction (PCR) reactions included 30 ng DNA, 1.5 mmol/L MgCl₂, standard concentrations of PCR reagents, and 0.5 U Taq polymerase (GIBCO-BRL) in a 20-µL reaction volume. After an initial denaturation step of 4 minutes at 95°C, the products were amplified using 40 cycles of 1 minute at 94°C, 1 minute at 57°C, and 1 minute at 72°C, followed by 5 minutes at 72°C and 5 minutes at 98°C. The amplified products were then digested overnight with 6 U of the Sau96I enzyme. Digested products were separated on 3% agarose gels, stained with ethidium bromide, and visualized with UV light. Genotypes are represented by a 147-bp band (W/W460 homozygote); bands of 147, 122, and 25 bp (G/W460 heterozygote); and bands of 122 and 25 bp (G/G460 homozygote).

A 268-bp fragment surrounding the GNß3 825C/T variant was amplified with the use of the forward primer 5'TGACCCACT-TGCCACCCGTGC-3'and the reverse primer 5'-GCAGCAG-CCAGGGCTGGC-3'. PCR reactions included 30 ng DNA, 1.0 mmol/L MgCl₂, standard concentrations of PCR reagents, and 0.5 U Taq polymerase (GIBCO-BRL) in a 20-µL reaction volume. After an initial denaturation step of 5 minutes at 94°C, the products were amplified using 35 cycles of 1 minute at 94°C, 45 seconds at 60°C, and 1 minute at 72°C, followed by 7 minutes at 72°C. The amplified products were then digested overnight with 1 U of the BsaJI enzyme. Digested products were separated on 3% agarose gels, stained with ethidium bromide, and visualized with UV light. Genotypes are represented by a band of 256 bp (825T/T homozygote); bands of 256, 152, and 104 bp (825C/T heterozygote); and bands of 152 and 104 bp (825C/C homozygote).

Statistical Analysis
Allele frequencies were estimated by gene counting. Agreement of the ADD1 G/W460 and GNß3 825C/T genotype frequencies with Hardy-Weinberg equilibrium expectations was tested with a ² goodness-of-fit test. The proportions, means, and SEs of established stroke risk factors^{39 40 41} were reported as weighted results for the cerebral infarct cases, MRI-CRS, incident clinical ischemic stroke cases, and CRS. Multivariable logistic regression models were used to assess the relationship between subclinical cerebral infarct case status and the ADD1 G/W460 or GNß3 825C/T polymorphisms. Cox proportional hazards models were used to estimate the hazard rate ratios (HRRs) of incident clinical ischemic stroke between those with or without the ADD1 W460 or GNß3 825T alleles. The SUDAAN software package was used to adjust for sampling strategy in the logistic regression and Cox proportional hazards models.⁴² For incident clinical ischemic stroke cases, the follow-up time interval was defined as the time between the initial clinical visit and the date of the first ischemic stroke. For the CRS, follow-up continued until December 31, 1996, the date of death, or the date of last contact if lost to follow-up, whichever came first. Race-stratified logistic regression and Cox proportional hazards models included age and sex as covariates. Race was also included as a covariate in ethnically unstratified analyses. The established stroke risk factors^{39 40 41} evaluated as potential confounders in the logistic regression and Cox proportional hazards models included diabetes and smoking status, systolic and diastolic blood pressure, use of antihypertensive medication, von Willebrand factor, and waist-to-hip ratio. Odds ratios (ORs) and HRRs were calculated with the assumption of a codominant effect of the W460 and 825T alleles. Covariates were assessed for statistical significance in the models by the Wald ² statistic.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References Introduction  References

 
Group-specific proportions, means, and SEs for multiple established stroke risk factors are presented in Table 1. Mean values for all variables shown differed significantly between the subclinical cerebral infarct cases and the MRI-CRS. The subclinical cerebral infarct cases had a significantly greater frequency of blacks and individuals using antihypertensive medication than the MRI-CRS. Incident clinical ischemic stroke cases differed significantly from the CRS for all variables shown. The frequency of males, blacks, individuals using antihypertensive medication, diabetics, and smokers was significantly greater among clinical cases than in the CRS.

View this table: [in this window] [in a new window]   Table 1. Characteristics of Case and Comparison Groups

Allele and genotype frequencies for the ADD1 G/W460 and GNß3 825C/T polymorphisms are presented in Table 2. The ADD1 G/W460 genotype frequency distributions were in accordance with Hardy-Weinberg equilibrium expectations. The GNß3 825C/T genotypes were not in Hardy-Weinberg equilibrium in the total sample because of a marked difference in allele frequencies between white and black populations. After stratification by ethnicity, the GNß3 825C/T genotype distributions were in accordance with Hardy-Weinberg equilibrium expectations. Because of the marked allele frequency difference between whites and blacks for the GNß3 825C/T polymorphism, subsequent analyses were performed separately by racial group.

View this table: [in this window] [in a new window]   Table 2. Allele and Genotype Frequencies in Case and Comparison Groups

Results from the multivariable logistic regression models examining the association of the ADD1 W460 allele and the GNß3 825T allele with subclinical cerebral infarct prevalence are presented in Table 3. After adjustment for age, sex, and race (model 1), the ADD1 W460 allele was not significantly associated with subclinical stroke prevalence (OR, 0.84; 95% CI, 0.55 to 1.28). No significant association was determined after further adjustment for multiple stroke risk factors (OR, 0.81; 95% CI, 0.52 to 1.28) (model 2). Similarly, the 825T allele of the GNß3 gene was not found to be significantly associated with subclinical stroke after adjustment for age and sex (whites: OR, 0.76; 95% CI, 0.47 to 1.22; blacks: OR, 0.89; 95% CI, 0.52 to 1.50) or after further adjustment for established stroke risk factors (whites: OR, 0.89; 95% CI, 0.53 to 1.49, blacks: OR, 0.88; 95% CI, 0.51 to 1.51).

View this table: [in this window] [in a new window]   Table 3. Relationship Between Case Status and ADD1 W460 and GNß3 825T Alleles

Results from the Cox proportional hazards models used to estimate the HRR of incident clinical ischemic stroke between individuals with or without the variant alleles of the ADD1 and GNß3 genes are presented in Table 3. After adjustment for age, sex, and race (model 1), the ADD1 W460 allele (HRR, 1.00; 95% CI, 0.71 to 1.43) was not a significant predictor of clinical stroke. No significant association was determined after further adjustment for multiple stroke risk factors (HRR, 1.08; 95% CI, 0.72 to 1.63) (model 2). The GNß3 825T allele was significantly associated with incident clinical ischemic stroke in whites after adjustment for age and sex (HRR, 1.45; 95% CI, 1.05 to 2.00; P=0.02) and after further adjustment for multiple stroke risk factors (HRR, 1.68; 95% CI, 1.18 to 2.41; P<0.01). The GNß3 825T allele was not significantly associated with clinical stroke in blacks for either adjustment model.

All multivariable logistic regression and Cox proportional hazards models were additionally adjusted for measures of left ventricular hypertrophy. Addition of left ventricular hypertrophy to the models did not change the relationship between the ADD1 W460 and GNß3 825T alleles and subclinical and clinical stroke (data not shown).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References Introduction  References

 
We have investigated the association between variants of the ADD1 and GNß3 genes and clinical and subclinical stroke in white and black individuals selected from the large prospective ARIC Study. This investigation was motivated by the idea that genetic polymorphisms shown to be associated with essential hypertension may influence stroke risk by means of a common pathophysiological pathway. The W460 allele of the ADD1 gene was not significantly associated with subclinical or clinical stroke in this sample of middle-aged Americans. The 825T allele of the GNß3 gene was found to be significantly associated with incident clinical ischemic stroke in whites but not subclinical cerebral infarction. The association of the GNß3 8235T allele with incident clinical ischemic stroke was modest (HRR, 1.68) and did not appear to be mediated by established risk factors, including blood pressure. The GNß3 825T allele was not associated with subclinical or clinical stroke in blacks.

Animal model studies have provided insight into the role of genes in stroke susceptibility. The majority of these studies involve the spontaneously hypertensive rat (SHR) and the stroke-prone and stroke-resistant substrains. Stroke occurs in stroke-prone SHR after high blood pressure has developed, and incidence of stroke is increased if the animals are exposed to a specific dietary regimen involving reduced protein and potassium and increased sodium intake.^{43 44 45} In addition to providing evidence that, in general, genetic factors contribute to stroke development,⁴³ studies involving these animal models have demonstrated that stroke is attributable to both blood pressure–dependent and blood pressure–independent genetic factors. The fact that despite similar blood pressures, SHR and stroke-prone SHR have different predisposition to stroke is indicative of blood pressure–independent genetic risk factors for stroke.⁴⁵ Thus, it is not surprising that the GNß3 825C/T polymorphism, which has been shown to be associated with essential hypertension, is also associated with incident clinical ischemic stroke in whites independent of current blood pressure levels or hypertension status. These observations, if replicated in further studies, suggest a molecular mechanism that is common to the etiology of hypertension and stroke but perhaps does not unify these diseases by a single pathophysiological process.

We expected a magnitude of association for the gene variants among subclinical cerebral infarct cases similar to the incident clinical ischemic stroke cases. This assumption was based on previous reports that suggest that MRI-detected abnormalities of the brain are markers of subclinical cerebrovascular disease and share similar risk factors with clinically diagnosed stroke.^{34 35 46} We did not observe the same magnitude of association of the GNß3 825C/T polymorphism with subclinical stroke as we did with clinical stroke for white individuals. Lack of association between the GNß3 825C/T polymorphism and subclinical cerebrovascular disease, as measured by MRI, has been reported for Japanese populations.^{28 47} However, collective interpretation of these results may not be applicable because of differences in GNß3 825T allele frequency between white and Japanese populations.^{28 48} One explanation for the difference observed between clinical and subclinical disease is potential survival bias, such that individuals with the at-risk GNß3 825T allele suffered preferential mortality before the MRI examination. However, this is unlikely in light of the moderate size of the observed allele effect. Although similarities between MRI-detected subclinical cerebrovascular disease and clinical stroke exist, there are also important differences,⁴⁹ and these differences may be at least partially responsible for the observed variability in the associations between the GNß3 polymorphism and incident clinical ischemic stroke and subclinical cerebral infarction.

The differences observed between whites and blacks in the association of the GNß3 825T allele with incident clinical ischemic stroke are difficult to interpret. Quantitative and qualitative measures of blood pressure phenotypes were not significantly different among GNß3 genotypes, in both whites and blacks, for all analysis groups (data not shown). Hence, the differential relationship between the GNß3 825C/T polymorphism and incidence of ischemic stroke among whites and blacks is not explained by a difference in the pattern of blood pressure phenotypes among GNß3 825C/T genotypes between these ethnic groups. Any comparison is also confounded by the large difference in allele frequencies for the GNß3 825C/T polymorphism between whites and blacks. As has been observed by others,^{32 50 51} the frequency of the 825T allele is significantly greater in populations of mixed African ancestry compared with populations of mixed European ancestry.

In summary, the 825C/T polymorphism of the GNß3 gene of stroke is significantly associated with increased risk of incident clinical ischemic stroke in white participants of the ARIC Study. This association did not appear to be mediated by established stroke risk factors, specifically blood pressure levels or hypertension status. Further characterization of whether the GNß3 gene contributes to increased stroke risk will be useful for the potential early identification of individuals at increased risk for stroke as a complication of hypertension and for developing a better understanding of the etiology and pathophysiology of the disease. This study underscores the need for timely, well-designed, and comprehensive genomic studies of the occurrence of stroke in humans to identify and localize stroke susceptibility genes.

	Acknowledgments

 
This study was supported 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. The authors thank participants in the ARIC Study for their important contributions. We also wish to acknowledge the valuable contributions made by the ARIC staff at the following collaborating institutions: University of North Carolina at Chapel Hill; University of North Carolina, Forsyth County; Wake Forest University, Winston-Salem, NC; University of Mississippi Medical Center, Jackson; University of Minnesota, Minneapolis; Johns Hopkins University, Baltimore, Md; and University of Texas, Houston.

Received August 17, 2000; revision received November 15, 2000; accepted December 21, 2000.

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Editorial Comment

Candidate Genes for Stroke: If Elected, Will They Serve?

Robert A. Hegele, MD, FRCPC, FACP, Guest Editor

Blackburn Cardiovascular Genetics Laboratory, Robarts Research Institute, London, Ontario, Canada

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References Introduction  References

 
Finding genes that underlie susceptibility to a complex disease like stroke is important for several reasons. First, genome-based assays could add value to decision-making algorithms for diagnosis, prognosis, prediction of susceptibility, and/or patient stratification for particular therapies. Additionally, since the completion of the first draft of the human genome map, the range of experimental approaches to find new mechanisms underlying stroke has been extended. Genomics has the potential to generate new biological knowledge much more rapidly than other scientific methodologies. This information will specify new molecular targets for classical pharmacological and/or novel interventions. Of course, the clinical utility of any new diagnostic or therapeutic modality will require formal evaluation with clinical trials.

The 2 main strategies to expose disease genes have been called "positional cloning" and the "candidate gene approach."^R1 Briefly, positional cloning begins without any a priori understanding of the gene products involved in the disease process. The genome is screened with DNA markers to identify chromosomal segments that are statistically correlated with disease. The genomic region containing the disease gene can be further narrowed by other methods. Statistical arguments can help to establish whether a gene so identified is actually a disease gene. Functional studies can also make the case for causation. While nongeneticists view this approach as "backward," positional cloning moves forward from the geneticist’s perspective.

In contrast, the candidate gene approach typically uses information from prior cellular, biochemical, or physiological functional studies to target a gene. Molecular markers are developed, and the relationship between the gene and disease is evaluated. Nongeneticists might see this as a "forward" approach for identifying disease genes, since the evidence for a functional role of the protein must have already been established by nongenetic experiments. However, the applicability of this approach when defined strictly is limited to genes whose products have been functionally evaluated. Thus, traditional candidate genes are those whose dysfunction is expected to cause a disease because of evidence from nongenetic experiments. However, newer technologies, such as DNA microarrays, provide new approaches to specify candidate genes in model systems. Thus, the definition of what constitutes a "candidate gene" may become even broader in the future.

In the accompanying article, the ARIC investigators report the results of a well-designed association analysis of 2 candidate genes for stroke. The disease phenotypes were prevalent subclinical stroke, ie, asymptomatic subjects with cerebral infarcts on MRI, and incident ischemic stroke, ie, symptoms of embolic or thrombotic brain infarction. Common single nucleotide polymorphisms (SNPs) in GNß3, which encodes the third form of the G-protein ß subunit polypeptide, and ADD1, which encodes -adducin, were analyzed in stroke subjects and controls. These genes were selected as "candidates" because of their reported roles in hypertension susceptibility.

GNß3 encodes a member of the large G-protein family of heptohelical receptors, whose activation is transduced into changes in intracellular function. Transduction via pertussis toxin–sensitive G-proteins in lymphoblasts and fibroblasts is enhanced in patients with essential hypertension.^R2 Studies of the GNß3 gene demonstrated a common SNP, designated 825C/T, which alters RNA splicing, resulting in deletion of 41 amino acids.^R3 Furthermore, the splice variant encoded by the GNß3 825T allele has enhanced activity in vitro.^R3 The GNß3 825T allele has been associated with hypertension,^R3 increased body mass,^{R4 R5} low birth weight,^R6 and weight retention after pregnancy.^R7 In the accompanying article Morrison and colleagues now report that the GNB3 825T allele is associated with an increased risk of clinical stroke in whites but not in blacks and that this association is independent of blood pressure.

The second candidate gene, ADD1, encodes -adducin, which is a heterodimeric cell-membrane skeletal protein. Evidence for an important role for -adducin comes from the Milan hypertensive rat strain, which develops renal hypertension and has a mutated adducin gene.^R8 A common human SNP in ADD1 changes the amino acid sequence of -adducin from glycine to tryptophan at residue 460. This variant, designated G/W460, alters renal tubular reabsorption in hypertensive subjects.^R9 However, the ADD1 W460 allele has not been consistently associated with hypertension and related phenotypes.^R9 Morrison and colleagues now report no association of the ADD1 W460 with stroke.

The findings of Morrison and colleagues in the accompanying article emphasize some of the challenges presented by genetic association studies. Both candidate genes were nominated because nongenetic experiments indicated that their products affect an intermediate phenotype—blood pressure—that is related to stroke risk. Furthermore, both SNPs alter protein structure, with functional consequences. However, the ADD1 W460 allele was not associated with stroke, and the association with the GNß3 825T allele was conditional, occurring only with clinical stroke and only in whites, but independent of blood pressure. This last attribute is particularly vexing, since the rationale for choosing GNß3 as a candidate had been its reported association with hypertension.

While the findings of Morrison and colleagues suggest a relationship between the GNß3 825T allele and stroke, the conditional nature of the association indicates the need for replication in other study samples, as the authors themselves point out. GNß3 is a good candidate, but more work is required before it can be elected to serve as a clinical marker of stroke risk. The limitations of association studies using candidate gene markers are well known and include the confounding effects of intersample differences in genetic background, environment, disease phenotype, and stratification or admixture artifacts.^R1 Despite such difficulties, association studies with candidate genes are likely to remain an important strategy to study genes that determine stroke susceptibility. The complexity of "stroke" theoretically means that each patient has an individual landscape of risk and specific trail of cellular and pathogenetic events that culminate in a unique clinical presentation. Fortunately, there are probably elements of stroke risk, causation, and pathogenesis that are common to many patients. This sustains hope that common genetic factors will have a predictable impact on stroke, although these factors are largely unspecified at present.

Received August 17, 2000; revision received November 15, 2000; accepted December 21, 2000.

	References

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3. Siffert W, Rosskopf D, Siffert G, Busch S, Moritz A, Erbel R, Sharma AM, Ritz E, Wichmann HE, Jakobs KH, Horsthemke B. Association of a human G-protein beta-3 subunit variant with hypertension. Nat Genet. 1998;18:45–48.

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7. Gutersohn A, Naber C, Muller N, Erbel R, Siffert W. G protein beta-3 subunit 825 TT genotype and post-pregnancy weight retention. Lancet. 2000;355:1240–1241.

8. Bianchi G, Tripodi G, Casari G, Salardi S, Barber BR, Garcia R, Leoni P, Torielli L, Cusi D, Ferrandi M, Pinna LA, Baralle FE, Ferrari P. Two point mutations within the adducin genes are involved in blood pressure variation. Proc Natl Acad Sci U S A. 1994;91:3999–4003.

9. Manunta P, Barlassina C, Bianchi G. Adducin in essential hypertension. FEBS Lett. 1998;430:41–44.

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