Pharmacogenetic Differences in Response to Albuterol between Puerto Ricans and Mexicans with Asthma
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美国呼吸和危急护理医学 2005年第3期
University of California, San Francisco
Lung Biology Center, San Francisco General Hospital, San Francisco, California
San Juan Veterans Administration Medical Center, University of Puerto Rico School of Medicine
Pediatric Pulmonary Program of San Juan, San Juan, Puerto Rico
Brigham and Women's Hospital, Boston, Massachusetts
The Harlem Lung Center, Harlem Hospital and Columbia University, New York, New York
Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico
ABSTRACT
Background: In the United States, Puerto Ricans and Mexicans have the highest and lowest asthma prevalence, morbidity, and mortality, respectively. Ethnic-specific differences in the response to drug treatment may contribute to differences in disease outcomes. Genetic variants at the 2-adrenergic receptor (2AR) may modify asthma severity and albuterol responsiveness. We tested the association of 2AR genotypes with asthma severity and bronchodilator response to albuterol in Puerto Ricans and Mexicans with asthma. Methods: We used both family-based and cross-sectional tests of association with 8 2AR single nucleotide polymorphisms in 684 Puerto Rican and Mexican families. Regression analyses were used to determine the interaction between genotype, asthma severity, and bronchodilator drug responsiveness. Results: Among Puerto Ricans with asthma, the arginine (Arg) 16 allele was associated with greater bronchodilator response using both family-based and cross-sectional tests (p = 0.00001eC0.01). We found a strong interaction of baseline FEV1 with the Arg16Glycine (Gly) polymorphism in predicting bronchodilator response. Among Puerto Ricans with asthma with baseline FEV1 < 80% of predicted, but not in those with FEV1 > 80%, there was a very strong association between the Arg16 genotype and greater bronchodilator responsiveness. No association was observed between Arg16Gly genotypes and drug responsiveness among Mexicans with asthma. Conclusions: Ethnic-specific pharmacogenetic differences exist between Arg16Gly genotypes, asthma severity, and bronchodilator response in Puerto Ricans and Mexicans with asthma. These findings underscore the need for additional research on racial/ethnic differences in asthma morbidity and drug responsiveness.
Key Words: asthma genetics 2-adrenergic receptor gene Latinos pharmacogenetic
Studies of racial/ethnic health disparities often define populations based on U.S. census definitions, which categorize Latinos as a single ethnic group. Latinos are the largest minority population in the United States, and Latino children represent the largest demographic group of all U.S. children (1). Although the terms "Hispanic" or "Latino" American describe a common language and cultural heritage, these terms do not refer to race, uniform ethnicity, or a common genetic background. The two largest Latino ethnic groups in the United States, Mexicans (63%) and Puerto Ricans (11%) (1), are genetically complex and comprised of various proportions of Native American, African, and European genetic origins (2). The relative proportions of these ancestral populations to the contemporary Latino gene pool make each Latino ethnic group genetically unique (2).
According to U.S. vital statistics, asthma prevalence, morbidity, and mortality are the highest among Puerto Ricans and the lowest among Mexicans for a total of a fourfold difference in asthma burden between these two ethnic groups (3, 4). Although there are many potential explanations for this observation, including environmental and socioeconomic factors, one potential explanation is that the genetic predisposition to asthma or to greater asthma severity differs among subgroups within the Latino population. These subgroups may have genetic variants, which may explain the variation in asthma severity and response to drug therapy between Latino ethnic groups.
Albuterol, a short-acting 2-adrenergic receptor (2AR) agonist, is the most commonly prescribed treatment for asthma worldwide (5). 2AR mediates physiologic responses, including bronchial smooth muscle relaxation (bronchodilation), vasodilatation, and lipolysis (6). Genetic variations at the 2AR gene may play a role in the observed ethnic-specific differences in asthma severity and bronchodilator drug responsiveness.
Polymorphisms in the 2AR gene have been inconsistently associated with varying degrees of bronchodilatory response to -agonists (7eC12) and with tachyphylaxis (13, 14). In one study, specific haplotypes, as opposed to individual single nucleotide polymorphisms (SNPs), were significantly associated with drug responsiveness (12). Consequently, there is no clear consensus regarding the extent to which genetic variants in the 2AR gene may influence asthma severity or bronchodilator drug responsiveness.
We used a family-based genetic association study to determine whether polymorphisms (SNPs and haplotypes) in the 2AR gene are associated with ethnic-specific differences in asthma severity and bronchodilator response in Puerto Rican and Mexican subjects with asthma participating in the Genetics of Asthma in Latino Americans (GALA) Study. We selected a family-based approach because it removes the effect of population stratification, a potentially important source of confounding (15). Spurious associations between genetic variants and phenotypes may arise as a result of population stratification when noneCfamily-based approaches are used (16). Moreover, racial and ethnic background can influence genetic variation and genotypic relative risk (17). Some of the results of this study have been previously reported in the form of an abstract (18).
METHODS
Study Participants
Recruitment and pulmonary function testing are described elsewhere and in detail in the online supplement (19). Briefly, 684 subjects with asthma (probands) and their biological parents were enrolled in the San Francisco Bay Area (SF), New York City (NY), Puerto Rico (PR), and Mexico City (MX). Clinical characteristics of all probands are shown in Table 1. Ethnicity was self-reported. Probands were enrolled only if both biological parents and all four biological grandparents were of Puerto Rican (for the NY and PR sites) or Mexican (for the SF and MX sites) ethnicity.
Pulmonary Function Tests and IgE Measurements
Spirometry was performed according to American Thoracic Society (ATS) standards (20). Pulmonary function test results are shown in Table 1 and are expressed as a percentage of the predicted normal value using age-adjusted prediction equations from Hankinson (21).
Probands with asthma were classified as having "mild" or "moderate-severe" asthma based on their baseline FEV1 expressed as percent of predicted or Pre-FEV1 levels. Subjects with a Pre-FEV1 greater or less than 80% of predicted were categorized as having "mild" and "moderate-severe" asthma, respectively. A quantitative measure of bronchodilator responsiveness was calculated as FEV1, which is the relative percent change in Pre-FEV1 after albuterol administration. Total plasma IgE was measured in duplicate using Uni-Cap technology (Pharmacia, Kalamazoo, MI).
Genotyping
Genotyping details are described further in the online supplement. Eight 2AR SNPs (eC709, eC654, eC47, +46, +79, +252, +491, and +523) were genotyped and used to assign haplotypes.
Statistical Analysis
Statistical analyses are further described in the online supplement. Mendelian inconsistencies were identified using PEDCHECK (http://watson.hgen.pitt.edu/register/docs/pedcheck.html) (22). Hardy-Weinberg equilibrium was calculated by means of 2 goodness-of-fit tests. The degree of linkage disequilibrium (LD) was estimated by using the r2 statistic (23).
Family-based tests (24) and family-based tests for associating haplotypes (25) were used to assess the association between individual SNPs and haplotypes, respectively, with quantitative measures of asthma-related traits. Quantitative phenotypes included: asthma severity (defined by Pre-FEV1), bronchodilator responsiveness (defined by FEV1, relative % of predicted), and IgE levels. Qualitative phenotypes included: Pre-FEV1 greater or less than 80%, FEV1 greater or less than 12%, and IgE levels greater or less than 100 IU/ml.
To determine the magnitude of the interaction between genotype/haplotype, Pre-FEV1, ethnicity, and drug responsiveness, we used linear regression models (among subjects with asthma only) to test for an association between genotypes or haplotypes and bronchodilator responsiveness. In each model, the most common homozygous genotype served as the reference group with heterozygotes and the less common homozygote treated as two separate predictors. Other independent variables included in the model were Pre-FEV1, age, birthplace, sex, regular or as-needed use of 2-receptor agonist, use of long acting 2-receptor agonist, use of steroids, albuterol dose used for spirometry, environmental tobacco smoke exposure, and, for selected analysis, an interaction term between genotype and Pre-FEV1 (genotypePre-FEV1). This interaction term was considered because the response to albuterol and effect of genotype on the response may be more substantial in subjects with severe airway obstruction (low initial FEV1) as opposed to subjects with less airway obstruction at the time of pulmonary function testing. To correct for the effect of population stratification, cross-sectional analyses were also adjusted for individual admixture using 44 unlinked ancestry informative genetic markers (see online supplement). All cross-sectional analyses were performed using STATA 8.0 S/E statistical software (College Station, TX).
RESULTS
Allele Frequencies, Hardy-Weinberg Equilibrium, and LD
Of the 684 GALA Study trios, we obtained genotyping information for 667 trios, which were used for these analyses. The allele frequencies of eight 2AR SNPs in the combined (Puerto Rican and Mexican together), Puerto Rican, and Mexican probands are listed in Table 2. All SNPs were found to be in conformance with Hardy-Weinberg equilibrium with the exception of SNP eC47 in combined (p value = 0.03) and SNPs +252 and +523 in Mexican parents (p values 0.05 and 0.03, respectively). However, with eight SNPs tested in combined and separate populations, these deviations could result from multiple testing. We did not find the rare allele of SNP eC709 in any of our samples and therefore this SNP was excluded from further analyses. For purposes of consistency, the haplotypes discussed below continue to list eC709 position, although it was invariant in our populations.
Pairwise LD differed between Puerto Ricans and Mexicans among the seven SNPs (Figure 1). SNP pairs eC654/+46, eC47/+79, and +252/+523 were in very tight LD (r2 > 0.7) in both populations. LD was stronger in the Mexican population (mean r2 = 0.5) for SNPs eC654, +46, +252, and +523 than in the Puerto Rican population (mean r2 = 0.3). In contrast, LD was stronger in the Puerto Rican population (mean r2 = 0.22) for SNPs eC47, +46, and +79 than in the Mexican population (mean r2 = 0.11).
Association Analysis of 2AR with Asthma Severity and Bronchodilator Response
Single SNP analysis of asthma qualitative and quantitative traits.
Association results differed when analyzed in the combined population (Table 3) versus separately in the Puerto Rican and Mexican populations (Table 4). Therefore, all subsequent analyses were performed in each ethnic group separately. Family-based analyses were used to test for associations between individual SNPs and Pre-FEV1, FEV1, and log IgE. Among Puerto Ricans, significant associations (p = 0.002eC0.04) were found for SNPs eC654, eC47, +46, and +79 and Pre-FEV1 and FEV1 (Table 4). Among Mexicans, glutamine (Gln) 27 was modestly associated (p = 0.04) with FEV1 (Table 4). There were no significant associations between individual SNPs and IgE levels in either Puerto Ricans or Mexicans.
Because family-based analyses suggested an association between SNPs and bronchodilator response, we tested the clinical magnitude of the effect of the arginine (Arg) 16 glycine (Gly) (Arg16Gly) polymorphism on bronchodilator response by cross-sectional analysis in Puerto Ricans and Mexicans separately. Regression analysis showed a highly significant association between the number of Arg16 alleles and bronchodilator response in all Puerto Ricans with asthma (p = 0.002) and in those with Pre-FEV1 values less than 80% of predicted (p = 0.0001) Figure 2. This association was independent of age and albuterol dosage used for performing spirometry. Among all Puerto Rican subjects, the mean values of FEV1 stratified by codon 16 were as follows: individuals with Arg16/Arg16 (10.46 ± 2.44%), with Arg16/Gly16 (6.13 ± 0.74%), and with Gly16/Gly16 (3.85 ± 0.99%). When the Puerto Rican probands were stratified based on Pre-FEV1 values less than or greater than 80%, Arg16 was significantly associated with higher levels of bronchodilator response among Puerto Ricans with asthma with Pre-FEV1 values less than 80%: individuals with Arg16/Arg16 (24.9 ± 6.86%) (n = 23), with Arg16/Gly16 (8.8 ± 1.2%) (n = 70), and with Gly16/Gly16 (4.3 ± 2.2%) (n = 39). The 95% confidence interval for the difference of 20.6 ± 4.5% that we observed in bronchodilator drug responsiveness between Arg16/Arg16 and Gly16/Gly16 genotypes was 11.7eC29.5%. Using an additive model, we found a mean increase of approximately 3% in FEV1 values per each Arg16 allele (p = 0.001) in all Puerto Ricans with asthma and of approximately 8.2% in Puerto Ricans with Pre-FEV1 values less than 80% (p = 0.00001). In this model, the estimated variance in FEV1 explained by Pre-FEV1 and Arg16Gly is 13.7 and 13.5%, respectively, among Puerto Ricans with asthma with Pre-FEV1 values less than 80%. However, when the interaction term (genotype for SNP +46Pre-FEV1) was added to the model, Pre-FEV1 and Arg16Gly interacted to explain 44.4% of the variance in FEV1. This model suggested a substantially higher negative impact of Gly16 on FEV1 for subjects with more severe asthma as defined by lower values of Pre-FEV1. We did not see any association between Arg16Gly genotypes and FEV1 values among Puerto Ricans with asthma with Pre-FEV1 values greater than 80% (p = 0.87, Figure 2) and Mexicans with asthma with Pre-FEV1 values less than 80% (p = 0.72) or greater than 80% (p = 0.16). The results of these cross-sectional analyses did not change when corrected for population stratification (see online supplement for further details).
Haplotype-based Association Analysis
A total of seven, five, and six different haplotypes (frequency of > 1%) were observed in the Puerto Rican, Mexican, and both populations combined, respectively (Table 2). Haplotype associations differed between Puerto Ricans and Mexicans. However, haplotype results were consistent with single SNP results for Puerto Ricans and Mexicans, respectively, and analyses of the three most common haplotypes are presented in Table 5. Haplotype 1 (carrying the Arg16 allele) and haplotype 2 (carrying the Gly16 allele) were overtransmitted in Puerto Rican subjects with FEV1 values greater and less than 12%, respectively. We did not find any association between the haplotypes and FEV1 values among Mexicans (Table 5).
The three most common eight SNP haplotypes identified in the Puerto Rican and Mexican populations could be distinguished from each other using only two SNPs, (+46 and +79), which code for codon 16 and codon 27, respectively. Phase for the two SNP haplotypes was imputed for both Puerto Rican and Mexican probands using the Phamily Phase program (http://archimedes.well.ox.ac.uk/pise/phamily.html) (26). The two SNP haplotypes were assembled as pairs and six common haplotype pairs with greater than 1% frequency were observed in Puerto Ricans and Mexicans with asthma. Among Puerto Rican probands, the mean FEV1 values for the six common haplotype pairs are shown in Figure 3. Comparisons were made for haplotype pair Arg16/Gln27 homozygote, which had the highest FEV1, and other haplotype pairs.
Among Puerto Ricans with asthma with a Pre-FEV1 of less than 80%, two SNP haplotype pairs were significantly associated with FEV1 (p = 0.009). Subjects homozygous for Arg16/Gln27 had the highest FEV1 (24.4 ± 6.9%) and those with Gly16/Gln27 homozygotes had the lowest (3.9% ± 6.5%) (Figure 3). No association was observed between two SNP haplotypes pairs among Puerto Rican subjects with Pre-FEV1 values greater than 80% (p = 0.55) and Mexican subjects with Pre-FEV1 values either less than 80% (p = 0.21) or greater than 80% (p = 0.39).
DISCUSSION
Although most studies of racial/ethnic health disparities often categorize Latinos as a single ethnic group, this classification masks important ethnic-specific differences in health outcomes and therapeutic effectiveness. Despite this classification, it is well accepted that in all regions of the United States, Puerto Ricans have higher asthma morbidity and mortality rates than other Latino ethnic groups. This observation has not so far been satisfactorily explained by environmental or socioeconomic factors. Our results demonstrate that Puerto Ricans with asthma have an ethnic-specific genetic predisposition to more severe asthma. Specifically, our study of 2AR polymorphisms shows that the Arg16 allele is significantly associated with asthma severity (Pre-FEV1) and bronchodilator responsiveness ( FEV1) in Puerto Rican but not in Mexican subjects with asthma. The large size and clinical diversity of these two study populations have allowed us to address many outstanding questions relating to the clinical relevance of 2AR polymorphisms in terms of asthma severity and bronchodilator drug responsiveness. Most importantly, however, this comprehensive family-based study of 2AR polymorphisms demonstrates striking ethnic-specific pharmacogenetic differences between Puerto Rican and Mexican subjects with asthma. Despite the fact that Puerto Ricans and Mexicans are classified as the same population, "Hispanic or Latino," our results demonstrate that there are different patterns of LD, haplotypes, and genetic associations between these two ethnic groups. To our knowledge, this is the first report of an ethnic-specific pharmacogenetic association for subjects with asthma.
Martinez and colleagues previously demonstrated that when compared with Gly16 homozygotes, Arg16 homozygotes and Arg16Gly heterozygotes were, respectively, 5.3 and 2.3 times more likely to respond to albuterol, respectively. (8). Our results demonstrate that Arg16Gly is an important modifier of asthma severity and drug responsiveness among Puerto Ricans. These results corroborate and extend previous reports of associations between 2AR polymorphisms and asthma (8, 9, 27, 28). Among Puerto Ricans with asthma with low baseline FEV1 values, for whom the 2-agonist response is probably most crucial, there was approximately a 20% difference in FEV1 values between the Arg16 and Gly16 homozygotes, a highly significant and clinically important difference. The intermediate value of heterozygotes is consistent with an additive model, which corroborates a dose-response effect of this allele in Puerto Ricans.
We did not see a convincing association between 2AR genotype and drug responsiveness among Mexicans with asthma. The lack of a clear association may be due to several factors, including differences in LD patterns between Puerto Ricans and Mexicans. For example, the Arg16Gly allele may be in LD with another important risk allele in Puerto Ricans, but not in LD with this risk allele in Mexicans. There may also be ethnic-specific genetic and/or environmental modifiers that attenuate the effect of the Arg16Gly allele in Mexicans. By design, neither environmental nor cultural differences were a primary focus of the GALA study, and therefore, could be confounders.
Drysdale and coworkers demonstrated that haplotypes but not individual SNPs were associated with bronchodilator responsiveness (12). Our results differ in that both individual SNPs and haplotypes modulate asthma severity and bronchodilator drug responsiveness. Specifically, Drysdale and coworkers demonstrated that Drysdale haplotype 6 (corresponding to GALA haplotype 2) was associated with increased measures of bronchodilator drug responsiveness, whereas Drysdale haplotype 4 (corresponding to GALA haplotype 1) was associated with lower measures of bronchodilator drug responsiveness. In contrast, our results demonstrate that GALA haplotype 1 (Drysdale haplotype 4) was associated with increased measures of bronchodilator responsiveness, whereas GALA haplotype 2 (Drysdale haplotype 6) was associated with lower levels of bronchodilator responsiveness. There are several important differences between our study and the study of Drysdale and coworkers, however, which might help to explain differences in our respective results. Drysdale and coworkers studied 121 unrelated Caucasian subjects with asthma who all had FEV1 values greater than 12% from baseline. Our study was a family-based study and included probands with asthma from two populations, Puerto Ricans (n = 393) and Mexicans (n = 274). Unlike case-control and case-only genetic association studies, family-based studies are robust to the effects of population stratification (16). It is unlikely that inadequate sample size precluded the replication of the previously reported association. We analyzed data from 667 family trios for a total of 2,001 individuals, which exceeds the sample size (n = 121) that Drysdale and coworkers used for their study. Although it is possible that the limited sample size precluded Drysdale and coworkers from finding associations between individual SNPs and asthma related phenotypes, there might be other potential reasons why our results differed from those reported previously. One important difference is that we studied different populations, and racial/ethnic-specific differences in environmental or genetic risk factors may account for the observed differences (17). Drysdale and coworkers performed their study exclusively in Caucasians from the United States, whereas we studied Latinos recruited from Mexico City, Puerto Rico, and the continental United States.
In contrast to the results of Drysdale and coworkers, our results corroborate recently published results from the Childhood Asthma Management Program (CAMP), a large family-based study primarily consisting of subjects with mild asthma (10). Similar to the GALA Study, the CAMP Study was a family-based study and included genotype data of 8 2AR SNPs in 700 complete trios (mother, father and child with asthma); 516 white, 70 African American, 56 Hispanic, and 56 listed as "other." An important difference between the CAMP and GALA Studies is that we directly compared two Latino ethnic groups consisting of subjects with mild and moderate-severe asthma. Results from GALA and CAMP differ in that CAMP demonstrated association of Pre-FEV1 values of less than 80% with SNPs eC654, eC47, +46, and +79 and association of bronchodilator responsiveness only with SNP +523. The most significant individual haplotype results were for GALA haplotype 2 (corresponding to CAMP haplotype 3), which had lower bronchodilator response measures in both CAMP and GALA Mexicans.
We have previously demonstrated that Puerto Ricans with asthma have significantly more airway obstruction, more health care utilization, and lower bronchodilator response to albuterol than Mexicans with asthma (19). Although our results do not entirely explain the significant disparities in asthma prevalence, morbidity, and mortality among Latino ethnic groups, our study raises several provocative issues regarding ethnic-specific pharmacogenetic differences between these two populations. This large family-based study consists of two well characterized, ethnically diverse populations of Puerto Ricans and Mexicans with asthma from four geographically diverse locations. Studies performed in these populations may have important public health implications because Latino children represent the largest demographic group of all U.S. children (1) and because asthma morbidity and mortality are highest amongst Puerto Ricans and the lowest amongst Mexicans for a fourfold difference in asthma burden between these two ethnic groups (3, 4). The fact that Puerto Ricans and Mexicans have the highest and lowest asthma prevalence, morbidity, and mortality, respectively, underscores the clinical significance and public health implications of these findings.
Our study demonstrates that the two largest Latino ethnic groups in the United States have different patterns of LD, haplotypes, and pharmacogenetic associations, and, therefore, should be considered as separate groups in future drug trials and pharmacogenetic studies of asthma. Ethnic-specific pharmacogenetic differences among populations with asthma have not been previously reported and thus merit further investigation.
Acknowledgments
The authors thank the families and the subjects for their participation and the numerous health care providers and community clinics for their support and participation in the GALA Study. In addition, they thank the primary clinical centers of the investigators, participating community clinics, and hospitals: La Clinica de La Raza, Oakland, CA; UCSF-Children's Hospital of Oakland Pediatric Clinical Research Center, Oakland, CA; General Clinical Research Center, SFGH, San Francisco, CA; Alliance Medical Center, Healdsburg, CA; Santa Clara Valley Medical Center, San Josee, CA; Fair Oaks Family Health Center, Redwood City, CA; Clinica de Salud del Valle de Salinas, Salinas, CA; Natividad Medical Center, Salinas, CA; Asthma Education and Management Program, Community Medical Centers, Fresno, CA; Diagnostic Health Centers of: Corozal, Naranjito, Catano, Orocovis, Barranquitas and San Antonio Hospital of Mayaguez; Morris Heights Health Center, Bronx, NY; Paterson School Board, Paterson, NJ; Eva's Clinic, Paterson, NJ; Lincoln Medical Center, Bronx; Harlem Hospital Center, NY; and the Metropolitan Hospital Center, New York, NY. They also thank Carmen Jimenez, Yannett Marcano, Pedro Yapor, M.D., Alma Ortiz, M.D., Lisandra Perez, M.D., Daniel Navarro, M.D., and Sheila Gonzalez, M.D., for their assistance with recruitment and Pui Yan Kwok, M.D., Ph.D., for his assistance with genotyping.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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Lung Biology Center, San Francisco General Hospital, San Francisco, California
San Juan Veterans Administration Medical Center, University of Puerto Rico School of Medicine
Pediatric Pulmonary Program of San Juan, San Juan, Puerto Rico
Brigham and Women's Hospital, Boston, Massachusetts
The Harlem Lung Center, Harlem Hospital and Columbia University, New York, New York
Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico
ABSTRACT
Background: In the United States, Puerto Ricans and Mexicans have the highest and lowest asthma prevalence, morbidity, and mortality, respectively. Ethnic-specific differences in the response to drug treatment may contribute to differences in disease outcomes. Genetic variants at the 2-adrenergic receptor (2AR) may modify asthma severity and albuterol responsiveness. We tested the association of 2AR genotypes with asthma severity and bronchodilator response to albuterol in Puerto Ricans and Mexicans with asthma. Methods: We used both family-based and cross-sectional tests of association with 8 2AR single nucleotide polymorphisms in 684 Puerto Rican and Mexican families. Regression analyses were used to determine the interaction between genotype, asthma severity, and bronchodilator drug responsiveness. Results: Among Puerto Ricans with asthma, the arginine (Arg) 16 allele was associated with greater bronchodilator response using both family-based and cross-sectional tests (p = 0.00001eC0.01). We found a strong interaction of baseline FEV1 with the Arg16Glycine (Gly) polymorphism in predicting bronchodilator response. Among Puerto Ricans with asthma with baseline FEV1 < 80% of predicted, but not in those with FEV1 > 80%, there was a very strong association between the Arg16 genotype and greater bronchodilator responsiveness. No association was observed between Arg16Gly genotypes and drug responsiveness among Mexicans with asthma. Conclusions: Ethnic-specific pharmacogenetic differences exist between Arg16Gly genotypes, asthma severity, and bronchodilator response in Puerto Ricans and Mexicans with asthma. These findings underscore the need for additional research on racial/ethnic differences in asthma morbidity and drug responsiveness.
Key Words: asthma genetics 2-adrenergic receptor gene Latinos pharmacogenetic
Studies of racial/ethnic health disparities often define populations based on U.S. census definitions, which categorize Latinos as a single ethnic group. Latinos are the largest minority population in the United States, and Latino children represent the largest demographic group of all U.S. children (1). Although the terms "Hispanic" or "Latino" American describe a common language and cultural heritage, these terms do not refer to race, uniform ethnicity, or a common genetic background. The two largest Latino ethnic groups in the United States, Mexicans (63%) and Puerto Ricans (11%) (1), are genetically complex and comprised of various proportions of Native American, African, and European genetic origins (2). The relative proportions of these ancestral populations to the contemporary Latino gene pool make each Latino ethnic group genetically unique (2).
According to U.S. vital statistics, asthma prevalence, morbidity, and mortality are the highest among Puerto Ricans and the lowest among Mexicans for a total of a fourfold difference in asthma burden between these two ethnic groups (3, 4). Although there are many potential explanations for this observation, including environmental and socioeconomic factors, one potential explanation is that the genetic predisposition to asthma or to greater asthma severity differs among subgroups within the Latino population. These subgroups may have genetic variants, which may explain the variation in asthma severity and response to drug therapy between Latino ethnic groups.
Albuterol, a short-acting 2-adrenergic receptor (2AR) agonist, is the most commonly prescribed treatment for asthma worldwide (5). 2AR mediates physiologic responses, including bronchial smooth muscle relaxation (bronchodilation), vasodilatation, and lipolysis (6). Genetic variations at the 2AR gene may play a role in the observed ethnic-specific differences in asthma severity and bronchodilator drug responsiveness.
Polymorphisms in the 2AR gene have been inconsistently associated with varying degrees of bronchodilatory response to -agonists (7eC12) and with tachyphylaxis (13, 14). In one study, specific haplotypes, as opposed to individual single nucleotide polymorphisms (SNPs), were significantly associated with drug responsiveness (12). Consequently, there is no clear consensus regarding the extent to which genetic variants in the 2AR gene may influence asthma severity or bronchodilator drug responsiveness.
We used a family-based genetic association study to determine whether polymorphisms (SNPs and haplotypes) in the 2AR gene are associated with ethnic-specific differences in asthma severity and bronchodilator response in Puerto Rican and Mexican subjects with asthma participating in the Genetics of Asthma in Latino Americans (GALA) Study. We selected a family-based approach because it removes the effect of population stratification, a potentially important source of confounding (15). Spurious associations between genetic variants and phenotypes may arise as a result of population stratification when noneCfamily-based approaches are used (16). Moreover, racial and ethnic background can influence genetic variation and genotypic relative risk (17). Some of the results of this study have been previously reported in the form of an abstract (18).
METHODS
Study Participants
Recruitment and pulmonary function testing are described elsewhere and in detail in the online supplement (19). Briefly, 684 subjects with asthma (probands) and their biological parents were enrolled in the San Francisco Bay Area (SF), New York City (NY), Puerto Rico (PR), and Mexico City (MX). Clinical characteristics of all probands are shown in Table 1. Ethnicity was self-reported. Probands were enrolled only if both biological parents and all four biological grandparents were of Puerto Rican (for the NY and PR sites) or Mexican (for the SF and MX sites) ethnicity.
Pulmonary Function Tests and IgE Measurements
Spirometry was performed according to American Thoracic Society (ATS) standards (20). Pulmonary function test results are shown in Table 1 and are expressed as a percentage of the predicted normal value using age-adjusted prediction equations from Hankinson (21).
Probands with asthma were classified as having "mild" or "moderate-severe" asthma based on their baseline FEV1 expressed as percent of predicted or Pre-FEV1 levels. Subjects with a Pre-FEV1 greater or less than 80% of predicted were categorized as having "mild" and "moderate-severe" asthma, respectively. A quantitative measure of bronchodilator responsiveness was calculated as FEV1, which is the relative percent change in Pre-FEV1 after albuterol administration. Total plasma IgE was measured in duplicate using Uni-Cap technology (Pharmacia, Kalamazoo, MI).
Genotyping
Genotyping details are described further in the online supplement. Eight 2AR SNPs (eC709, eC654, eC47, +46, +79, +252, +491, and +523) were genotyped and used to assign haplotypes.
Statistical Analysis
Statistical analyses are further described in the online supplement. Mendelian inconsistencies were identified using PEDCHECK (http://watson.hgen.pitt.edu/register/docs/pedcheck.html) (22). Hardy-Weinberg equilibrium was calculated by means of 2 goodness-of-fit tests. The degree of linkage disequilibrium (LD) was estimated by using the r2 statistic (23).
Family-based tests (24) and family-based tests for associating haplotypes (25) were used to assess the association between individual SNPs and haplotypes, respectively, with quantitative measures of asthma-related traits. Quantitative phenotypes included: asthma severity (defined by Pre-FEV1), bronchodilator responsiveness (defined by FEV1, relative % of predicted), and IgE levels. Qualitative phenotypes included: Pre-FEV1 greater or less than 80%, FEV1 greater or less than 12%, and IgE levels greater or less than 100 IU/ml.
To determine the magnitude of the interaction between genotype/haplotype, Pre-FEV1, ethnicity, and drug responsiveness, we used linear regression models (among subjects with asthma only) to test for an association between genotypes or haplotypes and bronchodilator responsiveness. In each model, the most common homozygous genotype served as the reference group with heterozygotes and the less common homozygote treated as two separate predictors. Other independent variables included in the model were Pre-FEV1, age, birthplace, sex, regular or as-needed use of 2-receptor agonist, use of long acting 2-receptor agonist, use of steroids, albuterol dose used for spirometry, environmental tobacco smoke exposure, and, for selected analysis, an interaction term between genotype and Pre-FEV1 (genotypePre-FEV1). This interaction term was considered because the response to albuterol and effect of genotype on the response may be more substantial in subjects with severe airway obstruction (low initial FEV1) as opposed to subjects with less airway obstruction at the time of pulmonary function testing. To correct for the effect of population stratification, cross-sectional analyses were also adjusted for individual admixture using 44 unlinked ancestry informative genetic markers (see online supplement). All cross-sectional analyses were performed using STATA 8.0 S/E statistical software (College Station, TX).
RESULTS
Allele Frequencies, Hardy-Weinberg Equilibrium, and LD
Of the 684 GALA Study trios, we obtained genotyping information for 667 trios, which were used for these analyses. The allele frequencies of eight 2AR SNPs in the combined (Puerto Rican and Mexican together), Puerto Rican, and Mexican probands are listed in Table 2. All SNPs were found to be in conformance with Hardy-Weinberg equilibrium with the exception of SNP eC47 in combined (p value = 0.03) and SNPs +252 and +523 in Mexican parents (p values 0.05 and 0.03, respectively). However, with eight SNPs tested in combined and separate populations, these deviations could result from multiple testing. We did not find the rare allele of SNP eC709 in any of our samples and therefore this SNP was excluded from further analyses. For purposes of consistency, the haplotypes discussed below continue to list eC709 position, although it was invariant in our populations.
Pairwise LD differed between Puerto Ricans and Mexicans among the seven SNPs (Figure 1). SNP pairs eC654/+46, eC47/+79, and +252/+523 were in very tight LD (r2 > 0.7) in both populations. LD was stronger in the Mexican population (mean r2 = 0.5) for SNPs eC654, +46, +252, and +523 than in the Puerto Rican population (mean r2 = 0.3). In contrast, LD was stronger in the Puerto Rican population (mean r2 = 0.22) for SNPs eC47, +46, and +79 than in the Mexican population (mean r2 = 0.11).
Association Analysis of 2AR with Asthma Severity and Bronchodilator Response
Single SNP analysis of asthma qualitative and quantitative traits.
Association results differed when analyzed in the combined population (Table 3) versus separately in the Puerto Rican and Mexican populations (Table 4). Therefore, all subsequent analyses were performed in each ethnic group separately. Family-based analyses were used to test for associations between individual SNPs and Pre-FEV1, FEV1, and log IgE. Among Puerto Ricans, significant associations (p = 0.002eC0.04) were found for SNPs eC654, eC47, +46, and +79 and Pre-FEV1 and FEV1 (Table 4). Among Mexicans, glutamine (Gln) 27 was modestly associated (p = 0.04) with FEV1 (Table 4). There were no significant associations between individual SNPs and IgE levels in either Puerto Ricans or Mexicans.
Because family-based analyses suggested an association between SNPs and bronchodilator response, we tested the clinical magnitude of the effect of the arginine (Arg) 16 glycine (Gly) (Arg16Gly) polymorphism on bronchodilator response by cross-sectional analysis in Puerto Ricans and Mexicans separately. Regression analysis showed a highly significant association between the number of Arg16 alleles and bronchodilator response in all Puerto Ricans with asthma (p = 0.002) and in those with Pre-FEV1 values less than 80% of predicted (p = 0.0001) Figure 2. This association was independent of age and albuterol dosage used for performing spirometry. Among all Puerto Rican subjects, the mean values of FEV1 stratified by codon 16 were as follows: individuals with Arg16/Arg16 (10.46 ± 2.44%), with Arg16/Gly16 (6.13 ± 0.74%), and with Gly16/Gly16 (3.85 ± 0.99%). When the Puerto Rican probands were stratified based on Pre-FEV1 values less than or greater than 80%, Arg16 was significantly associated with higher levels of bronchodilator response among Puerto Ricans with asthma with Pre-FEV1 values less than 80%: individuals with Arg16/Arg16 (24.9 ± 6.86%) (n = 23), with Arg16/Gly16 (8.8 ± 1.2%) (n = 70), and with Gly16/Gly16 (4.3 ± 2.2%) (n = 39). The 95% confidence interval for the difference of 20.6 ± 4.5% that we observed in bronchodilator drug responsiveness between Arg16/Arg16 and Gly16/Gly16 genotypes was 11.7eC29.5%. Using an additive model, we found a mean increase of approximately 3% in FEV1 values per each Arg16 allele (p = 0.001) in all Puerto Ricans with asthma and of approximately 8.2% in Puerto Ricans with Pre-FEV1 values less than 80% (p = 0.00001). In this model, the estimated variance in FEV1 explained by Pre-FEV1 and Arg16Gly is 13.7 and 13.5%, respectively, among Puerto Ricans with asthma with Pre-FEV1 values less than 80%. However, when the interaction term (genotype for SNP +46Pre-FEV1) was added to the model, Pre-FEV1 and Arg16Gly interacted to explain 44.4% of the variance in FEV1. This model suggested a substantially higher negative impact of Gly16 on FEV1 for subjects with more severe asthma as defined by lower values of Pre-FEV1. We did not see any association between Arg16Gly genotypes and FEV1 values among Puerto Ricans with asthma with Pre-FEV1 values greater than 80% (p = 0.87, Figure 2) and Mexicans with asthma with Pre-FEV1 values less than 80% (p = 0.72) or greater than 80% (p = 0.16). The results of these cross-sectional analyses did not change when corrected for population stratification (see online supplement for further details).
Haplotype-based Association Analysis
A total of seven, five, and six different haplotypes (frequency of > 1%) were observed in the Puerto Rican, Mexican, and both populations combined, respectively (Table 2). Haplotype associations differed between Puerto Ricans and Mexicans. However, haplotype results were consistent with single SNP results for Puerto Ricans and Mexicans, respectively, and analyses of the three most common haplotypes are presented in Table 5. Haplotype 1 (carrying the Arg16 allele) and haplotype 2 (carrying the Gly16 allele) were overtransmitted in Puerto Rican subjects with FEV1 values greater and less than 12%, respectively. We did not find any association between the haplotypes and FEV1 values among Mexicans (Table 5).
The three most common eight SNP haplotypes identified in the Puerto Rican and Mexican populations could be distinguished from each other using only two SNPs, (+46 and +79), which code for codon 16 and codon 27, respectively. Phase for the two SNP haplotypes was imputed for both Puerto Rican and Mexican probands using the Phamily Phase program (http://archimedes.well.ox.ac.uk/pise/phamily.html) (26). The two SNP haplotypes were assembled as pairs and six common haplotype pairs with greater than 1% frequency were observed in Puerto Ricans and Mexicans with asthma. Among Puerto Rican probands, the mean FEV1 values for the six common haplotype pairs are shown in Figure 3. Comparisons were made for haplotype pair Arg16/Gln27 homozygote, which had the highest FEV1, and other haplotype pairs.
Among Puerto Ricans with asthma with a Pre-FEV1 of less than 80%, two SNP haplotype pairs were significantly associated with FEV1 (p = 0.009). Subjects homozygous for Arg16/Gln27 had the highest FEV1 (24.4 ± 6.9%) and those with Gly16/Gln27 homozygotes had the lowest (3.9% ± 6.5%) (Figure 3). No association was observed between two SNP haplotypes pairs among Puerto Rican subjects with Pre-FEV1 values greater than 80% (p = 0.55) and Mexican subjects with Pre-FEV1 values either less than 80% (p = 0.21) or greater than 80% (p = 0.39).
DISCUSSION
Although most studies of racial/ethnic health disparities often categorize Latinos as a single ethnic group, this classification masks important ethnic-specific differences in health outcomes and therapeutic effectiveness. Despite this classification, it is well accepted that in all regions of the United States, Puerto Ricans have higher asthma morbidity and mortality rates than other Latino ethnic groups. This observation has not so far been satisfactorily explained by environmental or socioeconomic factors. Our results demonstrate that Puerto Ricans with asthma have an ethnic-specific genetic predisposition to more severe asthma. Specifically, our study of 2AR polymorphisms shows that the Arg16 allele is significantly associated with asthma severity (Pre-FEV1) and bronchodilator responsiveness ( FEV1) in Puerto Rican but not in Mexican subjects with asthma. The large size and clinical diversity of these two study populations have allowed us to address many outstanding questions relating to the clinical relevance of 2AR polymorphisms in terms of asthma severity and bronchodilator drug responsiveness. Most importantly, however, this comprehensive family-based study of 2AR polymorphisms demonstrates striking ethnic-specific pharmacogenetic differences between Puerto Rican and Mexican subjects with asthma. Despite the fact that Puerto Ricans and Mexicans are classified as the same population, "Hispanic or Latino," our results demonstrate that there are different patterns of LD, haplotypes, and genetic associations between these two ethnic groups. To our knowledge, this is the first report of an ethnic-specific pharmacogenetic association for subjects with asthma.
Martinez and colleagues previously demonstrated that when compared with Gly16 homozygotes, Arg16 homozygotes and Arg16Gly heterozygotes were, respectively, 5.3 and 2.3 times more likely to respond to albuterol, respectively. (8). Our results demonstrate that Arg16Gly is an important modifier of asthma severity and drug responsiveness among Puerto Ricans. These results corroborate and extend previous reports of associations between 2AR polymorphisms and asthma (8, 9, 27, 28). Among Puerto Ricans with asthma with low baseline FEV1 values, for whom the 2-agonist response is probably most crucial, there was approximately a 20% difference in FEV1 values between the Arg16 and Gly16 homozygotes, a highly significant and clinically important difference. The intermediate value of heterozygotes is consistent with an additive model, which corroborates a dose-response effect of this allele in Puerto Ricans.
We did not see a convincing association between 2AR genotype and drug responsiveness among Mexicans with asthma. The lack of a clear association may be due to several factors, including differences in LD patterns between Puerto Ricans and Mexicans. For example, the Arg16Gly allele may be in LD with another important risk allele in Puerto Ricans, but not in LD with this risk allele in Mexicans. There may also be ethnic-specific genetic and/or environmental modifiers that attenuate the effect of the Arg16Gly allele in Mexicans. By design, neither environmental nor cultural differences were a primary focus of the GALA study, and therefore, could be confounders.
Drysdale and coworkers demonstrated that haplotypes but not individual SNPs were associated with bronchodilator responsiveness (12). Our results differ in that both individual SNPs and haplotypes modulate asthma severity and bronchodilator drug responsiveness. Specifically, Drysdale and coworkers demonstrated that Drysdale haplotype 6 (corresponding to GALA haplotype 2) was associated with increased measures of bronchodilator drug responsiveness, whereas Drysdale haplotype 4 (corresponding to GALA haplotype 1) was associated with lower measures of bronchodilator drug responsiveness. In contrast, our results demonstrate that GALA haplotype 1 (Drysdale haplotype 4) was associated with increased measures of bronchodilator responsiveness, whereas GALA haplotype 2 (Drysdale haplotype 6) was associated with lower levels of bronchodilator responsiveness. There are several important differences between our study and the study of Drysdale and coworkers, however, which might help to explain differences in our respective results. Drysdale and coworkers studied 121 unrelated Caucasian subjects with asthma who all had FEV1 values greater than 12% from baseline. Our study was a family-based study and included probands with asthma from two populations, Puerto Ricans (n = 393) and Mexicans (n = 274). Unlike case-control and case-only genetic association studies, family-based studies are robust to the effects of population stratification (16). It is unlikely that inadequate sample size precluded the replication of the previously reported association. We analyzed data from 667 family trios for a total of 2,001 individuals, which exceeds the sample size (n = 121) that Drysdale and coworkers used for their study. Although it is possible that the limited sample size precluded Drysdale and coworkers from finding associations between individual SNPs and asthma related phenotypes, there might be other potential reasons why our results differed from those reported previously. One important difference is that we studied different populations, and racial/ethnic-specific differences in environmental or genetic risk factors may account for the observed differences (17). Drysdale and coworkers performed their study exclusively in Caucasians from the United States, whereas we studied Latinos recruited from Mexico City, Puerto Rico, and the continental United States.
In contrast to the results of Drysdale and coworkers, our results corroborate recently published results from the Childhood Asthma Management Program (CAMP), a large family-based study primarily consisting of subjects with mild asthma (10). Similar to the GALA Study, the CAMP Study was a family-based study and included genotype data of 8 2AR SNPs in 700 complete trios (mother, father and child with asthma); 516 white, 70 African American, 56 Hispanic, and 56 listed as "other." An important difference between the CAMP and GALA Studies is that we directly compared two Latino ethnic groups consisting of subjects with mild and moderate-severe asthma. Results from GALA and CAMP differ in that CAMP demonstrated association of Pre-FEV1 values of less than 80% with SNPs eC654, eC47, +46, and +79 and association of bronchodilator responsiveness only with SNP +523. The most significant individual haplotype results were for GALA haplotype 2 (corresponding to CAMP haplotype 3), which had lower bronchodilator response measures in both CAMP and GALA Mexicans.
We have previously demonstrated that Puerto Ricans with asthma have significantly more airway obstruction, more health care utilization, and lower bronchodilator response to albuterol than Mexicans with asthma (19). Although our results do not entirely explain the significant disparities in asthma prevalence, morbidity, and mortality among Latino ethnic groups, our study raises several provocative issues regarding ethnic-specific pharmacogenetic differences between these two populations. This large family-based study consists of two well characterized, ethnically diverse populations of Puerto Ricans and Mexicans with asthma from four geographically diverse locations. Studies performed in these populations may have important public health implications because Latino children represent the largest demographic group of all U.S. children (1) and because asthma morbidity and mortality are highest amongst Puerto Ricans and the lowest amongst Mexicans for a fourfold difference in asthma burden between these two ethnic groups (3, 4). The fact that Puerto Ricans and Mexicans have the highest and lowest asthma prevalence, morbidity, and mortality, respectively, underscores the clinical significance and public health implications of these findings.
Our study demonstrates that the two largest Latino ethnic groups in the United States have different patterns of LD, haplotypes, and pharmacogenetic associations, and, therefore, should be considered as separate groups in future drug trials and pharmacogenetic studies of asthma. Ethnic-specific pharmacogenetic differences among populations with asthma have not been previously reported and thus merit further investigation.
Acknowledgments
The authors thank the families and the subjects for their participation and the numerous health care providers and community clinics for their support and participation in the GALA Study. In addition, they thank the primary clinical centers of the investigators, participating community clinics, and hospitals: La Clinica de La Raza, Oakland, CA; UCSF-Children's Hospital of Oakland Pediatric Clinical Research Center, Oakland, CA; General Clinical Research Center, SFGH, San Francisco, CA; Alliance Medical Center, Healdsburg, CA; Santa Clara Valley Medical Center, San Josee, CA; Fair Oaks Family Health Center, Redwood City, CA; Clinica de Salud del Valle de Salinas, Salinas, CA; Natividad Medical Center, Salinas, CA; Asthma Education and Management Program, Community Medical Centers, Fresno, CA; Diagnostic Health Centers of: Corozal, Naranjito, Catano, Orocovis, Barranquitas and San Antonio Hospital of Mayaguez; Morris Heights Health Center, Bronx, NY; Paterson School Board, Paterson, NJ; Eva's Clinic, Paterson, NJ; Lincoln Medical Center, Bronx; Harlem Hospital Center, NY; and the Metropolitan Hospital Center, New York, NY. They also thank Carmen Jimenez, Yannett Marcano, Pedro Yapor, M.D., Alma Ortiz, M.D., Lisandra Perez, M.D., Daniel Navarro, M.D., and Sheila Gonzalez, M.D., for their assistance with recruitment and Pui Yan Kwok, M.D., Ph.D., for his assistance with genotyping.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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