Pharmacogenetic Differences in Response to Albuterol between Puerto Ricans and Mexicans with Asthma

Pharmacogenetic Differences in Response to Albuterolbetween Puerto Ricans and Mexicans with AsthmaShweta Choudhry, Ngim Ung, Pedro C. Avila, Elad Ziv, Sylvette Nazario, Jesus Casal, Alfonso Torres,Jennifer D. Gorman, Keyan Salari, Jose R. Rodriguez-Santana, Monica Toscano, Jody Senter Sylvia,MariaElena Alioto, Richard A. Castro, Michael Salazar, Ivan Gomez, Joanne K. Fagan, Jorge Salas,Suzanne Clark, Craig Lilly, Henry Matallana, Moises Selman, Rocio Chapela, Dean Sheppard,Scott T. Weiss, Jean G. Ford, Homer A. Boushey, Jeffrey M. Drazen, William Rodriguez-Cintron,Edwin K. Silverman, and Esteban Gonzalez Burchard on behalf of the Genetics of Asthma in LatinoAmericans (GALA) Study

University of California, San Francisco; Lung Biology Center, San Francisco General Hospital, San Francisco, California; San Juan VeteransAdministration Medical Center, University of Puerto Rico School of Medicine; Pediatric Pulmonary Program of San Juan, San Juan, PuertoRico; Brigham and Women’s Hospital, Boston, Massachusetts; The Harlem Lung Center, Harlem Hospital and Columbia University, New York,New York; and Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico

Background: In the United States, Puerto Ricans and Mexicans havethe highest and lowest asthma prevalence, morbidity, and mortal-ity, respectively. Ethnic-specific differences in the response to drugtreatment may contribute to differences in disease outcomes. Ge-netic variants at the �2-adrenergic receptor (�2AR) may modifyasthma severity and albuterol responsiveness. We tested the associ-ation of �2AR genotypes with asthma severity and bronchodilatorresponse to albuterol in Puerto Ricans and Mexicans with asthma.Methods: We used both family-based and cross-sectional tests ofassociation with 8 �2AR single nucleotide polymorphisms in 684Puerto Rican and Mexican families. Regression analyses were usedto determine the interaction between genotype, asthma severity,and bronchodilator drug responsiveness. Results: Among PuertoRicans with asthma, the arginine (Arg) 16 allele was associated withgreater bronchodilator response using both family-based and cross-sectional tests (p � 0.00001–0.01). We found a strong interactionof baseline FEV1 with the Arg16Glycine (Gly) polymorphism in pre-dicting bronchodilator response. Among Puerto Ricans with asthmawith baseline FEV1 � 80% of predicted, but not in those with FEV1 �

80%, there was a very strong association between the Arg16 geno-type and greater bronchodilator responsiveness. No association wasobserved between Arg16Gly genotypes and drug responsivenessamong Mexicans with asthma. Conclusions: Ethnic-specific pharma-cogenetic differences exist between Arg16Gly genotypes, asthmaseverity, and bronchodilator response in Puerto Ricans and Mexi-cans with asthma. These findings underscore the need for additionalresearch on racial/ethnic differences in asthma morbidity and drugresponsiveness.

(Received in original form September 28, 2004; accepted in final form November 8, 2004)

Supported by National Institutes of Health grants: K23 HL04464, HL07185, andGM61390; National Center on Minority Health and Health Disparities, ExtramuralClinical Research Loan Repayment Program for Individuals from DisadvantagedBackgrounds, 2001–2003, to EGB; HL51823, HL074204, 3M01RR000083–38S30488, HL56443 and HL51831 to the Asthma Clinical Research Network;SFGH General Clinical Research Center M01RR00083–41, U01-HL 65899, UCSF-Children’s Hospital of Oakland Pediatric Clinical Research Center (M01 RR01271),Oakland, CA, Sandler Center for Basic Research in Asthma; and the Sandler FamilyFoundation.

Correspondence and requests for reprints should be addressed to: Esteban GonzalezBurchard, M.D., University of California, San Francisco, San Francisco, CA 94143-0833. E-mail: [email protected]

This article has an online supplement, which is accessible from this issue’s tableof contents at www.atsjournals.org

Am J Respir Crit Care Med Vol 171. pp 563–570, 2005Originally Published in Press as DOI: 10.1164/rccm.200409-1286OC on November 24, 2004Internet address: www.atsjournals.org

Keywords: asthma genetics; �2-adrenergic receptor gene; Latinos; phar-macogenetic

Studies of racial/ethnic health disparities often define popula-tions based on U.S. census definitions, which categorize Latinosas a single ethnic group. Latinos are the largest minority popula-tion in the United States, and Latino children represent thelargest demographic group of all U.S. children (1). Althoughthe terms “Hispanic” or “Latino” American describe a commonlanguage and cultural heritage, these terms do not refer to race,uniform ethnicity, or a common genetic background. The twolargest Latino ethnic groups in the United States, Mexicans(63%) and Puerto Ricans (11%) (1), are genetically complexand comprised of various proportions of Native American, Afri-can, and European genetic origins (2). The relative proportionsof these ancestral populations to the contemporary Latino genepool make each Latino ethnic group genetically unique (2).

According to U.S. vital statistics, asthma prevalence, morbid-ity, and mortality are the highest among Puerto Ricans and thelowest among Mexicans for a total of a fourfold difference inasthma burden between these two ethnic groups (3, 4). Althoughthere are many potential explanations for this observation, in-cluding environmental and socioeconomic factors, one potentialexplanation is that the genetic predisposition to asthma or togreater asthma severity differs among subgroups within the Lat-ino population. These subgroups may have genetic variants,which may explain the variation in asthma severity and responseto drug therapy between Latino ethnic groups.

Albuterol, a short-acting �2-adrenergic receptor (�2AR) ago-nist, is the most commonly prescribed treatment for asthmaworldwide (5). �2AR mediates physiologic responses, includingbronchial smooth muscle relaxation (bronchodilation), vasodila-tation, and lipolysis (6). Genetic variations at the �2AR genemay play a role in the observed ethnic-specific differences inasthma severity and bronchodilator drug responsiveness.

Polymorphisms in the �2AR gene have been inconsistentlyassociated with varying degrees of bronchodilatory response to�-agonists (7–12) and with tachyphylaxis (13, 14). In one study,specific haplotypes, as opposed to individual single nucleotidepolymorphisms (SNPs), were significantly associated with drugresponsiveness (12). Consequently, there is no clear consensusregarding the extent to which genetic variants in the �2AR genemay influence asthma severity or bronchodilator drug respon-siveness.

We used a family-based genetic association study to determinewhether polymorphisms (SNPs and haplotypes) in the �2ARgene are associated with ethnic-specific differences in asthma

564 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 171 2005

TABLE 1. CHARACTERISTICS AND PULMONARY FUNCTION RESULTS OF GENETICS OFASTHMA IN LATINO AMERICANS STUDY OF PROBANDS WITH ASTHMA INCLUDEDIN THE �2AR ANALYSIS

Comparison by Ethnicity

Puerto Ricans Mexicans(n � 393) (n � 274) p Value

CharacteristicAge, yr 12.0 (10:15) 13.1 (11:19) � 0.001Sex, % male 55.9 54.1 0.66BMI, kg/m2 21.2 (17:26) 23.8 (20:28) � 0.000Serum IgE, IU/ml 258.5 (92:628) 270.0 (99:615) 0.92Moderate-severe asthma, % 30.8 40.5 � 0.02

Baseline spirometryPre-FEV1, % of pred 83 (74:93) 90 (77:100) � 0.0001Pre-FEV1 � 80%, % 33.9 28.8 0.17

Bronchodilator responsiveness� FEV1, relative % pred 5.0 (0.6:10) 7.4 (4:12) � 0.0001IgE levels, IU/ml 258.5 (92:628) 270.0 (99:615) 0.91

Definition of abbreviations: BMI � body mass index; � FEV1 � the relative percent change in Pre-FEV1 after albuterol administration;pred � predicted; Pre-FEV1 � baseline FEV1 expressed as percent of predicted.

Values are expressed as median (25th:75th percentile), and were missing for some subjects. Analysis was done by Mann-Whitneyrank test.

severity and bronchodilator response in Puerto Rican and Mexi-can subjects with asthma participating in the Genetics of Asthmain Latino Americans (GALA) Study. We selected a family-basedapproach because it removes the effect of population stratifica-tion, a potentially important source of confounding (15). Spuri-ous associations between genetic variants and phenotypes mayarise as a result of population stratification when non–family-based approaches are used (16). Moreover, racial and ethnicbackground can influence genetic variation and genotypic rela-tive risk (17). Some of the results of this study have been pre-viously reported in the form of an abstract (18).

METHODS

Study Participants

Recruitment and pulmonary function testing are described elsewhereand in detail in the online supplement (19). Briefly, 684 subjects withasthma (probands) and their biological parents were enrolled in theSan Francisco Bay Area (SF), New York City (NY), Puerto Rico (PR),and Mexico City (MX). Clinical characteristics of all probands areshown in Table 1. Ethnicity was self-reported. Probands were enrolledonly if both biological parents and all four biological grandparents wereof Puerto Rican (for the NY and PR sites) or Mexican (for the SF andMX 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 inTable 1 and are expressed as a percentage of the predicted normalvalue 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 ofpredicted or Pre-FEV1 levels. Subjects with a Pre-FEV1 greater orless than 80% of predicted were categorized as having “mild” and“moderate-severe” asthma, respectively. A quantitative measure of bron-chodilator responsiveness was calculated as � FEV1, which is the relativepercent change in Pre-FEV1 after albuterol administration. Totalplasma IgE was measured in duplicate using Uni-Cap technology (Phar-macia, Kalamazoo, MI).

Genotyping

Genotyping details are described further in the online supplement.Eight �2AR SNPs (�709, �654, �47, �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-Weinbergequilibrium was calculated by means of �2 goodness-of-fit tests. The degreeof linkage disequilibrium (LD) was estimated by using the r 2 statistic (23).

Family-based tests (24) and family-based tests for associating haplo-types (25) were used to assess the association between individual SNPsand haplotypes, respectively, with quantitative measures of asthma-related traits. Quantitative phenotypes included: asthma severity (de-fined by Pre-FEV1), bronchodilator responsiveness (defined by � FEV1,relative % of predicted), and IgE levels. Qualitative phenotypes in-cluded: Pre-FEV1 greater or less than 80%, � FEV1 greater or less than12%, 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 linearregression models (among subjects with asthma only) to test for an associa-tion between genotypes or haplotypes and bronchodilator responsiveness.In each model, the most common homozygous genotype served as thereference group with heterozygotes and the less common homozygotetreated as two separate predictors. Other independent variables in-cluded 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 to-bacco smoke exposure, and, for selected analysis, an interaction termbetween genotype and Pre-FEV1 (genotype*Pre-FEV1). This interac-tion term was considered because the response to albuterol and effectof genotype on the response may be more substantial in subjects withsevere airway obstruction (low initial FEV1) as opposed to subjectswith less airway obstruction at the time of pulmonary function testing.To correct for the effect of population stratification, cross-sectionalanalyses were also adjusted for individual admixture using 44 unlinkedancestry informative genetic markers (see online supplement). All cross-sectional analyses were performed using STATA 8.0 S/E statisticalsoftware (College Station, TX).

RESULTS

Allele Frequencies, Hardy-Weinberg Equilibrium, and LD

Of the 684 GALA Study trios, we obtained genotyping informa-tion for 667 trios, which were used for these analyses. The allelefrequencies of eight �2AR SNPs in the combined (Puerto Ricanand Mexican together), Puerto Rican, and Mexican probandsare listed in Table 2. All SNPs were found to be in conformance

Choudhry, Ung, Avila, et al.: �2AR, Asthma, Latinos, and Pharmacogenetics 565

TABLE 2. ALLELE FREQUENCIES FOR �2-ADRENERGIC RECEPTOR SINGLE NUCLEOTIDE POLYMORPHISMS AND HAPLOTYPES(� 1% FREQUENCY) IN COMBINED (PUERTO RICAN AND MEXICAN), PUERTO RICAN, AND MEXICAN PROBANDS. THEALLELE FREQUENCIES FROM THE CHILDHOOD ASTHMA MANAGEMENT PROGRAM AND DRYSDALE STUDIES ARE LISTEDFOR COMPARISON

Frequency (%)

Drysdale DrysdaleAmino Acid Allele/ PR � Mex PR Mex CAMP* Drysdale African Hispanic-

SNP Position Alleles/Haplotype Change & Codon Haplotype Probands Probands Probands Parents Whites Americans Latinos

�709 C/A — C 100 100 100 98.0�654 A/G — A 37.9 37.0 39.1 36.0�47 C/T Arg19/Cys19 C 23.2 27.4 17.6 37.0�46 A/G Arg16/Gly16 A 43.9 45.2 42.1 40.0�79 G/C Glu27/Gln27 G 22.9 26.5 17.8 37.0�252 A/G — A 32.0 26.2 40.6 23.0�491 C/T Thr164/Ile164 C 98.5 98.0 99.2 99.0�523 C/A — C 70.0 75.5 62.3 80.0Haplotype 1 C A T A C G C C — 1 36.8 34.9 39.7 35.4 33.0 29.7 40.0Haplotype 2 C G T G C A C A — 2 26.8 21.9 34.2 18.2 13.2 31.3 13.3Haplotype 3 C G C G G G C C — 3 22.7 26.8 16.5 36.3 48.3 6.3 26.7Haplotype 4 C G T A C G C C — 4 7.1 8.9 4.2 0.7 29.7 10.0Haplotype 5 C G T G C A C C — 5 1.7 1.8 1.6 0.7 0.0 3.3Haplotype 6 C G T G C A T A — 6 1.3 1.6 N.P. 1.0 1.6 3.3Haplotype 7 C G C G C G C C — 7 N.P. 1.3 N.P. N.P. N.P. N.P.

Definition of abbreviations: Arg � arginine; CAMP � Childhood Asthma Management Program data; Cys � cysteine; Drysdale � Drysdale and coworkers data; Gln �

glutamine; Glu � glutamic acid; Gly � glycine; Ile � isoleucine; Mex � Mexican; N.P. � not present; PR � Puerto Rican; SNP � single nucleotide polymorphism;Thr � threonine.

* Includes combined allele frequency of whites, African Americans, Hispanics, and “others.”

with Hardy-Weinberg equilibrium with the exception of SNP�47 in combined (p value � 0.03) and SNPs �252 and �523 inMexican 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 notfind the rare allele of SNP �709 in any of our samples andtherefore this SNP was excluded from further analyses. For pur-poses of consistency, the haplotypes discussed below continueto list �709 position, although it was invariant in our populations.

Pairwise LD differed between Puerto Ricans and Mexicansamong the seven SNPs (Figure 1). SNP pairs �654/�46, �47/�79,and �252/�523 were in very tight LD (r 2 � 0.7) in both popula-

Figure 1. Linkage disequilibrium(LD) between �2-adrenergic recep-tor (�2AR) single nucleotide poly-morphisms (SNPs) in Puerto Ricanand Mexican subjects with asthma.The degree of LD was estimatedby using the r 2 statistic. The resultsare color coded as shown by thecolor bar.

tions. LD was stronger in the Mexican population (mean r 2 � 0.5)for SNPs –654, �46, �252, and �523 than in the Puerto Ricanpopulation (mean r 2 � 0.3). In contrast, LD was stronger in thePuerto Rican population (mean r 2 � 0.22) for SNPs �47, �46,and �79 than in the Mexican population (mean r 2 � 0.11).

Association Analysis of �2AR with Asthma Severity andBronchodilator Response

Single SNP analysis of asthma qualitative and quantitative traits.Association results differed when analyzed in the combined pop-ulation (Table 3) versus separately in the Puerto Rican andMexican populations (Table 4). Therefore, all subsequent analy-


TABLE 3. FAMILY-BASED ASSOCIATION ANALYSIS OF QUALITATIVE AND QUANTITATIVE ASTHMA TRAITS WITH�2-ADRENERGIC RECEPTOR SINGLE NUCLEOTIDE POLYMORPHISMS IN THE COMBINED POPULATION

SNPs �654 (A/G) �47 (C/T) �46 (A/G) �79 (G/C) �252 (A/G) �491 (C/T) �523 (C/A)

Amino Acid and Codon Arg19Cys Arg16Gly Glu27Gln

p Value

Quantitative traitsPre-FEV1 0.024 (G) 0.045 (G)� FEV1, relative % pred 0.024 (A) 0.011 (A)Log10 IgE level 0.047 (C)

Qualitative traitsPre-FEV1 � 80% predPre-FEV1 � 80% pred� FEV1 relative % pred � 12% 0.018 (A)� FEV1 relative % pred � 12% 0.038 (A)IgE level � 100 IU/mlIgE level � 100 IU/ml

Definition of abbreviations: Arg � arginine; � FEV1 � the relative percent change in Pre-FEV1 after albuterol administration; Gln � glutamine; Glu � glutamic acid;Gly � glycine; pred � predicted; Pre-FEV1 � baseline FEV1 expressed as percent of predicted; SNPs � single nucleotide polymorphisms.

For quantitative traits, the allele in parentheses was associated with a higher value of the trait. For qualitative traits, the allele in parentheses was overtransmitted toprobands with asthma. All other p values are not shown because they were not significant.

ses were performed in each ethnic group separately. Family-based analyses were used to test for associations between individ-ual SNPs and Pre-FEV1, � FEV1, and log IgE. Among PuertoRicans, significant associations (p � 0.002–0.04) were found forSNPs �654, �47, �46, and �79 and Pre-FEV1 and � FEV1 (Ta-ble 4). Among Mexicans, glutamine (Gln) 27 was modestly asso-ciated (p � 0.04) with � FEV1 (Table 4). There were no signifi-cant associations between individual SNPs and IgE levels ineither Puerto Ricans or Mexicans.

Because family-based analyses suggested an association be-tween SNPs and bronchodilator response, we tested the clinicalmagnitude 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 be-tween the number of Arg16 alleles and bronchodilator responsein all Puerto Ricans with asthma (p � 0.002) and in those withPre-FEV1 values less than 80% of predicted (p � 0.0001)Figure 2. This association was independent of age and albuteroldosage used for performing spirometry. Among all Puerto Rican

TABLE 4. FAMILY-BASED ASSOCIATION ANALYSIS OF QUALITATIVE AND QUANTITATIVE ASTHMA TRAITS WITH�2-ADRENERGIC RECEPTOR SINGLE NUCLEOTIDE POLYMORPHISMS IN PUERTO RICANS* AND MEXICANS†

SNPs �654 (A/G) �47 (C/T) �46 (A/G) �79 (G/C) �252 (A/G) �491 (C/T) �523 (C/A)

Amino Acid and Codon Arg19Cys Arg16Gly Glu27Gln

p Value

Quantitative traitsPre-FEV1 0.035 (G)* 0.048 (G)*� FEV1, relative % pred 0.003 (A)* 0.006 (A)* 0.043 (C) †

Log10 IgE levelQualitative traits

Pre-FEV1 � 80% predPre-FEV1 � 80% pred� FEV1 relative % pred � 12% 0.006 (A)* 0.044 (A)*� FEV1 relative % pred � 12%IgE level � 100 IU/mlIgE level � 100 IU/ml

For definition of abbreviations see Table 3.Only significant associations (p values � 0.05) are shown.For quantitative traits, the allele in the parenthesis was associated with higher value of the trait. For qualitative traits, the allele in the parenthesis was overtransmitted

to probands with asthma. All other p values for both ethnic groups are not shown because they were not significant.

subjects, the mean values of � FEV1 stratified by codon 16 wereas follows: individuals with Arg16/Arg16 (10.46 2.44%), withArg16/Gly16 (6.13 0.74%), and with Gly16/Gly16 (3.85 0.99%). When the Puerto Rican probands were stratified basedon Pre-FEV1 values less than or greater than 80%, Arg16 wassignificantly associated with higher levels of bronchodilator re-sponse among Puerto Ricans with asthma with Pre-FEV1 valuesless than 80%: individuals with Arg16/Arg16 (24.9 6.86%)(n � 23), with Arg16/Gly16 (8.8 1.2%) (n � 70), and withGly16/Gly16 (4.3 2.2%) (n � 39). The 95% confidence intervalfor the difference of 20.6 4.5% that we observed in broncho-dilator drug responsiveness between Arg16/Arg16 and Gly16/Gly16 genotypes was 11.7–29.5%. Using an additive model, wefound a mean increase of approximately 3% in � FEV1 valuesper each Arg16 allele (p � 0.001) in all Puerto Ricans withasthma and of approximately 8.2% in Puerto Ricans with Pre-FEV1 values less than 80% (p � 0.00001). In this model, theestimated variance in � FEV1 explained by Pre-FEV1 andArg16Gly is 13.7 and 13.5%, respectively, among Puerto Ricanswith asthma with Pre-FEV1 values less than 80%. However,


Figure 2. Responsiveness to �2AR agonist expressed as � FEV1 (relative% predicted) in different codon 16 genotypes among all Puerto Ricansubjects with asthma (solid bars) and Puerto Rican subjects with asthmawith baseline Pre-FEV1 values less than (open bars) and greater than(hatched bars) 80% of predicted.

when the interaction term (genotype for SNP �46*Pre-FEV1)was added to the model, Pre-FEV1 and Arg16Gly interacted toexplain 44.4% of the variance in � FEV1. This model suggesteda substantially higher negative impact of Gly16 on � FEV1 forsubjects with more severe asthma as defined by lower values ofPre-FEV1. We did not see any association between Arg16Glygenotypes and � FEV1 values among Puerto Ricans with asthmawith Pre-FEV1 values greater than 80% (p � 0.87, Figure 2) andMexicans 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 populationstratification (see online supplement for further details).

Haplotype-based Association Analysis

A total of seven, five, and six different haplotypes (frequencyof � 1%) were observed in the Puerto Rican, Mexican, andboth populations combined, respectively (Table 2). Haplotypeassociations differed between Puerto Ricans and Mexicans. How-ever, haplotype results were consistent with single SNP results

TABLE 5. FAMILY-BASED ASSOCIATION ANALYSIS OF QUALITATIVE AND QUANTITATIVEASTHMA TRAITS WITH THE THREE MOST COMMON �2 ADRENERGIC RECEPTOR HAPLOTYPESIN THE COMBINED PUERTO RICAN � MEXICAN, PUERTO RICAN, AND MEXICAN POPULATIONS

Haplotype 1 Haplotype 2 Haplotype 3

C A T A C G C C C G T G C A C A C G C G G G C C

p Value p Value p Value

PR � Mex PR Mex PR � Mex PR Mex PR � Mex PR Mex

Quantitative traitsPre-FEV1

� FEV1, relative % predLog 10 IgE level

Qualitative traitsPre-FEV1 � 80% predicted 0.029Pre-FEV1 � 80% predicted� FEV1, relative % pred � 12% 0.003� FEV1, relative % pred � 12% 0.023IgE level � 100 IU/ml 0.038IgE level � 100 IU/ml 0.015 0.028

Definition of abbreviations: � FEV1 � the relative percent change in Pre-FEV1 after albuterol administration; Mex � Mexican;PR � Puerto Rican; pred � predicted; Pre-FEV1 � baseline FEV1 expressed as percent of predicted.

For qualitative traits, the p values represent haplotypes overtransmitted to probands with asthma. Only significant associations(p values � 0.05) are shown; all other p values are not shown because they were not significant.

for Puerto Ricans and Mexicans, respectively, and analyses ofthe three most common haplotypes are presented in Table 5.Haplotype 1 (carrying the Arg16 allele) and haplotype 2 (car-rying the Gly16 allele) were overtransmitted in Puerto Ricansubjects with � FEV1 values greater and less than 12%, respec-tively. We did not find any association between the haplotypesand � FEV1 values among Mexicans (Table 5).

The three most common eight SNP haplotypes identified in thePuerto Rican and Mexican populations could be distinguished fromeach other using only two SNPs, (�46 and �79), which code forcodon 16 and codon 27, respectively. Phase for the two SNP haplo-types was imputed for both Puerto Rican and Mexican probandsusing the Phamily Phase program (http://archimedes.well.ox.ac.uk/pise/phamily.html) (26). The two SNP haplotypes were assembledas pairs and six common haplotype pairs with greater than 1%frequency were observed in Puerto Ricans and Mexicans withasthma. Among Puerto Rican probands, the mean � FEV1 valuesfor the six common haplotype pairs are shown in Figure 3. Compari-sons 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 lessthan 80%, two SNP haplotype pairs were significantly associatedwith � FEV1 (p � 0.009). Subjects homozygous for Arg16/Gln27had 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 pairsamong Puerto Rican subjects with Pre-FEV1 values greater than80% (p � 0.55) and Mexican subjects with Pre-FEV1 valueseither less than 80% (p � 0.21) or greater than 80% (p � 0.39).

DISCUSSION

Although most studies of racial/ethnic health disparities oftencategorize Latinos as a single ethnic group, this classificationmasks important ethnic-specific differences in health outcomesand therapeutic effectiveness. Despite this classification, it is wellaccepted that in all regions of the United States, Puerto Ricanshave higher asthma morbidity and mortality rates than otherLatino ethnic groups. This observation has not so far been satis-factorily explained by environmental or socioeconomic factors.


Figure 3. Responsiveness to �2AR agonist expressed as � FEV1 (relative% predicted) in different codon 16/codon 27 haplotype pairs amongall Puerto Rican subjects with asthma (solid bars) and Puerto Ricansubjects with asthma with baseline Pre-FEV1 values less than (open bars)and greater than (hatched bars) 80% of predicted.

Our results demonstrate that Puerto Ricans with asthma havean ethnic-specific genetic predisposition to more severe asthma.Specifically, our study of �2AR polymorphisms shows that theArg16 allele is significantly associated with asthma severity (Pre-FEV1) and bronchodilator responsiveness (� FEV1) in PuertoRican but not in Mexican subjects with asthma. The large sizeand clinical diversity of these two study populations have allowedus to address many outstanding questions relating to the clinicalrelevance of �2AR polymorphisms in terms of asthma severityand bronchodilator drug responsiveness. Most importantly, how-ever, this comprehensive family-based study of �2AR polymor-phisms demonstrates striking ethnic-specific pharmacogeneticdifferences between Puerto Rican and Mexican subjects withasthma. Despite the fact that Puerto Ricans and Mexicans areclassified as the same population, “Hispanic or Latino,” ourresults demonstrate that there are different patterns of LD, haplo-types, and genetic associations between these two ethnic groups.To our knowledge, this is the first report of an ethnic-specificpharmacogenetic association for subjects with asthma.

Martinez and colleagues previously demonstrated that whencompared with Gly16 homozygotes, Arg16 homozygotes andArg16Gly heterozygotes were, respectively, 5.3 and 2.3 timesmore likely to respond to albuterol, respectively. (8). Our resultsdemonstrate that Arg16Gly is an important modifier of asthmaseverity and drug responsiveness among Puerto Ricans. Theseresults corroborate and extend previous reports of associationsbetween �2AR polymorphisms and asthma (8, 9, 27, 28). AmongPuerto Ricans with asthma with low baseline FEV1 values, forwhom the �2-agonist response is probably most crucial, therewas approximately a 20% difference in � FEV1 values betweenthe Arg16 and Gly16 homozygotes, a highly significant and clini-cally important difference. The intermediate value of hetero-zygotes is consistent with an additive model, which corroboratesa dose-response effect of this allele in Puerto Ricans.

We did not see a convincing association between �2AR geno-type and drug responsiveness among Mexicans with asthma. Thelack of a clear association may be due to several factors, includingdifferences in LD patterns between Puerto Ricans and Mexicans.For example, the Arg16Gly allele may be in LD with anotherimportant risk allele in Puerto Ricans, but not in LD with thisrisk allele in Mexicans. There may also be ethnic-specific genetic

and/or environmental modifiers that attenuate the effect of theArg16Gly allele in Mexicans. By design, neither environmentalnor cultural differences were a primary focus of the GALAstudy, and therefore, could be confounders.

Drysdale and coworkers demonstrated that haplotypes butnot individual SNPs were associated with bronchodilator respon-siveness (12). Our results differ in that both individual SNPsand haplotypes modulate asthma severity and bronchodilatordrug responsiveness. Specifically, Drysdale and coworkers dem-onstrated that Drysdale haplotype 6 (corresponding to GALAhaplotype 2) was associated with increased measures of bron-chodilator drug responsiveness, whereas Drysdale haplotype 4(corresponding to GALA haplotype 1) was associated with lowermeasures of bronchodilator drug responsiveness. In contrast,our results demonstrate that GALA haplotype 1 (Drysdale hap-lotype 4) was associated with increased measures of bronchodila-tor responsiveness, whereas GALA haplotype 2 (Drysdale hap-lotype 6) was associated with lower levels of bronchodilatorresponsiveness. There are several important differences betweenour 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 sub-jects with asthma who all had � FEV1 values greater than 12%from baseline. Our study was a family-based study and includedprobands with asthma from two populations, Puerto Ricans (n �393) and Mexicans (n � 274). Unlike case-control and case-onlygenetic association studies, family-based studies are robust tothe effects of population stratification (16). It is unlikely thatinadequate sample size precluded the replication of the pre-viously reported association. We analyzed data from 667 familytrios for a total of 2,001 individuals, which exceeds the samplesize (n � 121) that Drysdale and coworkers used for their study.Although it is possible that the limited sample size precludedDrysdale and coworkers from finding associations between indi-vidual SNPs and asthma related phenotypes, there might beother potential reasons why our results differed from those re-ported previously. One important difference is that we studieddifferent populations, and racial/ethnic-specific differences inenvironmental or genetic risk factors may account for the ob-served differences (17). Drysdale and coworkers performed theirstudy exclusively in Caucasians from the United States, whereaswe studied Latinos recruited from Mexico City, Puerto Rico,and the continental United States.

In contrast to the results of Drysdale and coworkers, our resultscorroborate recently published results from the Childhood AsthmaManagement Program (CAMP), a large family-based study pri-marily consisting of subjects with mild asthma (10). Similar tothe GALA Study, the CAMP Study was a family-based studyand included genotype data of 8 �2AR SNPs in 700 complete trios(mother, father and child with asthma); 516 white, 70 AfricanAmerican, 56 Hispanic, and 56 listed as “other.” An importantdifference between the CAMP and GALA Studies is that wedirectly compared two Latino ethnic groups consisting of subjectswith mild and moderate-severe asthma. Results from GALA andCAMP differ in that CAMP demonstrated association of Pre-FEV1 values of less than 80% with SNPs �654, �47, �46, and�79 and association of bronchodilator responsiveness only withSNP �523. The most significant individual haplotype resultswere for GALA haplotype 2 (corresponding to CAMP haplo-type 3), which had lower bronchodilator response measures inboth CAMP and GALA Mexicans.

We have previously demonstrated that Puerto Ricans withasthma have significantly more airway obstruction, more healthcare utilization, and lower bronchodilator response to albuterolthan Mexicans with asthma (19). Although our results do notentirely explain the significant disparities in asthma prevalence,


morbidity, and mortality among Latino ethnic groups, our studyraises several provocative issues regarding ethnic-specific phar-macogenetic differences between these two populations. Thislarge family-based study consists of two well characterized, ethni-cally diverse populations of Puerto Ricans and Mexicans withasthma from four geographically diverse locations. Studies per-formed in these populations may have important public healthimplications because Latino children represent the largest demo-graphic group of all U.S. children (1) and because asthma mor-bidity and mortality are highest amongst Puerto Ricans and thelowest amongst Mexicans for a fourfold difference in asthmaburden between these two ethnic groups (3, 4). The fact thatPuerto Ricans and Mexicans have the highest and lowest asthmaprevalence, morbidity, and mortality, respectively, underscoresthe clinical significance and public health implications of thesefindings.

Our study demonstrates that the two largest Latino ethnicgroups in the United States have different patterns of LD, haplo-types, and pharmacogenetic associations, and, therefore, shouldbe considered as separate groups in future drug trials and phar-macogenetic studies of asthma. Ethnic-specific pharmacogeneticdifferences among populations with asthma have not been pre-viously reported and thus merit further investigation.

Conflict of Interest Statement : S.C. does not have a financial relationship with acommercial entity that has an interest in the subject of this manuscript; N.U. doesnot have a financial relationship with a commercial entity that has an interestin the subject of this manuscript; P.C.A. received $1,500 in lecture fees fromGlaxoSmithKline (GSK) in 2003, $1,500 in consulting fees from Aventis in 2004,and will receive $215,000 in research grants from GSK in 2004–2005; E.Z. doesnot have a financial relationship with a commercial entity that has an interest inthe subject of this manuscript; S.N. does not have a financial relationship with acommercial entity that has an interest in the subject of this manuscript; J.C. doesnot have a financial relationship with a commercial entity that has an interest inthe subject of this manuscript; A.T. does not have a financial relationship with acommercial entity that has an interest in the subject of this manuscript; J.D.G.will be receiving $195,000 from 2004–2007 as a recipient of the Pfizer ScholarsGrants in Clinical Epidemiology; K.S. does not have a financial relationship witha commercial entity that has an interest in the subject of this manuscript; J.R.R.-S.does not have a financial relationship with a commercial entity that has an interestin the subject of this manuscript; M.T. does not have a financial relationship witha commercial entity that has an interest in the subject of this manuscript; J.S.S.does not have a financial relationship with a commercial entity that has an interestin the subject of this manuscript; M.A. does not have a financial relationship witha commercial entity that has an interest in the subject of this manuscript; R.A.C.does not have a financial relationship with a commercial entity that has an interestin the subject of this manuscript; M.S. does not have a financial relationshipwith a commercial entity that has an interest in the subject of this manuscript;I.G. does not have a financial relationship with a commercial entity that has aninterest in the subject of this manuscript; J.K.F. does not have a financial relation-ship with a commercial entity that has an interest in the subject of this manuscript;J.S. does not have a financial relationship with a commercial entity that has aninterest in the subject of this manuscript; S.C. does not have a financial relationshipwith a commercial entity that has an interest in the subject of this manuscript;C.L. received support from Merck for lectures in 2003–2004; H.M. does not havea financial relationship with a commercial entity that has an interest in the subjectof this manuscript; M.S. does not have a financial relationship with a commercialentity that has an interest in the subject of this manuscript; R.C. does not havea financial relationship with a commercial entity that has an interest in the subjectof this manuscript; D.S. does not have a financial relationship with a commercialentity that has an interest in the subject of this manuscript; S.T.W. received agrant for $900,065 (Asthma Policy Modeling Study) from AstraZeneca from 1997to 2003, has been a coinvestigator on a grant from Boehringer Ingelheim toinvestigate a chronic obstructive pulmonary disease (COPD) natural history model,which began in 2003 but he received no funds for his involvement in this project,has been an advisor to the TENOR Study for Genentech for which he received$5,000 for 2003–2004, received a grant from Glaxo-Wellcome for $500,000for genomic equipment from 2000 to 2003, and was a consultant for RochePharmaceuticals in 2000 but received no financial remuneration for this consul-tancy; J.G.F. does not have a financial relationship with a commercial entity thathas an interest in the subject of this manuscript; H.A.B. received payment fromGSK for service on the Scientific Committee for a clinical trial (the GOAL Study),received payments as honoraria for lectures in 2003, and was responsible for theRegents of the University of California receiving from GSK $485,000 for researchconducted under his direction between 1999 and 2001, unrestricted gifts forresearch and training of $8,323 in 2002, $4,500 in 2003, $2,371 in 2004, and$7,390 for his service to the Scientific Advisory Board for this study; J.M.D. doesnot have a financial relationship with a commercial entity that has an interest inthe subject of this manuscript; W.R.-C. does not have a financial relationship with

a commercial entity that has an interest in the subject of this manuscript; E.K.S.received grant support and honoraria from GSK for a study of COPD geneticsand received a $500 Speaker Fee from Wyeth for a talk on COPD genetics; E.G.B.does not have a financial relationship with a commercial entity that has an interestin the subject of this manuscript.

Acknowledgment : The authors thank the families and the subjects for their partici-pation and the numerous health care providers and community clinics for theirsupport and participation in the GALA Study. In addition, they thank the primaryclinical centers of the investigators, participating community clinics, and hospitals:La Clinica de La Raza, Oakland, CA; UCSF-Children’s Hospital of Oakland PediatricClinical Research Center, Oakland, CA; General Clinical Research Center, SFGH,San Francisco, CA; Alliance Medical Center, Healdsburg, CA; Santa Clara ValleyMedical Center, San Jose, 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 Cen-ter, Bronx, NY; Paterson School Board, Paterson, NJ; Eva’s Clinic, Paterson, NJ;Lincoln Medical Center, Bronx; Harlem Hospital Center, NY; and the MetropolitanHospital Center, New York, NY. They also thank Carmen Jimenez, Yannett Mar-cano, 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 PuiYan Kwok, M.D., Ph.D., for his assistance with genotyping.

References

1. US Census Bureau U.S.DoC. United States Census 2000: United StatesDepartment of Commerce, 2000.

2. Hanis CL, Hewett-Emmett D, Bertin TK, Schull WJ. Origins of U.S.Hispanics: implications for diabetes. Diabetes Care 1991;14:618–627.

3. Carter-Pokras OD, Gergen PJ. Reported asthma among Puerto Rican,Mexican-American, and Cuban children: 1982 through 1984. Am JPublic Health 1993;83:580–582.

4. Homa DM, Mannino DM, Lara M. Asthma mortality in U.S. Hispanicsof Mexican, Puerto Rican, and Cuban heritage, 1990–1995. Am J RespirCrit Care Med 2000;161:504–509.

5. Nelson HS. Beta-adrenergic bronchodilators. N Engl J Med 1995;333:499–506.

6. Insel PA. Seminars in medicine of the Beth Israel Hospital, Boston:adrenergic receptors–evolving concepts and clinical implications. NEngl J Med 1996;334:580–585.

7. Ohe M, Munakata M, Hizawa N, Itoh A, Doi I, Yamaguchi E, HommaY, Kawakami Y. Beta 2 adrenergic receptor gene restriction fragmentlength polymorphism and bronchial asthma. Thorax 1995;50:353–359.

8. Martinez FD, Graves PE, Baldini M, Solomon S, Erickson R. Associationbetween genetic polymorphisms of the beta2-adrenoceptor and re-sponse to albuterol in children with and without a history of wheezing.J Clin Invest 1997;100:3184–3188.

9. Lima JJ, Thomason DB, Mohamed MH, Eberle LV, Self TH, JohnsonJA. Impact of genetic polymorphisms of the beta2-adrenergic receptoron albuterol bronchodilator pharmacodynamics. Clin Pharmacol Ther1999;65:519–525.

10. Silverman EK, Kwiatkowski DJ, Sylvia JS, Lazarus R, Drazen JM, LangeC, Laird NM, Weiss ST. Family-based association analysis of beta2-adrenergic receptor polymorphisms in the childhood asthma manage-ment program. J Allergy Clin Immunol 2003;112:870–876.

11. Lipworth BJ, Hall IP, Tan S, Aziz I, Coutie W. Effects of genetic polymor-phism on ex vivo and in vivo function of beta2-adrenoceptors in asth-matic patients. Chest 1999;115:324–328.

12. Drysdale CM, McGraw DW, Stack CB, Stephens JC, Judson RS, Nanda-balan K, Arnold K, Ruano G, Liggett SB. Complex promoter andcoding region beta 2-adrenergic receptor haplotypes alter receptorexpression and predict in vivo responsiveness. Proc Natl Acad SciUSA 2000;97:10483–10488.

13. Tan KS, McFarlane LC, Lipworth BJ. Effects of oral and inhaled cortico-steroid on lymphocyte beta2- adrenoceptor function in asthmatic pa-tients. Br J Clin Pharmacol 1997;44:565–568.

14. Israel E, Drazen JM, Liggett SB, Boushey HA, Cherniack RM, ChinchilliVM, Cooper DM, Fahy JV, Fish JE, Ford JG, et al. The effect ofpolymorphisms of the beta(2)-adrenergic receptor on the response toregular use of albuterol in asthma. Am J Respir Crit Care Med 2000;162:75–80.

15. Spielman RS, McGinnis RE, Ewens WJ. Transmission test for linkagedisequilibrium: the insulin gene region and insulin-dependent diabetesmellitus (IDDM). Am J Hum Genet 1993;52:506–516.

16. Ziv E, Burchard EG. Human population structure and genetic associationstudies. Pharmacogenomics 2003;4:431–441.

17. Burchard EG, Ziv E, Coyle N, Gomez SL, Tang H, Karter AJ, MountainJL, Perez-Stable EJ, Sheppard D, Risch N. The importance of race


and ethnic background in biomedical research and clinical practice.N Engl J Med 2003;348:1170–1175.

18. Burchard E, Choudhry S, Ung N, Avila PC, Ziv E, Nazario S, CasalJ, Torres A, Salari K, Rodriguez Santana J, et al. Pharmacogeneticdifferences in response to bronchodilators between Puerto Rican andMexican asthmatics [abstract]. American Society of Human Genetics,Toronto, Canada, 2004. Vol. 54th Annual.

19. Burchard EG, Avila PC, Nazario S, Casal J, Torres A, Rodriguez-SantanaJR, Toscano M, Sylvia JS, Alioto M, Salazar M, et al. Lower bronchodi-lator responsiveness in Puerto Rican than in Mexican asthmatic sub-jects. Am J Respir Crit Care Med 2003.

20. American Thoracic Society. Standardization of spirometry, 1994 update.Am J Respir Crit Care Med 1995; 152:1107–1136.

21. Hankinson JL, Odencrantz JR, Fedan KB. Spirometric reference valuesfrom a sample of the general U.S. population. Am J Respir Crit CareMed 1999;159:179–187.

22. O’Connell JR, Weeks DE. PedCheck: a program for identification of

genotype incompatibilities in linkage analysis. Am J Hum Genet 1998;63:259–266.

23. Ott J. Analysis of human genetic linkage. In: The Johns Hopkins Univer-sity Press, 3rd ed. Baltimore, 1999.

24. Laird NM, Horvath S, Xu X. Implementing a unified approach to family-based tests of association. Genet Epidemiol 2000;19:S36–S42.

25. Horvath S, Xu X, Lake SL, Silverman EK, Weiss ST, Laird NM. Family-based tests for associating haplotypes with general phenotype data:application to asthma genetics. Genetic Epidemiol 2004;26(1):61–69.

26. Stephens M, Smith NJ, Donnelly P. A new statistical method for haplo-types reconstruction from population data. Am J Hum Genet 2001;68:978–989.

27. Reihsaus E, Innis M, MacIntyre N, Liggett SB. Mutations in the geneencoding for the �2-adrenergic receptor in normal and asthmatic sub-jects. Am J Respir Cell Mol Biol 1993;8:334–339.

28. Summerhill E, Leavitt SA, Gidley H, Parry R, Solway J, Ober C. Beta(2)-adrenergic receptor Arg16/Arg16 genotype is associated with reducedlung function, but not with asthma, in the Hutterites. Am J RespirCrit Care Med 2000;162:599–602.

Pharmacogenetic Differences in Response to Albuterol between Puerto Ricans and Mexicans with Asthma

Documents

Transcript of Pharmacogenetic Differences in Response to Albuterol between Puerto Ricans and Mexicans with Asthma