INTRODUCTION

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Inhaled ₂-agonists, established for first-line bronchodilator therapy, are the most widely prescribed drugs in the initial treatment of patients with asthma (1). It is well recognized that there are significant interindividual variations in bronchodilator response (BDR), due presumably to both genetic and environmental factors. However, although twin and family studies suggest that individual responses to other pharmacological agents including aspirin, dicumarol, and phenylbutazone are primarily genetic rather than environmental (2), such studies have not been performed for BDR. Several potent drugs are now available to treat the growing number of cases of asthma in the United States. Albuterol, an intermediate-acting (3-6 h) bronchodilator (1), offers immediate relief to those who are suffering an asthma attack. However, the effectiveness of this treatment varies considerably across patients. A more thorough understanding into the causes of differential BDR would allow better patient-specific treatment and could ultimately lead to alternative therapies. Here, we set out to study the familial correlation of BDR as a first step in providing a theoretical basis for the genetic studies of BDR that are already taking place (5, 6).

To investigate the degree of familial clustering for BDR, we studied short-term response to albuterol in 1,161 index families with asthma from Anqing, China. Each four-subject family unit included a mother, a father, a first offspring aged 8-20, and subsequent offspring also aged 8-20. This design allowed us to investigate the relationships of BDR among first-degree relatives and, specifically, to determine whether the BDR of the mother, father, and first offspring each had a differential impact on the BDR of the subsequent offspring.

	METHODS

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Study Subjects

Family members of 1161 index families with asthma were recruited from eight counties (Zongyang, Huining, Qianshan, Tongcheng, Taihu, Wangjiang, Susong, and Yuexi) in Anqing, China. A detailed description of the study design and sample selection has been presented elsewhere (7). Selection criteria for the index family with asthma required (1) a never-smoking index patient with asthma, aged 8-20; (2) at least one never-smoking sibling of the index case with asthma, aged 8-20; (3) the availability of both parents; and (4) no more than one parent with a history of asthma.

Procedures

The survey was conducted between July 1, 1994 and January 26, 1998 by faculty members from Anhui Medical University and a team of locally hired interviewers fluent in the regional dialect. A letter explaining the study was sent to each eligible family. Local officials and health centers arranged for the interviews and measurements to take place at a central office at times convenient to the participants. The following procedures were carried out in accordance with the NIH Collaborative Agreement on Asthma Genetics: (1) a standardized questionnaire (modified ATS-DLD) assessing respiratory history and symptoms, occupational and smoking histories, home environment, and family history of asthma and other chronic or genetic diseases; (2) standardized pulmonary function testing satisfying the American Thoracic Society (ATS) performance and reproducibility criteria (8); (3) methacholine challenge testing for all subjects with FEV₁ values greater than 60% of predicted value; (4) bronchodilator testing; (5) skin testing of reactivity to 10 specific antigens along with a positive and negative control; and (6) drawing of venous blood for serum immunoglobulin E (IgE) level, phadeatop, eosinophil and leukocyte counts, and DNA extraction. Informed consent was obtained from all study participants. This study was approved by the Institutional Review Boards of the Brigham and Women's Hospital and the Harvard School of Public Health. Height and weight measurements were taken by standard methods for each subject after the removal of shoes and outerwear. Height was measured to the nearest 0.1 cm on a portable stadiometer. Weight was measured to the nearest 0.1 kg with the subject standing motionless on the scale.

BDR

To assess response to a bronchodilator, two puffs (180 µg) of the ₂-adrenergic agonist albuterol were administered using a metered dose inhaler via a spacer. Subjects were instructed to inhale a single puff of albuterol and hold their breath for 10 s and then exhale. This maneuver was repeated after 1 min and then 10 min following the second inhalation, standardized pulmonary function tests were performed. BDR was expressed as (postbronchodilator FEV₁  prebronchodilator FEV₁)/prebronchodilator FEV₁ × 100% (i.e., FEV₁% init), which is the most common method for expressing BDR (9). In our study, we used four separate predictive models for BDR of father, mother, first offspring, and the subsequent offspring based on a reference population that is limited to only healthy subjects with no physician-diagnosed asthma, and no asthma-related respiratory symptoms, including persistent wheeze, chronic cough, chronic phlegm, and shortness of breath. The preset thresholds for defining bronchodilator hyperresponsiveness (BDHR) were 4.74, 5.53, 4.83, and 5.49% (corresponding to the 90th percentile of each distribution in the reference population without asthma or respiratory symptoms) for father, mother, first offspring, and the subsequent offspring, respectively.

Statistical Methods

Our analyses were conducted in several stages. First, we calculate adjusted (i.e., residual) BDR values based on separate predictive models that accounted for the effects of age, age², height, height², weight, weight², smoking status, sex, baseline FEV₁, baseline FEV₁², asthma, wheeze, and allergy (reflected by the skin test). These adjusted BDR values were used to estimate Pearson correlation coefficients (r). Given the structure of our families, this allowed us to study the adjusted BDR correlation in the following relationships: (1) father-mother, (2) father-first offspring, (3) mother-first offspring, (4) father-subsequent offspring, (5) mother-subsequent offspring, and (6) first offspring-subsequent offspring.

Second, we used the multiple logistic regression model to evaluate the relationship of the BDHR status among the family members adjusting for the covariates. To correct for the intercorrelation in BDR among siblings of the same parents, we used the generalized estimation equation (GEE) method, which provides asymptotically correct standard error estimates, leading to improved confidence intervals and hypothesis tests (10). Because there is a familial dependency of BDR for members within the same family, standard statistical methods for independent data can yield correct point estimates, but confidence intervals and p values will be generally incorrect and possibly anticonservative if the correlation is not taken into account.

Finally, we used multiple logistic regression analysis to assess the risk of BDHR (i.e., hyperresponsiveness) among the subsequent offspring given the BDR status ("high" if above median, "low" otherwise) of other family members. Odds ratios for all main effects and two-way interactions were calculated with all relevant variables (i.e., risk factors of the subsequent offspring and BDR status of the mother, father, and first offspring) included in the model. For the purpose of odds ratio (OR) calculation, the reference category was always taken to be low BDR.

	RESULTS

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In all, our study included a total of 4,946 subjects who were from 1,161 index families with asthma. Clinical characteristics of the participants can be found in Table 1. Overall, fathers, on average, were 3 yr older and 10 cm taller than mothers, and their pre- and postbronchodilator FEV₁ values were about 640 ml greater. Smoking was much more frequent among fathers than mothers (fathers: 72.9%, mothers: 3.3%). The first offspring was on average 2.6 yr older than the subsequent offspring, and there were more female offspring in these index families with asthma.

The pairwise correlation of adjusted BDR values among family members was calculated. With adjustment for major confounding factors, the correlation coefficient (r) for father- first offspring, mother-first offspring, mother-subsequent offspring, and first offspring-subsequent offspring pairs was 0.096 (p < 0.01), 0.096 (p < 0.01), 0.088 (p < 0.01), and 0.165 (p < 0.01), respectively. The spouse-spouse correlation (r = 0.019) was not significant. Because the use of BDHR as a threshold variable was more appropriate than the use of BDR, we examined the associations of the adjusted BDHR status among family members (Table 2). The first sibling's BDHR status was significantly associated with the BDHR status of the subsequent sibling (OR = 2.66, 95% CI: 1.84-3.85) (Table 2).

Using the GEE, we further examined through multivariate logistic regression the independent association of the adjusted BDHR status of father, mother, and first offspring with the risk of BDHR of the subsequent offspring (Table 3), respectively. Among these families, the first sibling's BDHR status appeared to be associated with elevated risks of BDHR to the subsequent offspring, and the effect reached significance (OR = 2.62, 95% CI: 1.79-3.84). Thus, as seen earlier, the first and subsequent offspring are clearly the most correlated.

Finally, we found that the risk of BDHR to the subsequent offspring is related to the BDR status (high or low) of both parents and the first offspring (Figure 1). Among index families with asthma, the OR for the low (father)-high (first offspring) group was 1.97 (95% CI: 1.20-3.22), that for the high- low group was 0.94 (95% CI: 0.53-1.64), and that for the high- high group was 2.36 (95% CI: 1.50-3.73). The OR for the low (mother)-high (first offspring) group was 2.39 (95% CI: 1.38- 4.12), that for the high-low group was 1.44 (95% CI: 0.82- 2.55), and that for the high-high group was 3.10 (95% CI: 1.85-5.20) (Figure 1). Thus, the OR estimates for high-low and low-high groups were lower than those for high-high groups.

View larger version (39K): [in this window] [in a new window]   Figure 1.   The risk of BDHR in the subsequent offspring in asthma index families by BDR status of first-degree relatives (being "high" or "low" is defined by the median of the BDR values).

	DISCUSSION

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Because genetic variants have been shown to be important in explaining variation in the efficacy for other pharmaceuticals, it is reasonable to consider the possibility that genes also play a role in BDR. If such a genetic effect does exist, then BDR levels (adjusted for known confounders) will tend to be correlated within families. We, therefore, set out to determine the degree of familial correlation of BDR. To our knowledge, this represents the first such study of its kind. We conducted our investigation in a large, community-based population from Anqing, China. We found a significant familial aggregation (Table 2). However, no additive or multiplicative interactions were found between the influences of mother-first offspring and father-first offspring pairs on the BDR of the subsequent offspring (Figure 1).

₂-Agonists have been characterized as those that directly activate the receptor (albuterol), those that are taken up into a membrane depot (formoterol), and those that interact with a receptor-specific auxiliary binding site (salmeterol) (11). A few previous investigations (12, 13) have examined long-term responses to bronchodilators in patients with asthma. The focus of our investigation is on the familial aggregation of short-term effects of albuterol.

Bronchial responsiveness is an integrated physiological mechanism involving airway epithelium, nerves, mediators, and bronchial smooth muscle. There is no agreement on how the BDR should be expressed and it can be assessed in four different ways: (1) FEV₁: FEV₁ changes in liters after administration of a bronchodilator drug; (2) FEV₁% init: FEV₁ as a percentage of initial FEV₁; (3) FEV₁% pred: FEV₁ as a percentage of predicted FEV₁; and (4) FEV₁% (pred-init): FEV₁ as a percentage of the deficit between predicted and initial FEV₁ value (14). Each of these indices has problems associated with it. For example, FEV₁ is related to body size and hence to lung volume, so appropriate corrections need to be made, especially in children. Moreover, it has been argued that using FEV₁% init spuriously amplifies the recorded BDR in patients with a low FEV₁ (9). However, Waalkens and coworkers (15) point out that FEV₁% init, despite its drawbacks, has an important characteristic: it reflects the greater clinical benefit in a patient with poor initial FEV₁ compared with a patient with a relatively normal baseline FEV₁. In addition to controversy concerning indices of BDR, there is also no consensus on what constitutes BDHR (16). As a result, a variety of cutoff values have been used previously (14, 17). Nickerson and coworkers (21) proposed using an increase of 15% in FEV₁% pred as the threshold to define BDHR, and Martinez and coworkers (6) used 15.3% in FEV₁ %pred as the threshold for BDHR in their investigation. A couple of other studies (22, 23) used 9.0% in FEV₁% pred as the threshold. The ATS criteria (24) define a FEV₁% init  12% to be BDHR. There are sound rationales to use BDHR as opposed to BDR in dissecting its genetic components. BDR appeared inappropriate to express the response to bronchodilators, as some studies suggest that a portion of individuals show paradoxical decreases in lung function with albuterol (25). The most plausible explanation for these "negative" responses is simple random variability of a determination performed twice among those subjects who do not respond to bronchodilators. It is thus likely that most of the meaningful information in this variable is concentrated among subjects with responses beyond preset thresholds, and we therefore used BDHR instead of BDR in our data analysis. In our study, BDHR was defined as a threshold variable that corresponds to the 90th percentile of the reference population, which was limited only to those subjects without asthma or other respiratory symptoms. In addition, because BDR may vary with age or sex, we used four separate predictive models for mother, father, first offspring, and subsequent offspring to obtain four specific thresholds. These cutoff values appeared appropriate for the purpose of our study, as the ATS definition of BDHR is overly restrictive when applied in our study population.

When we tested for correlation of adjusted BDR values by calculating Pearson correlation coefficients, we found significant results for father-first offspring, mother-first offspring, mother-subsequent offspring, and first offspring-subsequent offspring pairs. Using a protocol for BDR measurement similar to ours, Martinez and coworkers (6) examined the association of two common polymorphisms (Arg16Gly and Gln27Glu) of the ₂-adrenergic receptor, an important potential candidate gene, with the variability of the response to albuterol in 269 children in the Tucson Children's Respiratory Study. Children who were homozygous for the Arg16 allele were found 5.3 times (95% CI: 1.6-17.7) more likely to show a positive response to albuterol than homozygotes for the Gly16 allele, with heterozygotes having an intermediate response (6). Furthermore, Lima and coworkers (5), who studied 16 clinically stable patients with moderate asthma, also found that albuterol-evoked FEV₁ was higher and the response was more rapid in Arg16 homozygotes compared with the cohort of carriers of the Gly16 variant (p < 0.05). Because we found a relatively low sibling-sibling correlation for BDR (0.088-0.165), it may be worthwhile to assess whether Arg16Gly polymorphism also plays an essential role in individual BDR variation in our Chinese populations.

In our study, correlation coefficients between biological relatives are significantly greater than zero, raising the possibility that genetic factors are critically involved in BDR regulation. Nevertheless, twin studies would be required to more specifically implicate genes and to determine the heritability of BDR. Success in major BDR gene identification beyond known obvious candidates is partly attributed to the appropriate definition of the BDR trait, which should be influenced by relatively few confounding factors. Risch and Zhang (28) pointed out that the power of linkage analysis is low when the sib pairs sampled are not extreme ones. Blackwelder and Elston (29) showed that the proportion of the total variance (heritability) in a trait attributable to a contributing locus would need to be large (~ 50%) to detect linkage in a reasonably sized sample by sib pair analysis when the sibs are sampled at random (i.e., irrespective of their trait values). Therefore, to maximize the power to detect major genes for BDR, it is key to ascertain only those sib pairs that are most informative. Individuals with intermediate values provide little information for linkage analysis. Thus, the enrollment of only the BDHR-concordant sib pairs may be suitable for studying BDR as a quantitative trait by using the genome-wide screening approach.

To date, there has been no segregation analysis conducted with respect to the BDR. Segregation analysis looks at the inheritance pattern of a disease in multiple pedigrees by exhaustively searching through known genetic disease models and finding the best fit. The closest model is then used for parametric linkage analysis, which is based on the method of maximum likelihood. Because of the genetic heterogeneity of BDR, the results of segregation analysis based on one population cannot be generalized to other populations. Moreover, because BDR represents a complex trait that may be caused by multiple interacting genes (i.e., epistasis), segregation analysis assuming a single-locus model gives incorrect results. It becomes very hard to estimate the number of loci influencing a complex trait except under very favorable conditions.

The major advantage of this study is a near absence of antiasthmatic medication use in rural China. Also, to verify that there is a familial component of BDR independent of asthma and allergy, the adjusted BDR used for the familial correlation analysis involved residuals obtained after controlling for asthma, wheeze, and allergy. This contrasts greatly with studies carried out in Westernized societies, and is significantly less confounded by other drug therapies. The major limitation of this study is that all study participants were exclusively Chinese, limiting its generalizability to other ethnic populations. Furthermore, the familial aggregation of BDR found in this study may result from either genetic factors or shared household environmental factors. Because previous studies (5, 6) have suggested gene variants may be associated with the variability of BDR, a candidate gene approach may provide important insights in dissecting the genetic basis of this complex phenotype.

In sum, our observations of familial clustering patterns of BDR in the index families with asthma indicate that genes are likely to play some roles in individual variation of BDR. Once genes underlying the variation of BDR are identified, we can make drug treatment more effective to treat subgroups of patients or to adjust bronchodilator drug doses differently, based on their genotypes. A well-recognized effect of the regular administration of ₂-agonists is the development of tolerance due to ₂-adrenergic receptor down-regulation. Lessening of BDR results particularly in shortening of the duration of bronchodilation (30, 31), and it occurs in patients with chronic obstructive pulmonary disease (32) and in those treated with the long-acting ₂-agonist (33). It is of interest to study the genetic basis of drug tolerance for long-acting ₂-agonists in the future. Alternative therapies may be used for those patients who are genetically susceptible to drug tolerance so that they obtain the optimal treatment to attain a successful outcome.