A m . J. H um . G enet. 69:1271–1277, 2001 A Genomewide Search for Quantitative-Trait Loci Underlying Asthma Xin Xu,1 Zhian Fang,3 Binyan Wang,1,3 Changzhong Chen,1,3 Wenwei Guang,3 Yongtang Jin,3 Jianghua Yang,3 Steve Lewitzky,4 Avram Aelony,4 Alex Parker,4 Joanne Meyer,4 Scott T. Weiss,1,2 and Xiping Xu1,2,3 1 3 Program for Population Genetics, Harvard School of Public Health, and 2Channing Laboratory, Brigham and Women’s Hospital, Boston; Institute of Biomedicine, Anhui Medical University, Hefei; and 4Genetics Division, Millennium Pharmaceuticals, Cambridge, MA A genomewide screen for quantitative-trait loci (QTLs) that underlie asthma was performed on 533 Chinese families with asthma, by the unified Haseman-Elston method. N ine asthma-related phenotypes were studied, including forced expiratory volume in 1 s (FEV1 ), forced vital capacity (FVC), airway responsiveness as indicated by methacholine (MTCH)-challenge test, serum total immunoglobulin E (TIgE), serum-specific immunoglobulin E, eosinophil count in peripheral blood, and skin-prick tests with three different allergens (cockroach, Dermatophagoides pteronyssinus, and D. farinae). Our study showed significant linkage between airway responsiveness to MTCH and D2S1780 on chromosome 2 (P ! .00002 ) and provided suggestive evidence (P ! .002 ) for six additional possible QTLs: D10S1435 and D22S685, for FEV1 ; D16S412, for FVC; D19S433, for airway responsiveness to MTCH; D1S518, for TIgE; and D4S1647, for skin reactivity to cockroach. N o significant or suggestive evidence of linkage for the other four traits was found. Introduction Asthma (M IM 600807) is a common clinical syndrome of reversible-airflow obstruction, characterized by airway hyperresponsiveness, airway inflammation, epithelial damage, and airway smooth-muscle hypertrophy (Sheffer 1995). Susceptibility to asthma is determined by the interaction of an unknown number of genetic and environmental factors. The genetics of asthma has been the subject of several recent reviews (see H olloway et al. 1999; Cookson and M offatt 2000; Palmer and Cookson 2000). Linkage analysis plays a pivotal role in the search for genes underlying asthma. Although clinical definitions play an important role in the diagnosis and treatment of asthma, it is quite conceivable that the best clinical definition does not coincide with the best definition for the mapping of genes that contribute to asthma. In comparison, intermediate phenotypes, in many cases, are quantitative, more reliable, and of less genetic heterogeneity, conferring better power in linkage studies. O n the basis of the definition of asthma, the asthma-related intermediate phenotypes can be broadly divided into two groups: the first group is related to lung function, including forced expiratory volume in 1 s Received June 20, 2001; accepted for publication September 27, 2001; electronically published O ctober 22, 2001. Address for correspondence and reprints: Dr. Xiping Xu, Program for Population Genetics, H arvard School of Public H ealth, 665 H untington Avenue, FXB-101, Boston, M A 02115. E-mail: xu@hsph .harvard.edu 䉷 2001 by The American Society of H uman Genetics. All rights reserved. 0002-9297/2001/6906-0013$02.00 (FEV1 ), forced vital capacity (FVC), and airway responsiveness to bronchorestrictors and to bronchodilators; the second group is related to inflammation and allergy, including serum total and serum-specific immunoglobulin E (TIgE and SIgE, respectively), skin-prick tests to aeroallergens, and peripheral-blood–eosinophil (EO S) count. Previous genomewide linkage studies suggested that several loci are probably linked to asthma or to related phenotypes, although few linkages reached the stringent genomewide significance level (Daniels et al. 1996; the Collaborative Study on the Genetics of Asthma 1997; O ber et al. 1998, 2000; Wjst et al. 1999; Dizier et al. 2000; Yokouchi et al. 2000; M athias et al. 2001). Furthermore, considerable inconsistency was observed among the previously published results, reflecting the complexity of asthma and, owing to relatively small sample sizes, the lack of power. In this study, we screened 2,551 subjects from 533 Chinese families with asthma, searching for loci linked to asthma-related intermediate phenotypes. To our knowledge, this is, thus far, the largest published whole-genome linkage study on asthma-related phenotypes. Subjects and M ethods Study Samples This study was conducted, in collaboration with Anhui M edical University and Anqing H ealth Bureau, in Anqing, China. The geographic characteristics of the Anqing population, as well as the procedure for ascer1271 1272 taining the study samples, have been described elsewhere (Xu et al. 1999a). In brief, by screening the Anqing population, we ascertained 2,752 index families with asthma, with the following criteria: (1) presence of at least two siblings with physician-diagnosed asthma who were ⭓8 years old, (2) availability of both parents, and (3) history of asthma in no more than one parent. In addition, we ascertained, from the same population, 270 reference families who were selected from 1992 census records by a two-stage random-sampling technique, in which the village was the first-stage sampling unit and the household was the second-stage sampling unit. O ur inclusion criteria for reference families were (1) family size of at least four, (2) availability of both parents, and (3) presence of at least two siblings who were ⭓8 years old. We used the reference families for assessing the effects that common covariates (e.g., age, gender, etc.) had on each asthmarelated phenotype. A multiple linear-regression model developed on the basis of the reference families was then used, prior to linkage analysis, to adjust phenotypic values in the families with asthma for fixed effects of these covariates. From the 2,752 index families, we selected 533 for genomewide-linkage study. This study has been approved by the H uman Subject Committees at Brigham and Women’s H ospital and at the H arvard School of Public H ealth. Each enrolled subject or, in the case of children, the subject’s parent or guardian has signed an informedconsent form. Evaluation of Phenotypes The following procedures were performed on the basis of protocols that are in accordance with those used in the N ational Institutes of H ealth Collaborative Study on the Genetics of Asthma (CSGA). Q uestionnaire adm inistration.—A modified American Thoracic Society (ATS) Division of Lung Disease questionnaire was administered specifically to assess respiratory history and symptoms, occupational and smoking histories, home environment, and family history of asthma or other chronic diseases. The questionnaire was divided into two forms—one for adults (aged 114 years) and one, to be completed by parents, for children (aged ⭐14 years). Pulm onary-function tests.— Standardized spirometry was performed on ATS “ Snowbird Guideline” –approved equipment (Schiller). Each subject was seated, with nose clip, and performed five to eight maneuvers, to obtain three acceptable tracings. For both FEV1 and FVC, the maximum of the three accepted measurements was believed to be more reproducible than the means (Tager et al. 1976) and was used as a quantitative phenotype in our subsequent analyses. A irw ay m ethacholine (M T CH )-challenge test.— M TCH -challenge test was performed for all subjects A m . J. H um . G enet. 69:1271–1277, 2001 with FEV1 160% of the predicted value, by a modified Chatham protocol (Chatham et al. 1982). In brief, each subject was challenged with the following five combinations of number of breaths and M TCH concentrations, in sequential order: one breath of 1 mg/ml, one breath of 5 mg/ml, four breaths of 5 mg/ml, one breath of 25 mg/ ml, and four breaths of 25 mg/ml. At each dose, two satisfactory spirometry maneuvers were obtained. Again, the greater of the two measurements was used in analyses. The test terminated either at the dose that produced a ⭓20% FEV1 drop (PD 20) from the baseline FEV1 or at the final dose. The individual airway responsiveness to M TCH was measured in dose-response–slope percentage (DRSP) defined as (FEV1b ⫺ FEV1t )/(FEV1b 7 [M TCH ]t ), where FEV1b is the baseline FEV1, FEV1t is FEV1 at the terminating dose of M TCH test, and [M TCH ]t is the cumulative dosage of M TCH inhaled during the test. The log10 transformation of DRSP, logDRSP, was approximately normally distributed in the general population and was used in our subsequent linkage analysis. For a small number of subjects, whose FEV1 did not drop during the test, logDRSP values were fixed at ⫺4.5. Sk in-prick test.— Skin-prick test was performed by a slightly modified semiquantitative puncture method developed by Santilli et al. (1980). In addition to histamine and saline controls, the following antigens were applied to the forearm of each subject: cockroach, D erm atophagoides pteronyssinus, D . farinae, house dust, mixed trees, mixed grass, tobacco leaf, polyvalent molds, artemisia, and silk. The diameters of the wheals were recorded; any subject with either a 12-mm wheal in response to the saline control or a !2-mm wheal in response to the histamine control was excluded from analyses. O f the 10 allergens tested in the population that we studied, cockroach, D . pteronyssinus, and D . farinae caused large wheals much more frequently than did the other 7 allergens. The population mean of the diameters of the wheals was 11.1 mm for cockroach, D . pteronyssinus, and D . farinae but !0.4 mm for the other allergens. For this reason, only skin reactions to cockroach, to D . pteronyssinus, and to D . farinae were selected for subsequent linkage analysis. M easurem ent of serum im m unoglobulin E levels.— TIgE levels were measured using a UniCAP system (Pharmacia Diagnostics). SIgE levels were determined using the Phadiatop kit (Pharmacia Diagnostics). The log10 transformation of TIgE, logTIgE, was approximately normally distributed in the general population and was used in our subsequent linkage analysis. EO S count.— EO S count was performed by use of a Coulter counter. The log10 transformation of EO S, logEO S, was approximately normally distributed in the general population and was used in our subsequent linkage analysis. 1273 Xu et al.: Genome Scan for Asthma-Related Q TLs Family Selection for Genome Scan and Genotyping O f the 2,752 enrolled families, 533 were selected for the genome scan; of these, 471 had at least two siblings with a PD 20 and 52 had one sibling with a PD 20 . The scan covered 22 autosomal chromosomes with 422 highly polymorphic microsatellite markers, with an average spacing of 7–8 cM . In brief, genomic DN A was extracted from whole blood by use of a commercially available kit (Gentra Systems). The genotyping was performed, by a fluorescent-based detection method, at M illennium Pharmaceuticals, by use of ABI377XL automated sequencers (Perkin-Elmer). Each genotype was double scored—that is, scored once by an expert technician (i.e., a human scorer) and once by a proprietary software package. Incongruities between the two scores were resolved by the human scorer. M arker data for each pedigree were checked for M endelian inheritance. Raw data for all observed deviations were reevaluated. Genotyping errors resulting in rare double recombination were cleaned up by the sib_clean program included in the ASPEX package (Schwab et al. 1995). The chromosomal orders and the intervals of markers were obtained from Weber’s linkage map (Center for M edical Genetics, M arshfield M edical Research Foundation) and were fine-tuned by the sib_map program included in the ASPEX package. Linkage Analysis The phenotypes tested for linkage included FEV1, FVC, logDRSP, logTIgE, SIgE, logEO S, and wheal size in skinprick test with three allergens (i.e., cockroach, D . pteronyssinus, and D . farinae). All the phenotypes were quantitative, with the exception of SIgE, which was treated as pseudoquantitative with values 0 and 1. Effects that factors such as age, gender, height, weight, and pack-years of smoking had on each phenotype were examined in a collection of 270 reference families who were randomly selected from the same community as were the families with asthma. In the reference population, the five aforementioned factors accounted for ∼75% of the variance in FEV1 and FVC but accounted for only ⭐5% of the variance in the other seven traits. For both FEV1 and FVC, a trait-prediction model was constructed by multiple linear regression, by use of the observations from the reference families, to adjust for the aforementioned covariates. The standardized residuals of FEV1 and FVC were then computed on the basis of data on the members of the families with asthma and were used in subsequent linkage analyses. The other seven traits were standardized using the mean and variance of the reference population prior to linkage analysis. The identical-by-descend (IBD) probabilities between a sib pair, at any arbitrary chromosomal location, were estimated by GEN EH UN TER version 2 (Kruglyak et al. 1996). Linkages to the asthma intermediate phenotypes were then analyzed by a unified H aseman-Elston (H E) method, by the computer program XWXW (Xu et al. 2000). The information content (I c) of IBD estimates varies according to marker heterozygosity, marker density, and family structure. For any IBD distribution in which z 0 , z 1 , and z 2 are the probabilities of sharing 0, 1, and 2 alleles IBD, respectively, the portion of IBD sharing has an expected value p̂ p (z 1 /2) ⫹ z 2 and 2 ˆ p Sip0 ˆ 2 . The variance is 0 a variance var(p) z i[(i/2) ⫺ p] when a marker is fully informative and is .125 under the null distribution (no marker genotype is available). The I c value for an estimate of IBD distribution was defined ˆ as I c p 1 ⫺ var(p)/.125 . I c takes the maximum value of 1 when an IBD estimate is unambiguous, and is 0 when no marker data are available. In our linkage analysis, we used sib-pair observations only when the I c values of IBD estimates were ⭓.1. Results O ur genome scan included 2,551 subjects from 533 families with asthma, from Anqing, China. O f the 533 families with asthma, only 15 are nonnuclear families who include either three generations or two marriages by one person (table 1). The phenotypic characteristics of these families with asthma, as well as of the reference families included in the construction of models for the prediction of phenotypic traits, are summarized in table 2. The distributions of the standardized traits in the nonfounders of the scanned families are depicted in figure 1. O ur linkage analysis suggested seven quantitativetrait loci (Q TLs) (P ! .002 ) for FEV1 , FVC, logDRSP, logTIgE, and skin reactivity to cockroach but did not suggest any Q TLs for logEO S, SIgE, and skin reactivity to D . pteronyssinus and to D . farinae. The suggestive Q TLs are as follows: D10S1435 and D22S685, at chromosomes 10 and 22, respectively, for FEV1 ; D16S412, at chromosome 16, for FVC; D2S1780 and D19S433, at chromosomes 2 and 19, respectively, for logDRSP; Table 1 Structure of 533 Genome-Scanned Families Category N o. N uclear family: 2 sibs 3 sibs 4 sibs 5 sibs 6 sibs 7 sibs 8 sibs 9 sibs N onnuclear family Total 241 177 66 16 13 1 2 2 15 533 1274 A m . J. H um . G enet. 69:1271–1277, 2001 Table 2 Phenotypic Characteristics of Genome-Scanned and Reference Families M EAN Ⳳ SD [Total N o.] VARIABLES Sex (% male) Age (years) H eight (m) Weight (kg) BM I (kg/m 2 ) Asthma a (% replying yes) FEV1 (liters) logDRSP (% /mg) logEO S (cells/mm 3) logTIgE (IU) SIgE (% positive) Skin reactivity (mm) to: Cockroach D . pteronyssinus D . farinae Asthma Index Family 48.8 30.7 Ⳳ 1.52 Ⳳ 47.0 Ⳳ 19.8 Ⳳ 25.7 2.55 Ⳳ ⫺2.10 Ⳳ 2.02 Ⳳ 2.16 Ⳳ 44.7 Random Control Family [2,551] 16.4 [2,549] .15 [2,547] 13.4 [2,547] 3.20 [2,547] [2,542] .90 [2,510] .86 [2,229] .49 [2,542] .68 [2,488] [2,501] 50.6 [1,021] 30.3 Ⳳ 15.5 [1,021] 1.54 Ⳳ .13 [1,021] 48.0 Ⳳ 12.4 [1,021] 19.8 Ⳳ 3.07 [1,021] 2.75 [1,018] 2.83 Ⳳ .79 [960] ⫺2.70 Ⳳ .73 [922] 1.99 Ⳳ .46 [1,020] 1.96 Ⳳ .72 [236] 36.7 [237] 1.67 Ⳳ 2.15 [2,099] 1.74 Ⳳ 2.33 [2,099] 1.09 Ⳳ 1.82 [2,099] 1.67 Ⳳ 2.02 [1,021] 1.71 Ⳳ 2.06 [1,021] 1.31 Ⳳ 1.83 [1,021] a Asthma was defined as a yes in reply to both “ H ave you ever had asthma?” and “ Was the asthma diagnosed by a physician?” D1S518, at chromosome 1, for logTIgE; and D4S1647, at chromosome 4, for skin reactivity to cockroach. The complete linkage-test results for these five traits are shown in figure 2, and the suggested Q TLs are summarized in table 3. It is noteworthy that the linkage between logDRSP and D2S1780 (P p .00002 ) reaches the stringent genomewide significance level (Lander and Kruglyak 1995). D iscussion The present study, which includes 2,551 subjects from 533 families, is the largest genomewide linkage study of asthma-related phenotypes thus far reported. In addition to the large sample size, our study takes advantage of extensively documented intermediate phenotypes related to asthma and of an improved H E linkage-analysis method. We also believe ours is the first report of a genomewide linkage study of pulmonary function (e.g., FEV1 and FVC). Phenotype assessment is critical in gene-mapping studies of complex disease. The lack of a standardized definition of the asthma phenotype makes this phenotype sensitive to misclassification and relatively unreliable for genetic studies. This is especially true in the sample that we studied, since the asthma statuses of the subjects were initially defined by local village physicians whose criteria for diagnosis of asthma may have substantially differed from one another’s. We have studied several algorithms for classification of asthma phenotypes in the Chinese population, on the basis of a combination of respiratory symptoms, increased airway re- sponsiveness, and a physician’s diagnosis of asthma (Celedon et al. 2000). H owever, it is not clear which algorithm is optimal for linkage analysis of our study sample. In comparison, the quantitative intermediate phenotypes in each subject were measured by the same team or laboratory and by the same procedures and instruments; hence, they are more reliable for linkage study. According to simulation studies and our own research experience, adjustment of traits with important covariates that are not related to the Q TL of interest substantially improves the power to detect linkage (Xu et al. 1999b). Although a few methods to directly adjust covariates in sib-pair linkage analysis have been suggested (Elston et al. 2000), they are ad hoc methods and are problematic in the presence of ascertainment bias. Alternatively, covariates can be adjusted prior to linkage analysis, if a good predictive model is available. By including reference families who were randomly selected from the same population as our study sample, we were able (1) to obtain unbiased estimates of the effects that covariates such as age, gender, height, weight, and smoking had on each trait of interest and (2) to make a corresponding adjustment to the trait. We applied this approach to the linkage analyses of both FEV1 and FVC, for which these covariates account for ∼75% of the total variance in the reference population. Without covariate adjustments, the significance levels of the three suggested Q TLs for FEV1 and FVC were substantially lower in the linkage test. The H E method and the variance-component (VC) method are the two methods widely used for testing linkage to quantitative traits. We chose the unified H E method for our linkage study, for the following three reasons: First, the sample distributions of many traits that we analyzed deviate substantially from normal; it has been shown that the H E method is more robust than the VC method when a trait is not normally distributed (Allison et al. 2000). Second, the VC method usually has more power than the H E method when multigeneration pedigrees are studied; since nuclear families Table 3 Significance of Linkage at Suggestive Q TLs PO IN TWISE P C H RO M O SO M E (M ARKER )a FEV1 FVC logDRSP logTIgE Cockroach 1 (D1S518) 2 (D2S1780) 4 (D4S1647) 10 (D10S1435) 16 (D16S412) 19 (D19S433) 22 (D22S685) .03 .006 … .0009 … … .002 .007 .07 … .05 .0006 .09 .03 … .00002 … … .02 .002 … .001 … .04 … … .06 … … … .0003 … … … … a The most significant linkage at a particular location is underlined. Xu et al.: Genome Scan for Asthma-Related Q TLs 1275 Figure 1 Distributions of standardized phenotypes in nonfounder members of genome-scanned families. The Y-axis is the count of nonfounder members. constitute the majority of our study samples, the advantages of using the VC method are limited. Third, compared to other versions of H E methods, the unified H E method allows for multiple sib pairs in a family and has greater power (Xu et al. 2000). Because of differences in marker heterozygosity, in marker density, and in family structure, the accuracy of sib-pair IBD estimates varies. Although we initially thought that weighing the sib-pair observations by their I c value might increase power, our simulations showed that giving the same weight to all observations with I c 1.1 has slightly better power in many situations (data not shown). As a result, we chose to include, in our linkage analysis, all sib-pair observations in which I c 1 .1 with equal weights. Our study demonstrates significant linkage of D2S1780 to airway responsiveness, as measured in logDRSP, and suggestive (P ! .002 ) linkage of six other chromosomal loci to one of the five quantitative traits FEV1 , FVC, logDRSP, logTIgE, and skin reactivity to cockroach (table 1276 A m . J. H um . G enet. 69:1271–1277, 2001 Figure 2 Complete genomewide-linkage–test results for five quantitative traits. The Y-axis is the ⫺logP of the linkage result; the vertical dotted lines separate the 22 autosomes indexed at the bottom. I c was calculated by GEN EH UN TER. 3). For some of these suggestive Q TLs, there is suggestive evidence of linkage not only to a single trait but also to other related traits, albeit at a reduced significance level; for example, there is suggestive evidence that D1S718 is linked not only to TIgE level (P p .001 ) but also to FEV1 (P p .03 ) and to FVC (P p .007 ). A number of genome scans for asthma and asthma-related phenotypes have been previously reported (Daniels et al. 1996; the Collaborative Study on the Genetics of Asthma 1997; O ber et al. 1998, 2000; Wjst et al. 1999; Dizier et al. 2000; Yokouchi et al. 2000; M athias et al. 2001). H owever, the results of these studies show considerable inconsistency, which presumably is due to differences in phenotypic definitions and study populations and, more likely, to a lack of power resulting from insufficient sample size. Such inconsistency also exists between our results and those previously reported. In our comparison, we regarded a previously reported result to be “ consistent” with our result if a previously reported linkage peak with P ! .01 was, regardless of the asthma traits tested, !20 cM from one of the seven Q TLs reported in this study; by this criterion, our suggested Q TLs on chromosomes 1, 10, and 19 are consistent with results reported by Wjst et al. (1999), Dizier et al. (2000), and the Collaborative Study on the Genetics of Asthma (1997) and O ber et al. (2000), respectively. We emphasize here that these “ consistent” linkages, although very encouraging, should not be regarded as positive replications, since comparison was made among many genome scans with different asthma-related traits and since, more important, few linkages reached the suggested genomewide significance level (Lander and Kruglyak 1995). Xu et al.: Genome Scan for Asthma-Related Q TLs Acknowledgments We wish to thank Anqing H ealth Bureau and Anqing H ospitals for their help and support. We also wish to thank Drs. N an Laird and L. J. Wei for helpful discussions and comments on the manuscript. This study was supported, in part, by N ational H eart, Lung, and Blood Institute grants H L56371 and H L66385 and by a grant from M illennium Pharmaceuticals. Electronic-Database Information The accession number and URLs for data in this article are as follows: ASPEX Package, The: Affected Sib-Pair Exclusion M apping, ftp://lahmed.stanford.edu/pub/aspex/doc/usage.html Center for M edical Genetics, M arshfield M edical Research Foundation, http://research.marshfieldclinic.org/genetics/ (for Weber’s linkage map) FBAT Web Page, The, http://www.biostat.harvard.edu/˜fbat/ default.html (for XWXW) GEN EH UN TER, http://www.fhcrc.org/labs/kruglyak/ Downloads/ O nline M endelian Inheritance in M an (O M IM ), http://www .ncbi.nlm.nih.gov/O mim/ (for asthma [M IM 600807]) References Allison DB, Fernández JR, H eo M , Beasley TM (2000) Testing the robustness of the new H aseman-Elston quantitative-trait loci–mapping procedure. 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