American Journal of Respiratory and Critical Care Medicine

A genome-wide search was conducted in 107 nuclear families with at least two siblings with asthma, as part of the French EGEA study. A two-stage analysis strategy was applied to the 107 families divided into two independent subsets of 46 and 61 families, where all regions detected in the first set of families were tested for replication in the second set. In addition, all regions reported by published genome scans in different populations were examined in the total sample. A total of 254 markers were typed in the first set of families and 70% of them in the second set. Linkage was investigated by model-free methods for asthma and four asthma-related phenotypes: bronchial responsiveness (BR), skin test response, total immunoglobulin E (IgE) levels, and eosinophil count. The two-stage analysis led to the detection of three regions: 11p13 for IgE, 12q24 for eosinophils, and 17q12–21 for asthma and skin tests. Among the regions reported by published genome screens, seven were found in the 107 French EGEA families: three being already detected by the two-stage analysis, 11p13 (p = 0.005), 12q24 (p = 0.0008), and 17q12–21 (p = 0.001), and four additional ones, 1p31 (p = 0.005) for asthma, 11q13 (p = 0.006) for IgE, 13q31 (p = 0.001) for eosinophils, and 19q13 (p = 0.02) for BR.

Asthma is a complex and heterogeneous disorder resulting from both genetic and environmental factors and presenting a wide spectrum of clinical manifestations. Asthma is associated with intermediate phenotypes including bronchial hyperresponsiveness (BHR) and atopy that represent biological and physiological markers of disease and can be measured objectively. Atopy is defined stricto sensu by positive skin prick tests to common allergens but also includes elevated total and allergen-specific immunoglobulin E (IgE) levels. Atopic disease is accompanied by blood eosinophilia, and large numbers of eosinophils are found in asthmatic airways. However, BHR and atopy are not specific to asthma. Most people with asthma have marked BHR but some degree of BHR has also been found in asymptomatic individuals (1, 2). Although most subjects with asthma are atopic, only a minority of atopic subjects have asthma (3). Thus, asthma, BHR, and atopy may result from common determinants but there may also be primary genetic alterations that are specific to each of these phenotypes. Candidate gene studies (4) and published genome screens (5– 8) for asthma, BHR, and/or atopy have detected a number of regions linked to one or a few of these phenotypes.

These published genome-wide searches have been conducted in different populations: (1) an Australian sample of 80 families selected from a population sample of 230 families to reduce the proportion of atopics with a replication sample of 77 British families ascertained through at least one asthmatic proband (5); (2) the American Collaborative Study on the Genetics of Asthma (6) including three family samples ascertained through at least two siblings with asthma from three ethnic groups: black (43 families), white (79 families), and Hispanic (18 families); (3) a sample of 361 Hutterite subjects from a founder population of European ancestry belonging to four related colonies selected for a high prevalence of asthma and atopy with a replication sample of 292 subjects (7); and (4) a sample of 97 European families, 83 of them from Germany and the remaining from Sweden, ascertained through two siblings with asthma (8). Whereas one of these studies (5) considered mainly quantitative traits associated with asthma, two others (6, 7) reported linkages to asthma only and to the specific IgE responsiveness subsequently (9, 10), and the German study (8) considered asthma and associated phenotypes including total and specific IgE levels, BHR, and eosinophil count, and more recently the specific IgE response to different allergens (11). Given the multiple regions detected by these published genome scans, only replication of linkages across studies will make it possible to point out the most likely regions involved in asthma and/or asthma-associated phenotypes to be further explored toward gene identification.

The Epidemiological Study on the Genetics and Environment of Asthma, Bronchial Hyperresponsiveness, and Atopy (EGEA) was designed to identify the genetic and environmental factors for asthma, BHR, and atopy (12, 13). As a first step toward gene identification, we conducted a genome-wide search in a subset of 107 French families of the EGEA study having at least two siblings with asthma. Linkage analyses were performed for asthma and four associated phenotypes (bronchial responsiveness [BR], positive response to a battery of skin prick tests, total serum IgE levels, and blood eosinophil count) using single and multipoint sib-pair analyses. We carried out a two-stage analysis by dividing our total sample of 107 families into two independent subsets and by testing for replication in the second family subset the potential linkages detected in the first subset. We also searched to replicate in the whole sample all regions reported by published genome screens for asthma and/or asthma-associated phenotypes.

Family Data

As part of the EGEA study, a sample of 119 families with at least two siblings with asthma and at most one parent with asthma was collected for the genome-wide search. This sample included 79 families from the main EGEA sample of 348 families selected through one proband with asthma and 40 additional families selected directly through two siblings with asthma in order to increase the number of such families for linkage analyses of asthma. The clinical characteristics of these two samples did not differ significantly (13). Siblings with asthma of our 119 families met the following criteria: currently older than 7 yr of age; born in France as well as his or her parents; and a positive response to at least one of two questions (Have you ever had attacks of breathlessness at rest with wheezing? Have you ever had an asthma attack?), associated with one of the following: presence of BHR (defined as a fall in baseline FEV1, the forced expiratory volume at 1 s, ⩾ 20% at ⩽ 4 mg methacholine), an increase of baseline FEV1 ⩾ 12% after bronchodilator use, hospitalization for asthma in life, or asthma therapy (12). After excluding families with insufficient DNA available or showing non-Mendelian transmission, the analyzed sample consisted of 107 families with a total of 493 individuals.

The protocol was approved by the ethical committee and subjects signed informed consent forms.

Clinical Evaluation

Information on respiratory and allergic symptoms, medical history, and environmental factors was recorded on a questionnaire. The following tests were performed.

Skin prick tests were done to 11 allergens (i.e., cat, Dermatophagoı̈des pteronyssinus, Cladosporium herbarum, Alternaria tenuis, timothy grass, olive, birch, Parieteria judaica, ragweed, Aspergillus, and Blatella Germanica).

Total immunoglobulin E concentration was measured by radioimmunoassay (Phadebas PRIST technique; Pharmacia Diagnostics, AB, France) in one central laboratory (14).

Total and differential white blood cell counts were assessed by manual or automating reading according to the usual method in each center.

Basal spirometry was conducted according to the ECRHS protocol (15) with slight modifications. The methacholine bronchial challenge test was performed in subjects with FEV1 > 80% predicted who did not decrease their FEV1 by more than 10% postdiluent.The test was stopped before the maximum cumulative dose if FEV1 decreased by at least 20% as compared with the postdiluent value and/or if the maximal dose was reached (4 mg in most subjects). Among the 279 genotyped siblings of the 107 families, 159 (57%) completed a methacholine challenge. Methacholine challenge could not be performed in one center for practical reasons (67 siblings), because of FEV1 < 80% predicted (20 siblings) and other reasons including contraindications and refusals (33 siblings). In those without methacholine challenge, bronchodilation was performed in 73% of them.

Phenotypes Analyzed

Asthma was defined as indicated above for the selection of the 107 families for linkage analysis. Intermediate phenotypes included total serum IgE levels, skin prick test (SPT) response (a positive response to at least one allergen corresponding to a difference of weal diameter ⩾ 3 mm with the negative control), bronchial responsiveness (BR) measured by the slope of the dose–response curve (percentage of FEV1 decrease after and before inhalation of methacholine divided by dose of inhaled methacholine), and blood eosinophil count (number/mm3). All quantitative traits were log transformed before linkage analysis. Because a few BR slope measures were less than or equal to zero, a constant was added to each value before the log transformation.

Genotypes

Genomic DNA was isolated from peripheral blood leukocytes by phenol/chloroform extraction. The initial screen included 254 autosomal microsatellite markers with an average spacing of 13 cM and an average heterozygosity over all loci of 0.78. Markers tested in the second set of families (i.e., belonging to all regions detected in the first set of families and to regions reported by published genome screens) represented 70% of markers of the initial screen. Genotypes at these loci were determined by fluorescence-based semiautomatic methods (16). Genotypes were scored using GENESCAN and GENOTYPER (ABI) softwares. Non-Mendelian marker data were identified by the UNKNOWN program. Allele size differences within families rather than exact alleles were determined using information on the parents' DNA, which was available in 97% of families. Potentially incorrect genotypes were reexamined and retested if necessary. Genotyping could be determined in 90% of all subjects with DNA available. We checked that marker maps computed from our data were consistent with published maps.

Linkage Analysis

Linkage analysis strategy. The large number of markers and phenotypes tested can lead to false positive linkages due to multiple tests. Use of stringent thresholds to conclude a significant linkage, as proposed by Lander and Kruglyak (17), provides very low rates of false positives at the expense of high rates of false negative results. It has been shown that a two-stage analysis using liberal thresholds such as 0.05 or 0.01 in two independent sets of families provides an important decrease of the rate of false negatives for a slight increase in the rate of false positives (18). We applied a two-stage analysis strategy by replicating in a second set of families the chromosomal regions detected in a first set. The total sample of 107 families was therefore divided into two subsets of families, the first set consisting of the first ascertained 46 families and the second set of the remaining 61 families. We retained only the regions detected in the two independent sets of families for the same phenotype. Chosen criteria for regions of potential linkages in the first family set were either a p value ⩽ 0.01 or p values ⩽ 0.05 for at least two adjacent markers with the same phenotype or for one marker with at least two phenotypes. These markers plus two flanking ones on each side were typed in the second set of 61 families. Regions replicated in the second set had to show a p value ⩽ 0.05 for the same phenotype.

Our second strategy was to search for previously published regions that could be replicated in our whole sample of 107 families. Those regions reported by genome-wide searches for the phenotypes studied here were the following: 1p (8, 10, 19), 2q (6), 4q (5), 5q (6, 7), 6p (5, 6, 8, 10), 7p (5), 11p (6), 11q (5), 12q (6-8), 13q (5, 6), 14q (6), 16q (5), 17q (6), 19q (6, 7) and 21q (6, 7). Note that the regions 2pter and 9q found by the German screen (8) and the region 16p found by the genome scan of positive SPT to at least one allergen in Hutterites (10) were not tested for replication in our whole sample as these results were not published at the time of genotyping our families.

Single point analysis was first applied to the two strategies mentioned above. All chromosomal regions detected by the two-stage analysis and/or reported in the literature and replicated in our whole family sample were then examined by multipoint linkage analysis.

Linkage analysis methods. Linkage analyses were performed by model-free methods that do not require specification of the underlying genetic model for the trait being investigated. Quantitative traits (IgE, BR-slope, eosinophil count) were analyzed using the Haseman– Elston method (20), where the squared sib-pair differences for the trait are regressed upon the estimated proportion of marker alleles the sib-pairs share identical by descent (IBD). The regression coefficient is zero under the null hypothesis of absence of linkage whereas it is negative if there is linkage. This is tested by a one-sided t test with number of degrees of freedom equal to the total number of sib-pairs minus two. Binary traits (asthma, SPT) were analyzed by a likelihood-based method applied to the whole sibship of affecteds, previously described (21-23) and implemented in the program SIBPAIR (24). The likelihood contribution for meioses from a heterozygous parent with n affected offsprings of which m inherited one marker allele and n − m the other is equal to [αm(1 − α)n−m + αn−m(1 − α)m]. For the whole family, the contribution is the product of the two parental contributions. The product of the likelihoods over all families is maximized over α with α being the probability for an affected sibling to receive the marker allele transmitted with the disease allele. Test for linkage is performed using a likelihood ratio test statistic, Λ = 2Ln[L(α)/L(α = 0.5)] with α being equal to 0.5 under the null hypothesis of no linkage and α > 0.5 under the hypothesis of linkage. The statistic Λ is distributed asymptotically as a mixture distribution of 0.5 χ2 0df and 0.5 χ2 1df, and ZMLB = Λ1/2 is a one-sided standard normal deviate. This maximum likelihood binomial (MLB) approach was shown to be more powerful than affected sib-pair tests (23).

Multipoint analyses using the whole marker information on a given chromosome were conducted with the program GENEHUNTER 2.0 (25) for the Haseman–Elston method and a modified version of GENEHUNTER for the maximum likelihood binomial approach (26).

Description of the Two Family Subsets Analyzed

Table 1 shows the clinical characteristics of genotyped siblings in the 107 families and in the two sets of families. The distributions of all characteristics are similar in the two sets of families and show no significant difference. In the 279 genotyped siblings of the 107 families, 59% of siblings are males and the mean age is 14.5 ± 7.6 (SD) yr. Mean age of first attack of asthma in the 221 siblings with asthma of the whole sample is 5.3 ± 5.13 (SD) yr (5.0 ± 5.04 in the first set and 5.6 ± 5.21 in the second one), with 95% having an age of onset < 16 yr and 5% having an age of onset ranging between 16 and 24 yr. Table 2 gives the distribution of sibships according to the number of genotyped siblings, being affected for binary traits (asthma or SPT) and having known measures for quantitative traits.

Table 1. CHARACTERISTICS OF GENOTYPED SIBLINGS

Total (107 Families)First Set (46 Families)Second Set (61 Families)
No. of genotyped sibs279118161
Mean age, yr (SD)14.5 (7.6)13.7 (5.7)15.1 (8.7)
Child/adult (< 16/⩾ 16 yr), % child70.674.6 67.7
Sex, % males59.559.3 59.6
Asthma, %83.786.3 81.8
SPT, %80.581.9 79.5
Mean log BR slope (SD)1.05 (0.90)1.12 (0.88)0.95 (0.93)
Mean log eosinophil count, Nb/mm3 (SD)2.51 (0.35)2.55 (0.37)2.47 (0.34)
Mean log IgE, IU/ml (SD)2.39 (0.65)2.34 (0.67)2.43 (0.64)

Definition of abbreviations: BR = bronchial responsiveness; IgE = immunoglobulin E; SPT = skin prick test.

Table 2. DISTRIBUTION OF FAMILIES ACCORDING TO THE NUMBER OF GENOTYPED SIBS AVAILABLE FOR LINKAGE ANALYSES TO ASTHMA AND ASSOCIATED TRAITS IN THE TWO SETS

No. of Sibs*
Set 1Set 2
2345Total2345Total
Asthma38 7104653 80061
SPT25 8303636 74147
BR slope24 7303414 53123
Eosinophil count2813414634176259
IgE2613514533196260

Definition of abbreviations: BR = bronchial responsiveness; IgE = immunoglobulin E; SPT = skin prick test.

*No. of affected sibs in the case of binary traits (asthma and SPT).

Regions Detected in the First Set of Families

Table 3 shows the results of single point linkage analysis in the first set of families. This table displays the regions leading to either a p value ⩽ 0.01 or p values ⩽ 0.05 for at least two adjacent markers with the same phenotype or for one marker with at least two phenotypes. Twenty regions were detected with these criteria. Markers were assumed to detect different regions of linkage if all p values were nonsignificant over a region of > 20 cM between two markers with significant linkage. Eight of these regions (5p15.2, 7p15–q22, 7q36, 8q22, 10p15, 12q21–24, 13q12–31, 17q12–21) led to p values ⩽ 0.005.

Table 3. REGIONS DETECTED IN THE FIRST SET OF FAMILIES WITH EITHER A p VALUE  ⩽  0.01 OR p VALUES  ⩽  0.05 FOR AT LEAST TWO PHENOTYPES OR AT LEAST TWO ADJACENT MARKERS

MarkerPositionθ* AsthmaSlopeEos countSPTIgE
D1S238q25.30.050.02
D2S152q32.10.140.050.04
D2S72q33.20.02
D2S126q36.30.020.01
D3S1303q21.10.040.040.03
D5S268p15.20.001
D7S513p21.30.160.02
D7S493p15.30.02
D7S484p15.20.160.0020.03
D7S519p11.20.110.020.0070.009
D7S502q11.10.100.020.03
D7S669q11.210.100.03
D7S524q11.230.090.010.002
D7S527q22.10.0070.007
D7S483q36.20.150.0005
D7S550q36.30.002
D8S503p23.20.040.05
D8S285q11.210.060.04
D8S260q12.10.03
D8S273q22.10.010.0080.005
D8S88q22.20.100.02
D8S257q22.30.140.006
D8S281q24.110.05
D10S189p15.10.002
D11S922p15.50.210.02
D11S569p15.30.01
D11S907p130.006
D11S916q14.10.100.01
D11S901q210.01
D12S92q21.20.140.040.007
D12S95q21.330.130.003
D12S338q23.30.180.030.02
D12S366q24.310.070.0003
D12S342q24.320.05
D13S192q12.120.060.03
D13S120q12.120.240.03
D13S153q14.30.140.002
D13S279q21.310.100.05
D13S170q31.10.003
D15S118q15.30.006
D15S207q26.30.01
D17S250q120.070.0030.001
D17S934q21.10.050.04
HOX2Bq21.20.090.030.02
D21S219q22.10.050.03
D21S167q22.20.01

Definition of abbreviations: Eos = eosinophil; IgE = immunoglobulin E; SPT = skin prick test.

*Recombination fraction between this marker and the following one.

Indicates two different regions (markers were assumed to detect different regions of linkage if all p values were nonsignificant over a region of > 20 cM between two markers with significant linkage).

Replication in the Second Set of Families

Table 4 presents the regions that were detected in the first set of families and leading to a p value ⩽ 0.05 by single point linkage analysis in the second set for the same phenotype. Two regions were replicated: the region 11p13 for IgE with p = 0.04 in the second set and p = 0.002 in the whole sample, and 17q12–21 for asthma and SPT with p values ranging between 0.05 and 0.002 in the second set of families and between 0.03 and 0.002 in the whole sample for three linked markers. Moreover, replication of linkage of eosinophil count to 12q24 was almost reached in the second set (p = 0.07) and the p value in the whole sample was 0.0006.

Table 4. REGIONS DETECTED IN THE FIRST SET OF FAMILIES AND REPLICATED IN THE SECOND SET WITH A p VALUE*  ⩽  0.05 FOR THE SAME PHENOTYPE

MarkerPositionPhenotypeFirst SetSecond SetPooled
D11S907p13IgE 0.007 0.04 0.002
D12S366q24.31Eosinophil count 0.0003 0.07 0.0007
D17S250q12Asthma 0.003 0.14 0.005
SPT 0.001 0.15 0.003
HOX2Bq21.2Asthma 0.03 0.05 0.008
SPT 0.07 0.03 0.008
D17S787q21.31Asthma0.30 0.005 0.02
SPT0.10 0.002 0.002

Definition of abbreviations: IgE = immunoglobulin E; SPT = skin prick test.

*  All p values ⩽ 0.05 are in bold type.

Replication of Regions Reported by Published Genome Scans

Table 5 shows the regions reported by previous genome scans, for phenotypes similar to those studied here, that is, asthma, BR, positive SPT response to at least one allergen, total IgE, and eosinophils, which were replicated in our total sample with a p value ⩽ 0.01. In addition to the cytogenetic assignment of these regions, Table 5 presents the genetic location of the linkage peaks (maxima of the test statistics). This location is expressed in cM from pter and was extracted from the Marshfield data base. We should note that it may be difficult to determine whether two peaks belong to the same or different regions, provided the magnitude of their confidence intervals, which often spans more than 20 cM.

Table 5. REGIONS DETECTED IN PUBLISHED GENOME SCREENS AND REPLICATED IN THE 107 FRENCH EGEA FAMILIES

Australian (5)* CSGA (6, 19)* Hutterites (7)* German (8)* French (EGEA)
 1p1p32 (76 cM), 1p34 (113 cM), IgE1p31 (94 cM), asthma,
 asthma, H, W p = 0.0006 (0.005)
11p11p15 (22 cM),11p13 (43 cM), IgE,
 asthma, W p = 0.002 (0.005)
11q11q13 (64–72 cM)11q13 (64–72 cM), IgE,
 IgE, SPT p = 0.008 (0.006)
12q12q14–21 (95 cM),12q15–21 (72 cM),12q13–21 (96 cM),12q24 (133 cM), eosinophils,
 asthma, W loose asthma BR slope, asthma p = 0.0007 (0.0008)
13q13q14–q31 (46 cM)13q32-ter (94 cM),13q31 (64 cM), eosinophils,
 atopy asthma, W p = 0.006 (0.001)
17q17p12–q12 (62 cM),17q12–q21 (62/75 cM), asthma/SPT,
 asthma, B p = 0.003 (0.002)/0.002 (0.001)
19q19q13 (68 cM),19q13 (78 cM),19q13 (88 cM), BR slope,
 asthma, W strict asthma p = 0.01 (0.02)

Definition of abbreviations: B = black group; BR = bronchial responsiveness; H = Hispanic group; IgE = immunoglobulin E; SPT = skin prick test; W = white group.

* Reference numbers.

  Cytogenetic location of linked regions and in parentheses the distance from pter of the linkage peak (maximum of the test statistic) retrieved from http://www.marshmed.org/genetics and http://cooke.gsf.de/asthmagen/main.cfm for markers localization and for linked regions.

p values obtained by single-point analysis and, in parentheses, those obtained by multipoint analysis.

The seven following regions, already reported by previous genome scans, met the replication criterion of p ⩽ 0.01 in our sample: (i) 1p31 for asthma (p = 0.0006), with a peak located half-way (20 cM on each side) between the two peaks reported, respectively, by a secondary analysis of asthma in the CSGA white families (19) and for IgE by the German scan (8); (ii) 11p13 for IgE levels (p = 0.0002), the peak being 20 cM distal to the peak reported for asthma in the CSGA white group (6); (iii) 11q13 for IgE (p = 0.008), which was found linked to IgE and SPT in the Australian sample (5), all peaks belonging to the same region (64–72 cM from pter); (iv) 12q24 linked to eosinophil count (p = 0.0007), with a peak about 37 cM distal to the 12q14–21 region reported for asthma in three samples (white group of CSGA [6], Hutterite pedigrees [7], and German families [8]); (v) 13q31 for eosinophil count (p = 0.006), linked to asthma in the CSGA white sample (6) and to atopy in Australian families (5), with the EGEA peak lying between the Australian and CSGA peaks (20 cM distal to the former and 30 cM proximal to the latter); (vi) 17q12–21 for asthma and SPT (p = 0.002) reported linked to asthma in the CSGA black families (6) with all peaks belonging to a 13 cM region; and (vii) 19q13 for BR slope (p = 0.01), reported for asthma in the CSGA white group (6) and the Hutterites (7), with all three peaks spanning a 20 cM region.

Multipoint Analysis

Multipoint linkage analysis plots are presented in Figure 1 for all phenotypes and chromosomes that were detected by single-point analyses. Most regions led to p values at the maximum multipoint test statistic that were of the same order of magnitude as those obtained by single-point analysis. However, the 13q region reached a higher significance level in multipoint than single-point analysis (multipoint p value = 0.001 versus single-point p value = 0.006) whereas the peak multipoint MLB score on 1p was smaller than the single-point score with D1S209 (multipoint p value = 0.005 versus single-point p value = 0.0006). We checked that the proportion of uninformative families, using the information content criterion as proposed by Kruglyak and coworkers (25), was higher for the two markers D1S197 and D1S216 located at 11.2 and 22.5 cM on each side of D1S209 than for D1S209 itself. However, this observation alone cannot explain our multipoint result as the families uninformative for D1S197 and D1S216 did not contribute much to the single-point statistic obtained with D1S209 in the whole sample.

Search for linkage in the EGEA sample was conducted using a two-stage analysis strategy and by replicating all regions reported by published screens in our whole sample. These two strategies, applied to single and multipoint linkage analyses of asthma and four associated phenotypes (BR, skin prick tests response, total serum IgE levels, and blood eosinophil count), led to the detection of a total of seven regions. Three of these regions were retained by the two-stage analysis: two regions, 11p for total serum IgE levels and 17q for asthma and SPT, were detected in the two independent subsets of families and replication of linkage of eosinophil count to 12q24 was almost reached in the second set (p = 0.07) with a p value in the whole sample of 0.0006. These regions have been reported in the literature (6, 27-29). Linkage results in our whole sample provided confirmatory evidence for four additional published regions: 1p, 11q, 13q, and 19q (5-8, 19, 30-32). We should note that none of the previous scans led to genome-wide significant levels, as proposed by Lander and Kruglyak (17). However, it is by accumulating evidence for linkage across studies that the most likely regions involved in asthma and asthma-related phenotypes can be identified. Many linkages detected at p values of 0.01 (or even greater) may be more meaningful that a single positive result with a p value less than 0.00001.

Examining the three following characteristics, (i) number of studies leading to positive linkages, (ii) degree of overlap of the detected regions, and (iii) similarity of linked phenotypes, can shed some light on these results. Among the seven regions mentioned above, four of them, 1p, 11p, 11q, and 17q, were reported by another genome scan in addition to EGEA, whereas the other three regions (12q, 13q, 19q) were found by at least two scans plus EGEA. The two 11p linkages concerned different phenotypes, IgE in EGEA and asthma in CSGA (6), with peaks distant from 20 cM, but this region was also detected by a recent analysis of the specific IgE response to birch, as part of the German scan (11). This latter result, belonging to the same region as the CSGA peak and 20 cM proximal to the EGEA linkage, was not shown in Table 5 as the specific response to allergens was not considered by our present analysis. The 11q and 17q linkages were found for similar phenotypes, respectively, IgE levels and SPT for 11q and asthma for 17q (plus SPT in the EGEA families) and belonged to the same region on each chromosome. The 11q region was also reported linked to atopy by candidate gene studies and by a recent linkage analysis of this region to the specific response to allergens in CSGA families (32). This region is known to include candidate genes, such as the beta chain of the high-affinity receptor for IgE (FCERB1), playing a central role in IgE-mediated allergic inflammation and shown to be associated with atopy, although the functional genetic variant remains to be identified (33). Tightly linked to FCERB1 is the CC16 gene coding for the Clara cell secretory protein, modulator of inflammation in the airways. The 17q region also contains a few candidate genes including the inflammatory cytokine cluster 1 (ICM1), the SCYA genes coding for small inducible cytokines, the signal transducer and activator of transcription 5 gene (STAT5A), and the colony-stimulating factor 3 (CSF3) and chemokine receptor 7 (CCR7) genes.

Linkages to 1p and 13q were detected by three genome scans but with different phenotypes and spanning at least a 40 cM region. Linkage and association of atopic asthma with markers of chromosome 13q were also recently found in Japanese families (34), the associated marker (D13S153) being tightly linked to the Australian peak and 20 cM proximal to the EGEA peak. The 19q linkages were reported by three genome-wide searches, for similar phenotypes (asthma/BR) and spanning a 20 cM region. This region contains the genes for Secretor status, Lewis blood groups, and the interleukin-11 gene (IL11). Finally, the 12q region has been detected by four scans, three of them reporting linkage to asthma (6-8) and one to eosinophils. The linked markers are spread over the whole chromosome 12q, on which candidate gene studies have also found linkages to asthma and IgE (27-29). Indeed, chromosome 12q contains many candidates genes including those for interferon-gamma (IFNG), mast cell growth factor (MGF), insulin-like growth factor-1 (IGF1), leukotriene A4 hydrolase (LTA4H), and the transcription factors (NFYB and STAT6). The linkage detected in the EGEA families is distal to most published linked regions, except that reported for asthma by a British candidate gene study that spans the same region as EGEA (29). This part of 12q includes the neuronal nitric oxide synthase gene (NOS1), known to have a key role in bronchomotor control in animals. A list of candidate genes in the chromosomal regions detected by our study is presented in Table 6.

Table 6. CANDIDATE GENES LOCATED IN CHROMOSOMAL REGIONS LINKED TO ASTHMA AND ASSOCIATED PHENOTYPES IN THE EGEA STUDY

Chromosomal LocationCandidate Gene(s)Function
1p35–p31Colony-stimulating factor 3 receptor (CSF3R)Receptor for colony-stimulating factor 3, involved in granulopoiesis
 during the inflammatory process
Interleukin-12 receptor, subunit B2 (IL12RB2)Receptor for interleukin-12, a Th1-associated cytokine
 produced by B cells and macrophages
11q12–q13Fc fragment of IgE high-affinity receptor (FCER1B)Responsible for initiating the allergic response acting as a
 trigger on mast and other cells
Clara cell secretory protein (CC16 or CC10)Modulator of inflammation in the airways
12q13–q24Signal transducer and activator of transcription 6 (STAT6)Involved in interleukin-4-induced commitment of CD4+ T cells
 to the Th2 type and IgE isotype switching in B cells
Interferon-gamma (IFNG)Promotes the differentiation of Th1 lymphocytes and inhibits
  differentiation and IL-4 production in Th2 cells
Mast cell growth factor (MGF or SCF )Controls proliferation of hematopoietic stem cells and mature
 mast cells
Leukotriene A4 hydrolase (LTA4H)Involved in prostaglandin metabolism and the inflammatory response
Insulin-like growth factor 1 (IGF1)Promotes the differentiation of both B and T lymphocytes
Nuclear transcription factor Y, beta chain (NFYB)Up-regulates the transcription of both IL-4 and HLAD genes
Nitric oxide synthetase 1 (NOS1)Acts as an inhibitory mechanism to cholinergic bronchoconstriction
 in the airways
13q22Endothelin receptor type B (EDNRB)Receptor for endothelins, potent vasoactive peptides
17q11–q21Inflammatory mediatory cytokine cluster 1 (IMC1)Mediation of inflammation
Signal transducer and activator of transcription 5A (STAT5)Involved in cytokine receptor-mediated homeostatic control
 of the hematopoietic system
Small inducible cytokines (SCYA5 or RANTES)Chemokine activating T cells and dentritic cells
Colony-stimulating factor 3 (CSF3)Stimulates the proliferation and differentiation of the progenitor
 cells for granulocytes
Chemokine receptor 7 (CCR7)Involved in recognition of chemoattractants such as IL-8, RANTES
19q13Fucosyltransferase 2 (FUT2)Controls the expression of ABH histoblood group in exocrine
 secretions (secretor status)
Fucosyltransferase 3 (FUT3)Lewis histoblood group
Interleukin-11 (IL11)Stimulates T-cell-dependent development of B cells

Definition of abbreviations: IgE = immunoglobulin E; IL = interleukin.

Whereas 11q, 12q, and 13q have been now replicated by many studies (33), 17q and 19q regions, which lead to consistent results in EGEA and other samples, appear to be new regions of interest that need to be further investigated. On another hand, other linkages reported by genome-wide searches on chromosomes 2q, 4q, 5q, 6p, 7p, 14q, 16q, and 21q (5-8) were not detected in our families.

Genome screens and candidate gene studies now show that a number of regions are potentially linked to asthma and asthma-related phenotypes and stress the difficulty of discriminating between false and true positive results. A few positive linkages have been identified consistently by different groups, including chromosomes 5q, 6p, 11q, 12q, and 13q (33), the three latter being replicated in the EGEA sample. However, discordance among other results may be partly explained by differences among populations made of various ethnic groups and living in different environments, differences among study designs, including sample sizes, type of family structure, and mode of ascertainment, differences in the definition of the phenotypes and the clinical characteristics of the samples, and differences in the methods of analysis. Multiple positive results may also reflect the complexity and heterogeneity of asthma and intermediate phenotypes, which may be controlled by many genes, some of them being common and other ones specific to these phenotypes.

To progress in the understanding of the genetic and environmental determinants of asthma and atopy, our future plans include extension of the genome scan to the whole EGEA sample and pooling our data with other European groups. Characterization of regions of major importance in large samples will be followed by linkage disequilibrium mapping toward gene identification. We will pay particular attention to get precise definition of the phenotypes and to adjust those phenotypes for relevant risk factors, concomitantly investigated in our data. Pooling data will also make it possible to use more sophisticated analyses that can take into account heterogeneity of disease, oligogenic models, and gene–environment interactions.

EGEA Cooperative Group

Respiratory epidemiology: I. Annesi-Maesano, F. Kauffmann (co-ordinator), M. P. Oryszczyn (INSERM U472, Villejuif); F. Neukirch, M. Korobaeff (INSERM U408, Paris).

Genetics: M. H. Dizier, J. Feingold (INSERM U155, Paris), F. Demenais (INSERM EPI 00-06 (ex-U358), Paris), M. Lathrop (Centre National de Génotypage, Evry, France).

Clinical centers: Grenoble: I. Pin, C. Pison; Lyon: D. Ecochard (deceased), F. Gormand, Y. Pacheco; Marseille: D. Charpin, D. Vervloet; Montpellier: J. Bousquet; Paris Cochin: A. Lockhart, R. Matran (now in Lille); Paris Necker: E. Paty, P. Scheinmann; Paris-Trousseau: A. Grimfeld.

Data management : J. Hochez (INSERM U155), N. Le Moual (INSERM U472).

Supported by convention INSERM-MSD, INSERM networks of clinical research (489012) and public health research (493009), and French Ministry of Research (ACCSV2/1A028A). Kits for IgE and Phadiatop determinations were kindly provided by Pharmacia.

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Correspondence and requests for reprints should be addressed to Marie-Hélène Dizier, Unité INSERM U535, Bâtiment INSERM Gregory Pincus, 80 rue du Général Leclerc, 94276 Le Kremlin Bicêtre Cedex, France. E-mail:

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