INTRODUCTION

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Asthma is a complex disorder involving immunologic, genetic, environmental, and other factors. Allergic sensitization appears to be the strongest identifiable predisposing factor for asthma (1). Nonallergic individuals, however, are also affected by asthma. Recent studies have suggested that the inflammatory reaction of the airways in asthma, accompanied by recruitment and activation of eosinophils and T lymphocytes, is the cardinal feature of the disease (2, 3).

Since family and twin studies have demonstrated an involvement of genetic components in the development of asthma and other allergic diseases (4, 5), much work has been done to find the responsible gene(s). Cookson and colleagues (6) reported linkage of a broad definition of atopy to marker D11S97 on chromosome 11q13. Subsequently, it was reported that variants in the high-affinity IgE receptor gene (FcRI), located centromeric to the D11S97 marker, play an important role in the triggering mechanism of allergic reactions in families in which atopy is maternally transmitted (7). Marsh and colleagues (8) showed a linkage between total serum IgE and markers on chromosome 5q31-q33, which contains the interleukin-3 (IL-3), IL-4, IL-5, IL-13, and granulocyte-macrophage colony stimulating factor (GM-CSF) genes. Postma and coworkers (9) reported the linkage of bronchial hyperresponsiveness (BHR) to chromosome 5q31-q33. Since cytokines encoded by the genes in this region play a crucial role in IgE production and the inflammatory process associated with BHR (3, 10), it is likely that the atopy-related candidate gene(s) is present on chromosome 5q31-q33.

Asthma is a genetically complex disease, and genetic heterogeneity in this disease is likely. Few studies of linkage between asthma and atopy in non-Caucasian populations have been reported. In addition, linkage studies of asthma in childhood have not been reported. In this study, we investigated linkages of asthma and atopic phenotypes to the markers on chromosomes 5q31-q33 and 11q13 among Japanese.

	METHODS

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Subjects

Probands of the families studied were among asthmatic children visiting the Pediatric Allergy Clinic of the University Hospital of Tsukuba. Families for the study were ascertained between February and October 1996. A full verbal and written explanation of the study was given to all of the family members interviewed, and 68 families (306 members) who were so inclined gave informed consent and were included in this study. Informed consent for subjects younger than school age was given by their parents. The study was approved by the Committee of Ethics of the University of Tsukuba.

Each family member was questioned about allergic symptoms, and physical examination was done by one or the other of two pediatricians. Asthma was diagnosed in subjects according to the criteria of the U.S. National Institutes of Health, with minor modifications (11). Patients had to show both of the following characteristics: (1) recurrent episodes of wheezing and shortness of breath during the preceding year; and (2) wheezing and dyspnea that were reversible, either spontaneously or with the use of a bronchodilator. Since wheezing is often associated with viral respiratory infection in young children (12), subjects older than 3 yr of age were evaluated for their asthma phenotype. Patients treated with systemic steroids were excluded from the study. The mean age of the probands and their siblings was 10.5 yr (range: 1 to 29 yr). All of the probands (68 subjects) and 33 (32%) of the siblings (a total of 101 of 171 siblings) had asthma. Young-adult patients included in the study had had chronic asthma since childhood. The mean age of the parents was 41.6 yr (range: 28 to 72 yr), and 22 (16%) had asthma or a past history of asthma. The geometric mean total serum IgE was 670 U/ml (range: 1.7 to 4,180 U/ml) for children and 227 U/ml (range: 2.7 to 2,053 U/ml) for parents.

Determination of Total Serum IgE and Specific IgE Antibody Levels

Total serum IgE levels were determined with the CAP System (Pharmacia, Uppsala, Sweden). Levels of IgE specific to Dermatophagoides farinae, D. pteronyssinus, cat and dog danders, Cryptomeria japonica, and orchard grass were also determined with the CAP System.

Molecular Methods

DNA was extracted from peripheral blood leukocytes collected in ethylene diamine tetraacetic acid (EDTA). Polymerase chain reaction (PCR) was performed, using primers for the markers on chromosomes 5q31-q33 and 11q13. The PCR cocktail contained 10 mM Tris-HCl, pH 9.0; 50 mM KCl; 1.5 mM MgCl₂; 0.1% Triton X-100; 200 µM of each of deoxynucleotide triphosphates (dNTPs); 0.25 U Taq DNA polymerase (Promega Ltd., Madison, WI); (-³²P) cytosine triphosphate, and approximately 40 ng of template DNA in a total volume of 10 µl, and the mixture was overlayed with 20 µl of mineral oil. PCR was performed in a 96-well plate, using a programmable thermal cycler (PTC-100; MJ Research, Inc., Watertown, MA) at 93° C for 5 min, followed by 25 cycle at 93° C for 30 s; annealing at a temperature of 57° C for 45 s; and an extension at 72° C for 45 s; followed by treatment at 72° C for 3 min. The PCR primers used were D5S393 and D5S436 from Genom Data Base, IL-4 and IL-9 from Marsh and colleagues (8), and FcRI from Daniels and Shirakawa (13). This mixture was denatured and run on a 6% urea polyacrylamide gel at 60 W for 2 h to 4 h. The gels were dried and autoradiographed with a Fujix BAS2000 Imaging Analyzer (Fuji Photo Film, Tokyo, Japan). Genotypes were determined from two independent readings of each autoradiograph. Individuals genotyping the families were blinded to the clinical data.

Linkage Analysis

An atopic phenotype of each proband was determined by either or both of the following criteria: a total serum IgE level more than 1 SD above the geometric mean for normal Japanese, and/or increased serum levels of specific IgE for one or more of the following allergens: D. farinae, D. pteronyssinus, cat and dog danders, C. japonica, and orchard grass. A positive response was taken as a radioallergosorbent test (RAST) score of 2 or greater (0.7 UA/ml). By this definition, 85 atopy-affected sib-pairs, as well as 46 asthma-affected sib-pairs, were analyzed. There were 27, 3, and 1 families with 2, 3, and 5 asthmatic sibs, respectively, and 27, 14, 1, and 1 families with 2, 3, 4, and 5 atopic sibs, respectively. Linkage analyses of affected (qualitative) sib-pairs were done with the SIBPAL program of the Statistical Analysis for Genetic Epidemiology (S.A.G.E.) statistical software package (14). In addition, linkage to IgE levels was calculated on the basis of a regression for the squared difference in log(total IgE) levels between sibs and the estimated proportion of marker alleles identical by descent according to the SIBPAL program. Sib-pairs were included in the linkage analysis only when marker genotypes were known. Families in which all members examined were of the same genotype were excluded from linkage analysis. By this procedure, 1 (IL-4) to 11 families (D5S436) were excluded. Sibships containing more than one pair of sibs were weighted (15). Allele frequencies of the markers were calculated from genotypes in the parents of the families studied.

	RESULTS

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Table 1 shows the results of linkage analysis of asthma and atopy as qualitative traits with the markers examined in the study. There was statistically significant evidence for linkage of asthma with the markers IL-4, IL-9, and D5S393 on chromosome 5q31-q33 (p = 0.0013, p = 0.018, and p = 0.0077, respectively) (Table 1). However, we did not find evidence for linkage of asthma with the marker FcRI on chromosome 11q13 (p = 0.43). As for atopy, there was also statistically significant evidence of linkage with the markers IL-4, IL-9, and D5S393 on chromosome 5q31-q33 (p = 0.006, p = 0.01, and p < 0.0001, respectively), but not with FcRI on chromosome 11q13 (p = 0.26).

Linkage analysis of log(total IgE) levels as a quantitative trait with the markers also supported linkage with the markers on chromosome 5q31-q33. The analysis was repeated without pairs excess differences (values > mean ± 3 SD), resulting in similar p values (Table 2).

	DISCUSSION

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The subjects examined in this study were children and young adults. Parametric linkage analysis was considered inappropriate because of incomplete penetration of atopy and asthma. We analyzed linkage with the affected sib-pair method in order to avoid misdiagnosing any subjects as falsely unaffected. The affected sib-pair method applied to a large number of small families also offers an advantage because they are likely to be more representative of the population than a small number of large families. However, the method provides decreased precision for determining the location of the genes contributing to susceptibility to atopy and asthma in comparison with parametric linkage analysis.

Since there is yet no readily applicable and validated biomarker for airway inflammation and asthma, definition of an asthma phenotype depends largely on patient symptoms. Cystic fibrosis (CF) and bronchiectasis in children, and chronic obstructive pulmonary disease (COPD) in middle-aged or older adults, have some of the same clinical manifestation as asthma (16). However, CF and bronchiectasis are rare in the Japanese. Since infants with viral respiratory tract infection often wheeze (12), we analyzed patients over 3 yr of age in order to evaluate the asthma phenotype in this study.

The present study provides supportive evidence for linkage of the asthma phenotype to markers on chromosomes 5q31-q33 in Japanese families. In addition, our results add evidence suggesting linkage of atopy to this genomic region. In contrast, we failed to find evidence of linkage of asthma or atopic phenotypes to the marker FcRI on chromosome 11q13.

The chromosome 5q region contains the genes for IL-3, IL-4, IL-5, IL-9, IL-13, GM-CSF, colony stimulating factor-1 receptor, and acidic fibroblast growth factor. IL-4 is a key cytokine involved in the regulation of IgE synthesis, providing a signal for immunoglobulin class-switching (17). Cytokines such as IL-3, IL-5 and GM-CSF are known to prolong eosinophil survival and to enhance its activity (18), resulting in progression of the inflammation of asthmatic airways. It is therefore likely that the chromosome 5q region contains nucleotide variants affecting the functions of these candidate genes or contains unknown genes leading to susceptibility to asthma and atopy.

Marsh and coworkers (8) first reported significant linkage of total serum IgE antibody concentrations to IL-4 or a nearby gene in chromosome 5q31.1 in 11 Caucasian Amish families. Studies in Dutch families, selected through a proband with asthma who was 45 yr of age or younger, showed linkage of total serum IgE levels to the markers on chromosome 5q (19, 20). The maximum score for the odds favoring genetic linkage (lod) was obtained with the genetic marker D5S436, which is distal to IL-4 by approximately 10 to 12 cM according to the Genome Data Base, with 9% recombination. It was also reported that BHR is coinherited with atopy in the same genomic region (9). To the contrary, studies in Australian and British random populations (21, 22) and in large atopic families in Minnesota (23) failed to find a linkage of IgE and/or BHR to chromosome 5q31-q33. These inconsistencies may be attributable to differences in populations, such as ethnicity and ascertainment of the families studied, and/or to differences in definition of phenotypes, although no definite data on the causes of these inconsistencies have been presented.

The evidence in our study of linkage of the asthmatic phenotype to chromosome 5q31-q33 is noteworthy. This may be associated with the fact that allergic factors contribute greatly to the development of asthma in patients such as ours. Epidemiologic studies, however, have shown that allergic sensitivities are commonly found in a considerable number of nonasthmatic subjects (24), suggesting that nonallergic factors are deeply involved in the expression of asthma even in allergic asthmatic individuals. Furthermore, it has been shown the bronchial reactivity of asthmatic patients is closely related to the degree of eosinophilic inflammation of their airways (10). Traits of BHR and increased levels of total IgE are coinherited and mapped to chromosome 5q31-q33 (9). It is therefore likely that plural loci in the chromosome 5q31-q33 region are synergistically related to asthma susceptibility.

Cookson and associates (6) have reported evidence of linkage to chromosome 11q13 markers, including FcRI, of the atopic phenotype, which was defined by positive skin-prick tests, positive RAST reactivities, and/or increased total serum IgE in British populations. Some studies confirmed the linkage to chromosome 11q13 (25, 26), but others failed to replicate this finding (27). Van Herwerden and colleagues (31) have shown linkage between FcRI and clinical asthma in the Australian population even in the absence of atopy. In Japanese populations, results confirming (26) and contradicting (29) linkage of the atopic phenotype to chromosome 11q13 have been reported. Our affected sib-pair analyses failed to detect linkage of asthma or atopy to FcRI. The inconsistency in the results of these various studies may be due to genetic heterogeneity and/or differences in linkage analytic methods.

The IL-4 gene and nearby genes on chromosome 5q31-q33 have been shown to be linked to the atopic phenotype and BHR in Caucasian populations, and with the present study, probably also in the Japanese. This study presents further evidence for a role of these loci in the etiology of asthma, and suggests similarities in the genetic basis of atopy in different ethnic groups.