Linkage between IgE receptor mediated histamine releasability from basophils and gene marker of chromosome 11q13

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1 Linkage between IgE receptor mediated histamine releasability from basophils and gene marker of chromosome 11q13 Yoon-Keun Kim, MD, a,c Sang-Heon Cho, MD, a,c Young-Yull Koh, MD, b,c Jee-Woong Son, MD, a,c Byung-Jae Lee, MD, a,c Kyung-Up Min, MD, a,c and You-Young Kim, MD a,c Seoul, Korea Background: The genetics of the regulation of the release of mediators involving the interaction of IgE with cells and their ability to release mediators have not been extensively investigated. With use of the candidate gene approach, it was reported that the gene regulating the β chain of the high-affinity receptor for IgE is on chromosome 11q13. Objective: To determine whether gene(s) in chromosome 11q13 may control the expression of maximal histamine release from basophil to anti-ige stimuli, linkage analysis between this phenotype and the gene marker of chromosome 11q13 was performed. Methods: Maximal histamine release to anti-ige and calcium ionophore A23187 and genotyping chromosome 11q13 with use of microsatellite marker (D11S97) were performed in 56 probands with asthma and 59 of their siblings. The linkage was analyzed by affected sib-pair analysis and the quantitative trait locus approach. Results: Maximal histamine release (mean ± SE) to anti-ige and A23187 was 43.3% ± 3.5% and 30.9 ± 3.4% in probands and 29.5% ± 2.6% and 22.2% ± 2.7 in siblings, respectively. Of 20 sib-pairs with the maximal histamine release to anti-ige more than 33% (mean plus 1 SD of nonasthmatic controls), 11 (55%) shared 2 D11S97 alleles, 9 (45%) shared 1 allele, and neither sib-pair shared identical alleles, which indicates a significant linkage of maximal histamine to anti-ige and gene marker of chromosome 11q13 (P =.02). The difference (mean ± SE) of the maximal histamine release to anti-ige between each proband and sibling was smaller in sib-pairs with 2 identical alleles than in those with 1 identical allele and with no identical allele (14.1% ± 2.6% vs 25.8% ± 3.1% vs 41.0% ± 4.9%). However, the difference (mean ± SE) to A23187 between each proband and sibling was not different among the 3 groups (9.7% ± 1.8% vs 17.9% ± 3.6% vs 10.4% ± 4.8%). Conclusion: Maximal histamine release from basophils to anti- IgE stimuli was linked to the gene marker of chromosome 11q13. (J Allergy Clin Immunol 1999;104: ) From the Departments of Internal Medicine a and Pediatrics, b Seoul National University College of Medicine, and the Institute of Allergy and Clinical Immunology, Seoul National University Medical Research Center, c Seoul, Korea. Supported by a research grant from the 1996 National R&D Program (MOST), Republic of Korea. Revision received May 19, 1999; accepted for publication May 19, Reprint requests: You-Young Kim, MD, Department of Internal Medicine, Seoul University College of Medicine, 28 Yungun-dong, Chongno-gu, Seoul, Korea. Copyright 1999 by Mosby, Inc /99 $ /1/ Key words: Basophil histamine release, chromosome 11q13, asthma, IgE-receptor, linkage analysis Allergic disorders are characterized pathophysiologically by enhanced IgE production, increased mediator release from inflammatory cells, and hypersensitivity of target organs. It is well known that IgE plays an important role in the development of allergic diseases and that IgE production is enhanced in the majority of patients with bronchial asthma. 1 IgE antibody response to antigen is the classic means of distinguishing allergic individuals from nonallergic subjects. 2 Allergic individuals have a higher serum IgE level and a higher number of IgE receptors per basophil than do normal subjects. 3 Although many of the manifestations of allergic disorders are mediated by antigen-ige antibody interactions, 4 others are not. 5 Therefore other immune and inflammatory abnormalities have been recruited to account for non-antigen-mediated pathophysiologic features: among these are end-organ hyperresponsiveness and abnormalities of releasability from inflammatory cells. 6 The term releasability implies that, in addition to the surface density of IgE molecules, biochemical events in basophils determine the capacity of basophils to release mediators in response to activating stimuli. 6 Measuring the amount of histamine release from basophils is used to evaluate mediator releasability. It was known that basophil release to anti-ige was significantly higher in asthmatic patients than in those with allergic rhinitis and in nonasthmatic control subjects. 7 Nevertheless, IgEdependent histamine release (HR) from basophils has not been clearly established as being under genetic control in asthma. Marone et al 8 estimated correlations in histamine release from basophils in response to anti-ige, f-met peptide, and the calcium ionophore A23187 in a twin study. The correlation among monozygotic twins was significantly greater than among dizygotic twins for anti-ige and A23187 but not for f-met peptide. These data suggest that anti-ige release and calcium ionophore release of histamine were determined in part by genetic factors, whereas genes probably had little effect on the release of histamine from f-met peptide. However, the genetic factors controlling IgE-receptor mediated HR from basophils or mast cells have not been evaluated. High-affinity receptors on basophils and mast cells have been identified and characterized. With use of the

2 J ALLERGY CLIN IMMUNOL VOLUME 104, NUMBER 3, PART 1 Kim et al 619 Abbreviations used ACMD: Albumin-calcium-magnesium-dextrose FcεRI: High-affinity IgE receptor HR: Histamine release PIPES: 1,4-Piperazinediethanesulfonic acid QTL: Quantitative trait locus SPT: Skin prick test candidate gene approach, Sandford et al 9 reported that the gene regulating the β chain of the high-affinity receptor for IgE (FcεRI-β) is on chromosome 11q13. The aim of this study was to evaluate the linkage between IgEreceptor mediated HR from basophils and gene marker of chromosome 11q13, where the FcεRI-β gene is located. METHODS Probands were recruited consecutively in the year 1996 from children between 4 and 15 years old attending the pediatric clinic at Seoul National University Children s Hospital. Asthmatic children had a history of episodic wheezing or breathlessness and showed reversible airway obstruction on treatment with a bronchodilator (more than 15% of predictive value of FEV 1 ) or bronchial hyperresponsiveness to methacholine (provocative concentration of methacholine needed to increase baseline lung resistance by 20% <12 mg/ml). Siblings of the probands were asked if they would participate in the study. The parents gave written informed consent for their children to participate in this study; only 3 eligible siblingpairs declined. Probands and their siblings underwent skin prick tests (SPTs) with 10 inhalant allergens common in Korea and gave blood specimens for the measurement of maximal HR from basophils, genotyping, and total serum IgE concentration. Maximal HR was also measured in the age-matched nonatopic nonasthmatic control subjects, who visited the pediatric clinic for nonallergic problems, to determine the affected phenotype of maximal HR. All control subjects had no clinical history of asthma, allergic rhinitis, and atopic dermatitis; had normal results on physical examination and spirometry; and had a negative skin response to common inhalant allergens on SPT. The study protocol was approved by the Hospital Ethics Committee. SPTs were performed with use of a panel of 10 allergens (Bencard). None of the subjects had received antihistamines orally for the 3 days preceding the study. The panel included house dust mite (Dermatophagoides pteronyssinus, Dermatophagoides farinae), cat fur, molds (Aspergillus spp, Alternaria spp), pollens (tree pollen mixture, grass pollen mixture, mugwort, and ragweed), and cockroach. A positive control of histamine (1 mg/m) along with a negative diluent control was included in all tests. After 15 minutes the mean diameter of a wheal formed by the allergen was compared with that formed by histamine. If the former was the same or larger than the latter (allergen/histamine ratio 1.0), the reaction was defined as positive. Total serum IgE levels were determined by ELISA. Polypropylene 96-well microtiter plates were precoated overnight at 4 C with mouse monoclonal antihuman IgE (Sigma, St Louis, Mo) in carbonate buffer of 1:3000 titer and were washed 3 times with 10 mmol/l PBS containing Tween-20 (0.05% Tween-20 PBS, ph 7.4). After washing, 3% BSA (Sigma) in 0.05% Tween-20 PBS was added and the plate was incubated for 1 hour to eradicate nonspecific background reaction; this was followed by 3 washes with 0.05% Tween-20 PBS, and 100 µl of serum, diluted 10-fold, was added to each well in duplicate for 1 2-hour incubation. After another 3 washes, peroxidase-conjugated antihuman IgE (optimally diluted at 1:1000 in 0.1% BSA 0.05% Tween-20 PBS) was added to each well and incubated for 1 hour at room temperature. After 5 washes, 100 µl of o-phenylene-diamine dihydrochloride (Sigma) was added to each well. The plate was incubated at 37 C for 1 hour and the reaction was terminated by the addition of 100 µl of 3 mol/l hydrogen sulfate. OD was determined at 490 nm in a Ceres 900 ELISA microtiter plate reader (Bio-Tek Instruments). Each sample was assayed in duplicate and the mean of results for each sample was taken. The minimum level of detection was 4 IU/mL. The coefficient of variation between assays was 14%. Peripheral blood leukocyte isolation Twenty milliliters of heparinized peripheral blood was collected from the antecubital vein of the subject involved and mixed with a solution consisting of 2 ml of 0.1 mmol/l EDTA and 5 ml of 6% dextran 2% dextrose 0.9% sodium chloride solution. This was followed by incubation for 90 minutes at room temperature. To obtain a pellet, supernatant containing plasma, leukocytes, and platelets was transferred to another tube and centrifuged at 4 C at 300g for 8 minutes; the pellet was washed twice with 10 ml of 1,4-piperazinediethanesulfonic acid (PIPES) buffer containing 4 ml of 0.1 mol/l EDTA. Finally, 5 ml of leukocyte suspension was prepared with PIPES albumin-calcium-magnesium-dextrose ((ACMD) buffer (ph 7.4) containing 0.03% albumin, 0.1 mol/l Ca ++, 0.1 mol/l Mg ++, and 1% dextrose. Maximal HR release to anti-ige and calcium ionophore A23187 After 0.2 ml of leukocyte suspension was added to 0.7 ml of PIPES-ACMD buffer, 0.1 ml of different concentrations of histamine-releasing stimulus were challenged for 15 minutes at 37 C. Supernatants were obtained after centrifugation at 4 C at 700g for 15 minutes and stabilized with 0.1 ml of 55% trichloroacetic acid; this was followed by freezing at 70 C for histamine assay. Spontaneous HR was measured with PIPES-ACMD buffer instead of stimulating agent, and total HR was measured after cell lysis. As stimuli 2 different agents, goat antihuman IgE (Sigma) and calcium ionophore A23187 (Sigma), diluted with PIPES-ACMD buffer at 3 different concentrations (anti-ige: 1:1000, 1:100, 1:10; calcium ionophore: 1 mmol/l, 3 mmol/l, 10 mmol/l) were used. Histamine assay An automated fluorospectrometric assay was used to measure the amount of histamine released. After alkalinization of the solution, histamine was extracted with butanol and liquified by 0.1N hydrochloric acid. Liquified histamine was condensed with 0.025% o-phthalaldehyde and stabilized by 0.1N phosphoric acid. With use of a fluorometer (primary filter 355 nm, secondary filter 455 nm), fluorescence was determined. HR was calculated according to the following equation: HR (%) = HR to stimuli Spontaneous HR Total HR Spontaneous HR 100 Maximal HR (%) was defined as the highest value of HR found at 3 different concentrations of anti-ige and A Maximal HR was defined to elevate if the value was higher than 33%, which was the mean plus 1 SD of nonatopic nonasthmatic control subjects. Genotype of chromosome 11q13 using microsatellite marker (D11S97) Genomic DNA was obtained from 5 ml of peripheral blood of all subjects by a DNA extraction kit (Guachem) as recommended by

3 620 Kim et al J ALLERGY CLIN IMMUNOL SEPTEMBER 1999 TABLE I. Characteristics of study subjects Probands (n = 56) Siblings (n = 59) Control subjects (n = 31) Male/female ratio 39:17 29:30 20:11 Age (y) (mean [range]) 8.9 (4-15) 9.0 (3-18) 9.0 (5-15) LogIgE (IU/mL) (mean ± SE) 2.51 ± 0.07* 2.38 ± ± 0.11 SPT (%) 78.5 (44/56) 57.4 (31/54) 0 HR-IgE (%) (mean ± SE) 43.3 ± 3.4* 29.5 ± ± 3.4 Affected HR-IgE (%) 50 (23/56) 45.8 (27/59) HR-Ca (%) (mean ± SE) 30.9 ± ± ± 4.7 LogIgE, Log (total serum IgE); SPT, positive test result (A/H ratio 1.0) to one or more of 10 common inhalant allergens; HR-IgE, maximal histamine release from basophils to anti-ige; Affected HR-IgE, maximal histamine release from basophils higher than 33%; HR-Ca, maximal histamine release from basophils to calcium ionophore A *P <.01 compared with nonatopic nonasthmatic controls. P <.05 compared with nonatopic nonasthmatic control subjects. TABLE II. D11S97 alleles shared by 20 affected sib-pairs with enhanced maximal histamine release from basophils to anti-ige No. of sib-pairs Percent Identical allele Observed* Expected Observed* Expected Two alleles One allele None *Sharing rate of D11S97 alleles in 20 sib-pairs with enhanced maximal histamine release from basophils ( 33%) was 75.5%, which indicates linkage between maximal histamine release after anti-ige stimuli and gene marker of chromosome 11q13 (chi-square test: P =.02). the supplier. Genotypes were determined by PCR for a microsatellite marker (D11S97) close to FcεR-I-β loci. The primer sequence was as follows: D11S97F: 5 -GGCAGGGTCCCTTTCAGCGTCTCATC- CACAGT-3 D11S97R: 5 -TTTTCAGGAGGTCAGGCTGTGGCTGTAG- GTCG-3 Each 35 µl reaction mixture contained 2.5 µl of human genomic DNA (200 ng), 0.5 µl of Taq polymerase (5 U/µL, Boehringer Mannheim), 1 µl of 10 pmol D11S97F and D11S97R, with the buffer recommended by the supplier (final concentration 1.5 mmol/l magnesium chloride). Amplification conditions were 30 cycles of 94 C for 30 seconds and 68 C for 7 minutes after initial denaturation at 94 C for 7 minutes. The reactions were terminated at 4 C after a final extension step at 68 C for 7 minutes. Amplified products were resolved on 1% agarose gels with ethidium bromide. Sizes of the alleles were determined by electrophoresis of the above products and size markers. D11S97 alleles were independently assigned by 2 investigators. Statistical analysis Results were expressed as mean ± SEM unless otherwise indicated. The chi-square test and the Student t test were used to assess statistical differences between the 2 groups, and analysis of variance was used to make comparisons among the 3 group means. Correlation between continuous variables was assessed by regression analysis. A P value of.05 or less was regarded as significant. Sib-pair analysis is a nonparametric test that enables an investigator to assess the evidence for linkage without having to define the mode of inheritance or other parameters. 10 The analysis is usually done on pairs of affected siblings within a family. A group of affected sibling pairs is genotyped for a particular marker, and if it is unlinked to the disease gene there is a 50:50 chance that each pair will share a specific marker allele. Any significantly greater level of allele sharing more than 50% indicates linkage of the marker to the disease gene. The significance of the deviation away from the expected degree of sharing can be assessed with the chi-square test. However, for complex genetic disorders in which multiple genes may contribute, it may not be possible to define a categorical phenotype. For the most part, the quantitative phenotypes that have been used as intermediate phenotypes of asthma and allergic diseases, such as total serum IgE and maximal HR, display a continuous distribution; the affected individuals are defined as those exceeding some threshold at the extreme of the distribution. The quantitative trait locus (QTL) approach allows the entire range of values to be used in the analysis, 11 and this approach can be used in sib-pair analysis. 12 For sib-pair analysis, the difference between siblings for a continuous variable can be used as the quantitative trait and compared with the sharing of alleles at one or more loci. 13 In this study linkage of maximal HR to anti-ige was assessed by both affected sib-pair analysis and the QTL approach, and, as for A23187, QTL sib-pair analysis was used because of difficulty in defining affected individuals. RESULTS Fifty-six probands and their 59 siblings participated in the study. Maximal HR to anti-ige and A23187 was also measured in 31 age-matched nonatopic nonasthmatic controls. Table I summarizes the clinical characteristics and maximal HR to anti-ige and A23187 of the probands, siblings, and control subjects. Mean age and sex ratio did not differ among the 3 groups. The mean (in International units per milliliter) of log (total serum IgE) was higher in the probands and siblings than in nonasthmatic controls (2.51 ± 0.07 vs 2.38 ± 0.10 vs 1.66 ± 0.11). As for SPT, 78.5% of the probands and 57.4% of the siblings showed positive skin responses to common inhalant allergens. The maximal HR (mean ± SD) to anti-ige was higher in probands than in nonasthmatic controls (43.8% ± 26.2% vs 18.1 ± 15.1%, P =.0001). The maximal HR to anti-ige was higher than 33% (mean + 1 SD of nonasthmatic controls) in 50% of the probands and 45.8% of the siblings. The maximal HR (mean ± SD) to A23187 was not different among the 3 groups (30.9% ± 25.2% vs 22.2% ± 21.0% vs 35.0% ± 27.2%). The maximal HR to

4 J ALLERGY CLIN IMMUNOL VOLUME 104, NUMBER 3, PART 1 Kim et al 621 anti-ige showed a significant association between the probands and the siblings (correlation coefficient 0.36, P =.005). As for A23187 stimuli, the association was more marked between the probands and the siblings (correlation coefficient 0.67, P =.0001). However, there were no significant associations between maximal HR to anti-ige and to A23187 in both the probands and the siblings (correlation coefficient 0.25, P >.05; correlation coefficient 0.20, P >.05, respectively). In 59 sib-pairs, 20 sib-pairs (33.8%) shared 2 D11S97 alleles, 28 (47.4%) shared 1 allele, and 11 (18.6%) shared neither allele. Table II shows D11S97 alleles shared by 20 affected sib-pairs whose maximal HR to anti-ige was higher than 33%. In these 20 sib-pairs, 11 sib-pairs (55%) shared 2 D11S97 alleles, 9 (45%) shared 1 allele, and none of affected sib-pairs shared neither allele. By affected sib-pair analysis, there was a significant linkage between maximal HR to anti-ige and D11S97 alleles (P =.02). Fig 1 shows the difference of maximal HR to anti-ige and to A23187 between each proband and sibling, respectively. As for anti-ige stimuli, the difference between sib-pairs with 2 identical alleles was smaller than with 1 identical allele and neither identical allele, and smaller between sib-pairs with 1 identical allele than with neither identical allele (14.1% ± 2.6% vs 25.8% ± 3.1% vs 41.0% ± 4.9%). As for calcium ionophore A23187, however, there were no significant differences among the 3 groups (9.7% ± 1.8% vs 17.9% ± 3.6% vs 10.4% ± 4.8%). DISCUSSION In this study we have found that maximal HR to anti- IgE stimuli is linked to the gene marker (D11S97) of chromosome 11q13 by the affected sib-pair analysis and the QTL approach. However, maximal HR to A23187 was not linked to D11S97 alleles by QTL sib-pair analysis. Allergic disorders are characterized pathophysiologically by enhanced IgE production, hypersensitivity of target organs, and increased mediator release from inflammatory cells. Human basophils and mast cells play a central role in allergic disease. On the membrane of basophils and mast cells, receptors are localized that are capable of binding with high-affinity to the Fc region of IgE antibodies. Basophils and mast cells release inflammatory mediators after binding of allergens to the IgE, which causes immediate and long-term physiologic effects. 14 The involvement of basophils has been proposed in allergic diseases such as anaphylaxis, allergic rhinitis, asthma, atopic dermatitis, and urticaria. 15 Recently, the appearance of basophils at sites of the allergic late phase that might indicate an important role of these cells in asthma has been described. 16 The concept of mediator releasability arose in the 1970s, when Lichtenstein and Conroy 17 suggested that in the pathogenesis of allergic disorders not only the amount of allergen-ige coupling signal but also the FIG 1. Difference of maximal histamine release from basophils to anti-ige and calcium ionophore A23187 between each proband and sibling. Asterisk, P <.05 compared with sib-pairs with 1 and no identical allele; two asterisks, P <.05 compared with sib-pairs with no identical allele. intrinsic property of the cell responding to the signal is important. Allergic inflammatory reactions involve the participation of many different cells. Peripheral blood basophils and tissue mast cells, for example, play an important role in allergic diseases by releasing potent inflammatory mediators such as histamine, leukotrienes, and prostaglandins on cross-linking of the membranebound IgE by an allergen. 17 Two kinds of stimuli induce histamine release from a basophil: IgE mediated and non IgE mediated. 18 Allergens and anti-ige induce histamine release by FcεRI on the cell surface; non-igemediated stimuli such as calcium ionophore A23187, complements, low-molecular-weight peptides, and lipids induce histamine release by non-ige-mediated pathways. In this study, to evaluate the pathways of IgE-mediated and non-ige-mediated histamine release, we measured HR from basophils to stimulation with anti-ige and calcium ionophore A23187, respectively. Histamine releasability is expressed by 2 index values such as sensitivity, expressed as the agonist s concentration needed to release 50% of total histamine released, and maximal HR to represent reactivity of mediator releasability. These 2 index values have been shown to be independent variables. 19 Maximal HR from basophils to anti-ige was higher in asthmatics than in nonasthmatic control subjects, but there were no significant differences in basophil sensitivity to anti-ige. 7 In this study the index of maximal HR was used to assess histamine releasability from basophils. This study showed that maximal HR to anti-ige was higher in asthmatic probands than in nonatopic nonasthmatic control subjects, but there were no significant differences in maximal HR to A This finding suggests that an IgE receptor mediated mechanism of mediator release plays an important role in the pathogenesis of asthma. IgE-dependent HR from basophils has not been clearly established as being under genetic control in asthma. An earlier twin study by Marone et al 8 estimated correlations in HR from basophils in response to anti-ige, f-met peptide, and calcium ionophore A The correlation

5 622 Kim et al J ALLERGY CLIN IMMUNOL SEPTEMBER 1999 among monozygotic twins was significantly greater than among dizygotic twins for anti-ige and A23187 but not for f-met peptide. These data suggest that HR to anti-ige and A23187 were determined in part by genetic factors, whereas genes probably had little effect on the release of histamine from f-met peptide. This study also showed that there was a significant positive correlation of maximal HR to anti-ige and A23187 between the probands and their siblings, although the association was more marked for A FcεRI on basophils have been identified and characterized. This receptor is composed of 3 subunits, α, β, and γ The α subunit, an integral membrane glycoprotein of the immunoglobulin supergene family, is responsible for ligand binding, whereas the γ subunit mediates both receptor assembly and signal transduction. Recent studies have begun to suggest a function for the β subunit of FcεRI. 21,22 The β subunit, like the γ subunit, contains a 19-amino acid motif in its cytoplasmic tail, found in many immune receptors. This motif, referred to as ARAM (antigen receptor activation motif) is both necessary and sufficient to mediate signaling through receptors. Thus the presence of a mutant β chain appears to influence signaling through this receptor. These data support the speculation that the mutation in the β subunit of FcεRI could result in modulation of FcεRI signaling, either by rendering the receptor more sensitive to ligand or by enhancing mediator release in response to receptor cross-linking. With use of the candidate gene approach, Sandford et al 9 reported that the gene regulating the β chain of the highaffinity receptor for IgE (FcεRI-β) is on chromosome 11q13. They described 2 code variants of FcεR-I-β 1181L and 1181L/V183L (originally Leu 181 and Leu 181/183). To the best of our knowledge, this is the first study to evaluate linkage between mediator releasability from basophils, intermediate phenotype of asthma and allergic diseases, and genotype of chromosome 11q13, in which the gene encoding FcεRI-β is located. This study showed that there was a significant linkage between maximal HR to anti-ige and gene marker of chromosome 11q13 by the affected and the QTL sib-pair analysis, but, as for A23187 stimuli, there was no significant linkage. This finding suggests that mutation(s) of gene(s) near chromosome 11q13, such as gene for FcεRI-β, contribute to control the expression of HR from basophils by a IgE receptor mediated mechanism. In conclusion, this study showed that maximal HR from basophils to anti-ige stimuli was linked to gene marker of chromosome 11q Ishizaka K, Ishizaka T. Human reaginic antibodies and immunoglobulin E. J Allergy 1968;42: Malveaux FJ, Conroy MC, Adkinson NF Jr, Lichtenstein LM. IgE receptors on human basophils: relationship to serum IgE concentration. J Clin Invest 1978;62: Sampson HA. Role of immediate food hypersensitivity in the pathogenesis of atopic dermatitis. J Allergy Clin Immunol 1983;71: McFadden ER. Pathogenesis of asthma. J Allergy Clin Immunol 1984;73: Lichtenstein SM, MacGlashan DW. The concept of basophil releasability. J Allergy Clin Immunol 1986;77: Casolaro V, Spadaro G, Marone G. Human basophil releasability. VI. Changes in basophil releasability in patients with allergic rhinitis or bronchial asthma. Am Rev Repir Dis 1990;142: Marone G, Poto S, Celestino D, Bonini S. Human basophil releaseability, III: genetic control of human basophil releasability. J Immunol 1986;137: Sandford AJ, Shirakawa T, Moffatt MF. Localisation of atopy and beta subunit of high-affinity IgE receptor (FceRI) on chromosome 11q. Lancet 1993;341: Shah S, Green JR. Disease susceptibility genes and the sib-pair method: a review of recent methodology. Ann Hum Genet 1994;58: Lander ES, Schork NJ. Genetic dissection of complex traits. Science 1994;265: Haseman JK, Elston RC. The investigation of linkage between a quantitative trait and a marker locus. Behav Genet 1972;2: Marsh DG, Neely JD, Breazeale DR, Ghosh B, Freidhoff IR, Ehrlich- Kautzky E, et al. Linkage analysis of IL4 and other chromosome 5q31.1 markers and total serum immunoglobulin E concentrations. Science 1994;264: MccGlashan DW, Lichtenstein LM. Studies of antigen binding on human basophil, I: antigen binding and functional consequences. J Immunol 1983;130: Marone G, Casolaro V, Cirillo R, Stellato C, Genovese A. Pathophysiology of human basophils and mast cells in allergic disorders. Clin Immunol Immunopathol 1989;50: Lichtenstein LM, Bochner BS. The role of basophils in asthma. Ann N Y Acad Sci 1991;629: Conroy MC, Adkinson NF, Lichtenstein LM. Measurement of IgE on human basophils: relation to serum IgE and anti-ige induced histamine release. J Immunol 1977;118: Lichtenstein LM. The mechanism of basophil histamine release induced by antigen and by the calcium ionophore A J Immunol 1975;114: MacGlashan DW Jr. Releasability of human basophils: cellular sensitivity and maximal histamine release are independent variables. J Allergy Clin Immunol 1993;91: Metzger H. The receptor with high affinity for IgE. Immunol Rev 1992;125: Alber G, Miller L, Jelsema CL, Varin-Blank N, Metzger H. Structurefunction relationships in the mast cell high affinity receptor for IgE: role of the cytoplasmic domains and of the beta subunit. J Biol Chem 1991;266: Jouvin MH, Adamczewski M, Numerof R, Letourneur O, Valle A, Kinet JP. Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor. J Biol Chem 1994;269: REFERENCES 1. Burrows B, Martinez FD, Halonen M, Barbee RA, Cline MG. Association of asthma with serum IgE levels and skin-test reactivity to allergens. N Engl J Med 1989;320:271-7.