The E237G polymorphism of the high-affinity IgE receptor chain and asthma

Zhang Xiaozhu, MD*; Zhang Weidong, MD; Qiu Diwen, MD; Sandford Andrew, PhD*; and Tan Wan Cheng, MD

Background: The chain of high-affinity IgE receptor (Fc RI ) has been proposed as a candidate gene for asthma and atopic diseases. Objectives: To determine the prevalence of the E237G polymorphism of the Fc RI gene and to investigate its association with asthma and total IgE levels in 3 Asian populations. Methods: A total of 291 asthmatic patients (141 Chinese, 68 Malay, and 82 Indian) and 355 asymptomatic blood donors (157 Chinese, 100 Malay, and 98 Indian) were recruited. The E237G genotype was determined by allele-specific polymerase chain reaction. Total serum IgE level was measured by enzyme-linked immunosorbent assay. Results: The G allele was more common in Chinese controls (17.9%) than in Malay (11.5%) (P .05) and Indian (9.2%) (P .01) controls. Genotypes with the G allele were more prevalent in asthmatic patients in the Chinese population (odds ratio, 1.97; 95% confidence interval, 1.053.77; P .04). Conclusions: There were interethnic differences in the frequencies of the G variant among Chinese, Malay, and Indian populations. The E237G polymorphism of Fc RI may be a risk factor for asthma in the Chinese population.
Ann Allergy Asthma Immunol. 2004;93:499503.

INTRODUCTION The high-affinity IgE receptor (Fc RI) is critical in the process of induction and maintenance of an allergic response in mast cells and basophils. This receptor is composed of 3 subunits ( , , and ). The subunit is an important modulator of the full signaling capacity of the entire Fc RI receptor, which not only amplifies the signal generated by the chains1 but also increases the expression level of surface Fc RI.2 The Fc RI gene is located in chromosome 11q13, a locus that was linked to atopy in a variety of linkage analyses.37 The Fc RI gene has been proposed as a candidate for atopic diseases. Several polymorphisms in the Fc RI gene have been identified, including 3 coding variants: I181L and I183V 8 in exon 6 and E237G in exon 7.9 The variant Leu181 was first identified in British allergic asthmatic probands, was maternally inherited in each, and showed a strong association with atopy.8 The I181L polymorphism has also been identified in Australian,10 Italian,11 and South African white and black12 populations and showed a high prevalence in Kuwaiti Arabs.13 The V183L polymorphism was found in only 1 patient, who also had the I181L variant.8 However, I181L and I183V were not detected in other population samples,14,15 especially in Asian populations.16 18

Associations were first observed between E237G and atopy, asthma,9 and bronchial hyperresponsiveness.9,19 Studies also demonstrated that the E237G polymorphism was more prevalent in Asian populations than in white populations.17,20 Studies performed in Japanese populations found associations between E237G and atopic asthma,17 elevated IgE levels,17,21 and allergic rhinitis.21 In this study, we determined the prevalence of E237G in Asians by studying a group of well-defined asthmatic patients and asymptomatic blood donors, and we evaluated the association of the polymorphism with asthma and serum total IgE levels. METHODS Subjects Two hundred ninety-one asthmatic patients (141 Chinese [Han], 68 Malay, and 82 Indian) and 355 unselected blood donors (157 Chinese [Han], 100 Malay, and 98 Indian) were recruited (Table 1). Ethnicity was defined by the study participants themselves. All the patients either were born in Singapore or had resided in Singapore for more than 30 years. All the asthmatic patients were adults who had physiciandiagnosed asthma and had multiple admissions for severe acute asthma to the National University Hospital of Singapore. They have marked airway responsiveness, as shown by documented evidence of more than 15% improvement in forced expiratory volume in 1 second after administration of 400 g of inhaled salbutamol or a 2-week course of 20 mg of oral prednisolone.22 A modified American Thoracic Society questionnaire concerning respiratory symptoms was administered to all asthmatic patients.23 Information on asthma medications, smok-

* The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, University of British Columbia, St. Pauls Hospital, Vancouver, British Columbia. Department of Medicine, National University of Singapore, National University Hospital, Singapore. Received for publication June 1, 2004. Accepted for publication in revised form August 9, 2004.

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Table 1. Characteristics of the Study Subjects Chinese Asthma (n 141) Age, mean SD, y Sex, M/F, No. Childhood onset of asthma, % Family history of asthma, % Positive skin prick test result, % 52 16* 64/77* 41.1 41.4 70 Control (n 157) 32 9 104/53 0 NA NA Asthma (n 68) 45 14* 25/43* 40.9 57.3 56.7 Malay Control (n 100) 34 9 55/45 0 NA NA Asthma (n 82) 50 17* 32/50* 42 57.5 63 Indian Control (n 98) 34 10 59/39 0 NA NA

Abbreviation: NA, not available. * There was a significant difference between asthmatic patients and controls in age and sex (P

.05).

ing status, and individual and family histories of pulmonary disease was also collected. A positive family history of asthma and childhood onset of asthma were ascertained by direct questioning of the patients. A positive family history was assumed if any parent or sibling had physician-diagnosed asthma. A diagnosis of childhood-onset asthma was assumed if the first physician-diagnosed asthma occurred at 12 years or younger. Skin prick testing for atopy to 19 common allergens, including house dust mite (Greer Laboratories, Lenoir, NC), was performed on all patients with asthma. Positive skin prick test results were defined as a wheal size of at least 5 mm greater than the negative control. This was the criterion used in the Lung Function Laboratory of the National University Hospital of Singapore, and it has been validated in published studies,24,25 which showed strong correlations between positive skin test results and elevated total and specific serum IgE levels. Control subjects were asymptomatic individuals who presented themselves as blood donors at the Singapore Blood Transfusion Service of National University Hospital of Singapore. None of the controls had a history of atopy or any respiratory disease. Fc RI E237G Genotyping Screening for the E237G variant was performed using the amplification refractory mutation system. The primers used in this study were B7FA1: TGG CCA GCT AGT CTG GTT

TGG TTT TCT GGA; B7FA2: GGA GCA TAT TAA GGT GGA CAG AAG CAG CAG; B7M1: ATT CAG CTA CTT ACA GTG AGT TGG AAG ACC CAG GCG G; and B7W2: CAC GTG ATT CTT ATA AAT CAA TGG GAG GAG ACA ATT.9 Amplified products were detected in a 2.5% (wt/vol) agarose gel stained with ethidium bromide. Thirty percent of the samples were genotyped twice to test the genotyping reproducibility. Total Serum IgE Measurement Total serum IgE levels were measured in all asthmatic patients by enzyme-linked immunosorbent assay (IBL GmbH, Hamburg, Germany) but not in the control groups because no serum samples were available from the blood donors. Statistical Analysis The Hardy-Weinberg equilibrium was tested in the control group using the 2 test. Allele frequencies between different groups were compared using 2 analyses. The associations between asthma, total serum IgE level, and genotypes were analyzed using logistic regression. Total serum IgE levels were considered to be continuous variables. Asthma was considered to be a binary variable. Because age and sex may be associated with these phenotypes, they were included as covariates in all models. The significance level was set at .05 for all analyses. The statistical analyses were performed using the JMP statistical software package (SAS Institute Inc, Cary, NC).

Table 2. Genotype and Allele Frequencies of the E237G Polymorphism in Asthmatic Patients and Controls* Chinese Genotype Asthma (n 141) 81 (57.4) 60 (42.6) 219 (77.7) 63 (22.3) Control (n 157) 108 (68.8) 49 (31.2) 258 (82.2) 56 (17.8) Asthma (n 68) 49 (72.1) 19 (27.9) 117 (86) 19 (14) Malay Control (n 100) 77 (77.0) 23 (23.0) 177 (88.5) 23 (11.5) Asthma (n 82) 71 (86.6) 11 (13.4) 152 (92.7) 12 (7.3) Indian Control (n 98) 80 (81.6) 18 (18.4) 178 (90.8) 18 (9.2)

E/E E/G E G

G/G

* Data are given as number (percentage). P .04; odds ratio, 1.97; 95% confidence interval, 1.053.77.

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Table 3. Genotypes of the E237G Polymorphism and Total Serum IgE Levels in Patients with Asthma Chinese (n Genotype % E/E E/G 57.9 42.1 140) % 72.1 27.9 Malay (n 68) % 86.4 13.6 Indian (n 81)

IgE, mean SE, IU/mL 247 248 30 30

IgE, mean SE, IU/mL 375 341 47 60

IgE, mean SE, IU/mL 367 446 36 65

G/G

RESULTS The characteristics of the study participants are summarized in Table 1. The distribution of allele and genotype frequencies is given in Table 2. The overall observed genotype distribution in each control group conformed to the expectations based on Hardy-Weinberg analysis. In this study, G/G homozygotes were very few, only 3 in Chinese asthmatic patients, 7 in Chinese controls, and 1 in Indian asthmatic patients. Therefore, in the statistical analyses, we combined G/G homozygotes with G/E heterozygotes as one group. Ethnic variation in the prevalence of the G allele was observed. In the control group, the G allele was more common in the Chinese (17.9%) than in the Malays (11.5%; P .05) and the Indians (9.2%; P .01). In addition, E/G G/G genotypes were significantly more prevalent in Chinese asthmatic patients (42.6%) compared with Chinese controls (31.2%; odds ratio, 1.97; 95% confidence interval, 1.053.77; P .04). However, there was no significant difference in genotype frequency between cases and controls among Malays and Indians. There was no significant difference between total serum IgE levels in different genotypic groups (Table 3). DISCUSSION The E237G polymorphism was not common in white populations. The frequency of patients with the G variant is usually approximately 3% to 5% in general white populations.9,15,20,26 In contrast, 237G was more prevalent in Asian populations. In this study, the G variant was found in 31.2% of Chinese controls, 23% of Malay controls, and 18.4% of Indian controls. It was also noted that the frequencies of G/G homozygotes were very low only 6 were detected in 646 study participants. This was similar to the results of a study17 performed in Japan in which no homozygote was identified among 300 study participants. In addition, this is the first report of the prevalence of the E237G polymorphism in the Malay and Indian populations and extends the previous information concerning the Chinese population. The high prevalence of the 237G variant in the Chinese population may imply its potential population contributable risk. Our results suggested that the E237G polymorphism in the chain of Fc RI may be a risk factor for asthma in the Chinese population. This observation was consistent with another study27 performed in southern China. In that study, the authors compared the frequency of the E237G polymorphism in 60 Chinese asthmatic patients and 65 Chinese controls. Contrastingly, we genotyped 141 Chinese asthmatic patients and 157 Chinese controls, more than double that

sample size. In addition, our study also included 2 other ethnic groups, although we did not find any significant association between the E237 polymorphism and asthma in either. It has been known that Fc RI is essential for the induction and maintenance of an allergic response. The chain, in particular, is critical to setting the level of cellular response to IgE and antigens through its 2 amplification capacities.1,2,28 Studies have observed that the combined signaling and expression amplification resulted in an estimated 12- to 30-fold amplification of downstream events.2 The polymorphism E237G is located in the intracellular tail of , just downstream of an immunoreceptor tyrosine-based activation motif essential for function,29 and it could affect Fc RI function. However, Donnadieu et al30 suggested that the E237G variant had no direct effect on the amplifier functions. Therefore, one possible explanation for the positive association in this study could be the linkage disequilibrium of E237G with other potential functional polymorphisms, eg, the polymorphisms found in the regulatory and promoter regions of the gene, which may affect receptor expression and be important in the pathogenesis of asthma.15,31,32 In addition, a recent study33 observed that the Fc RI gene encodes 2 protein isoforms. One is full-length chain, and the other is a truncated chain, which inhibits the cell surface expression of Fc RI, and the relative abundance of the 2 different isoforms of chain may control the level of Fc RI surface expression and affect susceptibility to allergic diseases. The E237G variant might affect the chain splicing, produce more full-length chain, and result in more functional IgE receptor and higher allergic response. It is also possible that the variant could change the binding affinity of the receptor and result in a prolonged allergic effect. A study9 performed in an Australian population found that 237G-positive individuals had a higher risk of having asthma compared with wild type (relative risk, 2.3). A study17 in a Japanese population suggested that the G allele of Fc RI was associated with atopic asthma, particularly childhood atopic asthma (odds ratio, 3.9). Our finding is consistent with these studies. There are 3 limitations to the present study. First, after classifying the study population into 3 ethnic groups, the numbers of individuals in each subgroup were relatively small. Given the genotype frequencies in asthmatic patients and controls in the Indian and Malay populations, we could detect a relative risk of 2.6 and 2.7 or greater, respectively, with 80% statistical power at the .05 significance level. If the relative risk was smaller than that we could not detect it in the given sample sizes. Second, although the controls in this

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study were volunteer blood donors with no history of asthma or atopy, we could not exclude the possibility that some of these individuals may develop clinical atopy or asthma in the future. Third, because the control subjects were volunteer blood donors, the age and sex distribution could not be precisely matched. To correct for this discrepancy, we used logistic regression to adjust for age and sex differences between asthmatic patients and controls. Although this adjustment is not as good as having sufficient matched controls, this is a well-accepted and validated approach that is commonly used in studies such as this. However, the strength of this study lies in the unique multiethnic comparison of asthmatic patients and controls in a socially, culturally, and geographically homogenous living environment. Singapore is located 2 north of the equator and has a tropical climate. The country consists of 3 major ethnic categories: Chinese (76.7%), Malays (14%), and Indians (7.9%). Most of the islands population lives in public housing tower blocks. Most Singaporeans described themselves as middle class. Singaporeans generally enjoy good health and high standards of nutrition and environmental sanitation, as indicated by their long life expectancy (76 years for men and 80 years for women) and low infant mortality rates. Therefore, genetic predisposition may play an important role in the development of asthma given the largely uniform nature of the relevant environmental factors. We found that 237G was associated with asthma in the Chinese population, indicating that this allele is a genetic risk factor for asthma in this ethnic group. The sample size (291 patients with asthma and 355 controls), although not large, still compared favorably with other candidate gene studies for asthma with ethnic comparisons. Green et al12 studied 93 white and black asthmatic patients and 95 white and black controls, and Zhu et al20 compared 281 white patients with 22 Asians. Thus, the significant difference in G allele frequency among different ethnic groups is likely to be accurate and reflective of the general population of Singapore. ACKNOWLEDGMENTS This work was supported by grant R-172-000-112-213 from National Medical Research of Singapore. We are grateful to Ms. Kelly Ngerng and Ms. Feng Yiping for their help in patient recruitment. REFERENCES
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Requests for reprints should be addressed to: Tan Wan Cheng, MD Department of Medicine National University of Singapore Lower Kent Ridge Road National University Hospital Singapore 119074 E-mail: mdctanwc@nus.edu.sg

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