INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CROSS-LINKING OF HIGH-AFFINITY IgE receptors (FcRIs) by IgE and multivalent antigens on the surface of mast cells and eosinophils is an important early step in the sequence of atopic reactions. FcRI is a heterotetrameric protein consisting of one alpha, one beta, and two gamma chains found exclusively on mast cells, eosinophils, basophils, monocytes, and Langerhans' cells (5, 8, 9, 11). The binding of IgE via the alpha chain of the receptor is an important factor regulating the number of FcRIs on the cell surface (4, 17).

The FcRI alpha chain gene is located on chromosome 1q23 in humans and consists of five exons and a 5'-untranslated region with known promoter activity. It is known that there is a direct relationship between the level of expression of FcRI and IgE levels in the blood of both humans and mice (4, 15, 17). We reasoned that naturally occurring DNA sequence variants in the FcRI alpha chain gene might be associated with IgE levels; thus factors that could modify the expression of FcRI could be associated with circulating IgE levels. To test this hypothesis, we used single-stranded conformational polymorphism (SSCP) analysis to screen genomic DNA derived from asthmatic subjects for sequence variation in the core promoter and all exons (including exon-intron boundaries) of the FcRI alpha chain gene. No coding region single nucleotide polymorphisms (SNPs) were identified. A total of three polymorphisms were identified, one of which showed an association with IgE levels in an asthmatic population.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Subjects. DNA was extracted by standard techniques from peripheral blood drawn from 389 patients with asthma diagnosed according to American Thoracic Society criteria (1). Patients consisted of 305 Caucasians and 84 African-Americans, whose only asthma medication was inhaled albuterol used on an as-needed basis. These patients were enrolled at over 20 centers in the United States as part of a drug treatment trial. They had disease of moderate severity, as indicated by an average forced expiratory volume in 1 s of 62.3% of predicted when tested at least 8 h after inhaled albuterol. Control DNA from 341 normal individuals (201 Caucasians and 140 African-Americans) without a history of asthma or atopy (as determined by responses to a questionnaire) was also obtained; these subjects were all enrolled in Boston.

PCR SSCP analysis. Genomic DNA was screened for mutations by SSCP analysis according to the method of Orita et al. (12) with minor modifications. Oligonucleotide primers (listed in Table 1) were designed from the published sequence (GenBank accession number L14075) (13). Forward and reverse primers were end labeled with [-³²P]ATP (DuPont NEN, Boston, MA) by using polynucleotide kinase (Boehringer Mannheim, Mannheim, Germany). PCR reactions were performed according to the methods previously described by our laboratory (2) with minor modifications. The radioactive amplified products were electrophoresed, transferred to chromatography paper (Whatmann, Maidstone, UK), and exposed to X-ray film after drying.

View this table: [in this window] [in a new window]   Table 1.   PCR primers for genotyping the FcRI alpha chain gene

Restriction fragment length polymorphism analysis. Restriction fragment length polymorphism analysis was used to genotype at the T/C-335 locus. PCR was performed with the following primers: forward 5'-CATATGACTAAGAGTTTGACTTAGG-3' and reverse 5'-GGCATAGGTCTAGCACAATC-3'. PCR products were digested with Mnl I (New England Biolabs, Beverly, MA), according to the manufacturer's instructions and resolved by electrophoresis. DNA with the homozygous T genotype produced a digest pattern consisting of 106 and 232 bp; the heterozygous T/C genotype produced a digest pattern consisting of 73, 106, 159, and 232 bp, and the homozygous C genotype produced a digest pattern consisting of 73, 106, and 159 bp. Selected samples (5 samples per each genotype) were confirmed by direct sequencing with complete concordance at each locus.

IgE levels. Total plasma IgE levels of asthmatic patients and controls were measured by using the UniCAP system (Pharmacia and Upjohn, Uppsala, Sweden), according to the manufacturer's instructions.

Stratification analysis. Evidence of population stratification between the high and low IgE groups was sought by the method of Pritchard and Rosenberg (14). Participants were genotyped at 40 candidate SNPs selected from the SNP consortium project website (http://snp.cshl.org/genome. shtml). SNPs were selected to be at least 20 Mb apart, and thus are unlinked. Under the null hypothesis of genotype frequencies in the high and low IgE groups at each marker locus, contingency tables were constructed for each homozygous-wild type vs. (heterozygous + homozygous) mutant genotype (recessive model), and a ² statistic was calculated. Because our interest is in whether loci show genotype-frequency differences as a group, the test statistics from each locus were summed as X = ₌₁ X, where X is the ² statistic computed at the ith marker locus and L is the set of unlinked marker loci typed in all individuals. Under the null hypothesis, H₀, X is ² distributed, with degrees of freedom equal to the sum of the degrees of freedom of individual loci. In the RESULTS section, the mean number (±SD) of genotypes at each locus is provided along with a distribution of ² values.

Statistical analysis other than for population stratification. Associations between allele frequency and asthma phenotype, IgE level, or race were determined by ² analysis. Consistency of genotype frequencies with Hardy-Weinberg equilibrium was tested with goodness of fit for a ² test on a contingency table of observed vs. predicted genotype frequencies. Results are expressed as fractions and considered statistically significant at the P < 0.05 level. Computations were performed with StatView 4.0 software (Abacus Concepts, Berkeley, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We screened 1.3 kb of the 5' flanking region and all five exons (1.4 kb), including exon-intron junctions, of FcRI alpha chain gene in 389 asthmatic patients for DNA sequence variants by PCR SSCP analysis. Three SNPs (Fig. 1) were detected; each was located in the 5' flanking region. No sequence variants were detected in the coding region of the FcRI alpha chain gene. Once variants were detected by SSCP and confirmed by direct sequencing, the 389 asthmatic patients and 341 normal controls were genotyped by restriction fragment length polymorphism (T/C-335) or SSCP (A/C-770 and G/A-664). Genotype data are summarized in Table 2. The most common polymorphism consisted of a T/C transition at 335 bp from the translation start site and had a minor allele (T) frequency of 0.36 for all genotyped individuals. The A/C transversion at 770 bp from the ATG start site had a minor allele (C) frequency of 0.045, and the G/A transition at 664 had a minor allele (A) frequency of 0.030. 

View larger version (67K): [in this window] [in a new window]   Fig. 1.   Three sequence variants were identified by single-stranded conformational polymorphism (SSCP) analysis in the FcRI alpha chain gene. A: detection of A/C-770 variant in 5' flanking region. Lanes 2 and 6 are heterozygous, whereas lanes 1, 3, 4, 5, 7, and 8 are wild type. B: detection of G/A-664 variant. Lane 2 is heterozygous, and lane 10 shows no wild-type allele; all other lanes are wild type. C: detection of T/C-335 variant in 5' flanking region. Lanes 1, 7, and 8 are T/C. Lanes 2, 3, 4, and 6 are TT. Lane 5 is CC.

View this table: [in this window] [in a new window]   Table 2.   Genotype frequencies of sequence variants in FcRI alpha chain gene

All sequence variants were in Hardy-Weinberg equilibrium, except those at G/A-664 in both Caucasian and African-American asthmatic patients when stratified by race; there were no significant differences in allele frequencies or genotypes between asthmatic patients and nonasthmatic patients. The frequency of the T/C-335 polymorphism differed significantly between Caucasians and African-Americans in both the asthmatic population (0.423 vs. 0.146, respectively; P < 0.0001) and the normal population (0.445 vs. 0.161, respectively; P < 0.0001). The allele frequencies of A/C-770 were marginally different between Caucasian asthmatic patients and African-American asthmatic patients (P = 0.039).

The total average IgE levels of the Caucasian and African-American asthmatic patients were 307.46 ± 441.83 and 401.02 ± 445.03 kU/l, respectively. The total IgE levels of Caucasian and African-American controls were 49.38 ± 100.75 and 145.64 ± 376.31 kU/l, respectively. There was substantial variance in the IgE levels (Fig. 2). To determine whether there was a relationship between genotype and IgE level, we examined the distribution of genotypes in the upper and lower quartiles of the IgE distribution. There was a greater proportion of genotype CC (T/C-335) than of TT or TC in the Caucasian patients in the lowest quartile of IgE level compared with Caucasian patients in the highest quartile (P = 0.0.025; Table 3). A similar difference was not observed in African-American asthmatic patients. There were no significant genotype-stratified differences in forced expiratory volume in 1 s, total eosinophil counts, or total IgE levels.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Total IgE levels in asthmatic patients and controls. A: total IgE levels of Caucasians. B: total IgE levels of African-Americans. There were significant differences between patients with asthma and normal controls (P < 0.0001), between Caucasian asthmatic patients and African-American asthmatic patients (P = 0.006), and between Caucasian controls and African-American controls (P < 0.0001).

View this table: [in this window] [in a new window]   Table 3.   Analysis of total IgE levels and genotype results of T/C-335 in FcRI alpha chain gene

Stratification analysis. The total number of genotypes for the stratification analysis of high and low IgE groups was 1,644, with an average of 41.1 ± 6.4 genotypes per locus. No evidence of stratification was detected within the high and low IgE groups ( = 30.6, P = 0.86). The distribution of each genotype as a function of ² value is provided (Fig. 3). Only 1 of 40 loci, other than 335, was significantly different between high and low IgE groups, and the strength of the association was greatest for the 335 locus; locus 335 had the highest ² value of all genotypes (² = 5.04, P = 0.025).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Histogram of 2 values from contingency tables comparing the number of subjects having two polymorphic alleles at a given locus with those having no or one polymorphic allele. Among the 40 test loci, only 1 had a 2 value exceeding 4, whereas the locus of interest in the alpha chain in the FcRI had the highest 2 value.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The alpha chain of the FcRI demonstrates a remarkable consistency of its DNA sequence in the coding region. Because on average at least 1 SNP per 1,000 of coding sequence is expected (3), this observation suggests that there have been evolutionary pressures to retain the intact sequence. Although the coding region was not polymorphic, we found three SNPs in the 5' flanking region of the FcRI alpha chain. The most common SNP, with an allele frequency of 0.47 in Caucasian asthmatic patients, demonstrated an association with IgE levels.

We used SSCP analysis, which has about a 70% efficiency in identifying variants (10), to screen for DNA sequence variants in the 5' untranslated region and coding exons of the gene for the alpha chain of the FcRI. We identified no sequence variants in the coding region of the gene. Because we screened 542 chromosomes, we had theoretical power of >99% to identify alleles with a frequency of 0.04 (7). Even if the <70% efficiency of SSCP reduced the effective number of chromosomes screened to 389, we still had 98% power to identify sequence variants at this minor allele frequency. We acknowledge, however, that there may be variants that are not resolved by SSCP and that may have escaped detection by this method.

The lack of DNA sequence variants in the coding region of the alpha chain of FcRI suggests that maintaining fidelity in this region of the genome is important to homeostasis. Sequence variants in the FcRI beta chain gene have been reported (6, 16) that may be associated with atopy or bronchial hyperresponsiveness. There are no data available on sequence variants in the gamma chain. An interpretation of these data is that binding of IgE by the alpha chain of the FcRI is an important event in atopic diathesis.

Although we found no coding region variants in the alpha chain, we found variants in the 5' untranslated region of the gene. Two of the variants were uncommon, with minor allele frequencies of <0.05, whereas the T/C transition at 335 was relatively common, with a minor T allele frequency of 0.47 in Caucasian asthmatic patients. The frequency of the T allele in African-American asthmatic patients was much lower (0.15). Thus this allele is one with marked differences in frequency among individuals of varying ethnicity.

In the entire cohort of Caucasian asthmatic patients, the T/C transition was not associated with IgE levels. That is, patients with a TT genotype did not have a different mean IgE level than patients with a CC genotype. We reasoned, however, that there may be multiple pathways leading to altered IgE levels and thus examined the population extremes to determine whether there was a significant difference in the proportion of genotypic assignments between Caucasian asthmatic patients with high and low IgE levels (Table 3). Our data show that there were nearly equal proportions of TT + TC and CC genotype among the individuals with the lowest IgE levels, whereas the proportion of patients with the CC genotype was lower among Caucasian asthmatic patients with the highest IgE levels. We speculate that, in Caucasian asthmatic patients, the CC genotype skews toward a lower IgE response. The same trend was not observed in African-American asthmatic patients, thus suggesting that the panel of genes controlling IgE levels differs in different ethnic groups.

Because case-control association studies may be confounded by population substructure, we subjected the DNA from patients in the upper and lower quartiles of IgE levels to an analysis to see whether there were underlying identifiable genetic differences between the two groups unassociated with the loci identified at the alpha chain of the FcRI. Our analysis shows that, among 40 polymorphic loci scored, only one, as would be expected, had a ² P value of <0.05 (Fig. 3). On the basis of this analysis, it is unlikely that our finding is spurious. Although a functional effect of the C/T transition has not been identified, our observation opens this region of the alpha chain of the FcRI to such study.

It is known that circulating IgE levels are closely related to the levels of expression of FcRI. Because the alpha chain is the binding unit for IgE, we speculate that the T/C transition at position 335 of the alpha chain of FcRI leads to lower IgE binding and represents a potential mechanism responsible for our finding. In following this line of reasoning, it seems reasonable to speculate that the microenvironmental availability of the alpha chain of the FcRI may participate in the regulation of IgE levels.